EMBO/soda-nel huggingface dataset:
GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring (2020)
Figure 7

Figure 7. GSK3ɑ spine anchoring requires tau.

A. Representative confocal images from EGFP-GSK3α FRAP experiments in organotypic slices cultured from C57 wild-type mice. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm.

B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in wild-type mouse slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines).

C, D. As A, B but in slices cultured from C57 tauKO mice. Scale bars = 1 µm.

E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 13, 11 and 12 spines for wt baseline, cLTD and recovery and 9, 7 and 5 spines for tauKO baseline, cLTD and recovery groups, respectively.

F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α in wild-type or tauKO slices in 'baseline', 'cLTD' or 'recovery' conditions. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.
GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring (2020)
Figure 8

Figure 8. Synaptic depression induced by GSK3ɑ activity requires tau.

A. Experimental scheme for B-F. Whole-cell patch-clamp recordings were made from nearby pairs of uninfected and infected neurons expressing EGFP-GSK3α in CA1 of organotypic hippocampal slices from C57 wt and tauKO mice. EPSCs were elicited with CA3 Schaffer collateral stimulation.

B. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α-expressing neurons (green traces) in C57 wt or tauKO slices.

C-F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in C57 wt (C) or tauKO slices (D), or at +40 mV (NMDAR-mediated EPSCs) in wt (E) or tauKO slices (F). Red circles represent the averages of all pairs in a given plot, and n denotes the number of cell pairs. In all cases, error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.

EXPANDED VIEW FIGURE LEGENDS
Glutamine deprivation alters the origin and function of cancer cell exosomes (2020)
Figure 2

Figure 2. Exosome biogenesis in Rab11-compartments of Drosophila secondary cells is ESCRT-dependent

(A-D) Wide-field fluorescence images of basal views through living SCs expressing a GFP-tagged form of Btl-GFP (yellow) in non-acidic compartments, with SC outline approximated by dashed white circles. Acidic compartments are marked by LysoTracker Red® (magenta). Boxed non-acidic compartments in merge images are magnified in A-D, Zoom. On the right, lower magnification confocal transverse images of fixed accessory gland (AG) lumens are shown with dotted lines indicating the basal side of the AG epithelial layer, which contains several fluorescent SCs (highlighted by arrows). (A) SC with no RNAi construct expressed (control) and AG lumen from same genotype. Btl-GFP-positive compartments containing fluorescent ILVs (in Btl-GFP panel), ILV membranes inside compartments (in Zoom panel) and secreted fluorescent puncta (AG lumen panel) are marked by arrowheads. (B) SC also expressing RNAi construct targeting ESCRT-0 subunit, Stam, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta (AG lumen) are strongly reduced. (C) SC also expressing RNAi construct targeting ESCRT-I subunit, Vps28, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta (AG lumen) are strongly reduced. (D) SC also expressing RNAi construct targeting ESCRT-III subunit, shrb, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta are strongly reduced.

(E) Bar chart showing the number of large (diameter greater than one micrometre) non-acidic Btl-GFP-positive compartments per SC. Data from 39 SCs (three per gland) are shown.

(F) Bar chart showing percentage of these large Btl-GFP compartments containing Btl-GFP-positive ILVs in control and ESCRT knockdown SCs. Data from 39 SCs (three per gland) are shown.

(G) Bar chart showing the total number of Btl-GFP fluorescent puncta in ten Z-planes from the AG lumen following ESCRT knockdown in SCs, compared to controls without knockdown. Data from at least 17 AG lumens per condition are shown.

Data information: All data are from six-day-old male flies shifted to 29°C at eclosion to induce expression of transgenes. Genotypes are: w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] with no knockdown construct (A), UAS-Stam-RNAi (HMS01429; B), UAS-Vps28-RNAi (v31894; C) or UAS-shrb-RNAi (v106823; D). Scale bars in A-D (5 µm); in A-D Zoom (1 µm); in A-D, AG lumen (20 µm). Data were analysed by one-way ANOVA. ****P < 0.0001, ***P < 0.001 relative to control. Bars and error bars in E-G denote mean +/- SD.
Glutamine deprivation alters the origin and function of cancer cell exosomes (2020)
Figure 3

Figure 3. Rab11a labels a distinct subset of multivesicular bodies and their intraluminal vesicles in HCT116 colorectal cancer cells

(A-D) Confocal images of fixed HCT116 cells, with boxed regions enlarged to the right. DAPI (grey) marks nucleus. Rab11a antibody is isoform-specific except in (F). (A) Rab11a (yellow) is located in compartments distinct from the late endosomal and lysosomal marker, LAMP2 (magenta). (B) CD63 (magenta) predominantly co-localises with the late endosomal and lysosomal marker, LAMP1 (yellow). (C) Rab11a (yellow) is located in compartments distinct from CD63 (magenta) under glutamine-replete conditions. (D) Rab11a (yellow) is located in compartments distinct from CD63 (magenta) under glutamine-depleted conditions.

(E) Super-resolution 3D-SIM image of fixed HCT116 cell expressing GFP-Rab11a (yellow), and stained with CD63 (magenta). DAPI (grey) marks nucleus. Boxed Rab11a-positive compartments, which frequently cluster, are magnified in Merge Zoom. This panel is further magnified in Merge Zoom x 2, revealing GFP-Rab11a (arrows in right panel) inside compartments.

(F) Wide-field fluorescence image of fixed HCT116 cells, stained with Rab11a (yellow) and Rab7 (magenta), expressing constitutively active GFP-tagged Rab5 (GFP-Rab5CA; cyan), which stalls endosomal maturation and produces enlarged Rab5-positive endosomes. One of these is boxed in the Merge and magnified in Merge Zoom, revealing internal puncta marked by Rab11a (arrows) and Rab7 (arrowheads) and limiting membrane subdomains of Rab11a (yellow arrowhead) and Rab7 (magenta arrowhead). DAPI (grey) marks nuclei.

Data information: Scale bars in A-F (5 µm), in Merge Zoom (1 µm), and in Merge Zoom x 2 (0.5 µm).
Glutamine deprivation alters the origin and function of cancer cell exosomes (2020)
Figure 4

Figure 4. Reduction in extracellular glutamine induces a switch to Rab11a-positive exosome secretion in HCT116 colorectal cancer cells

(A) Western blot analysis of putative exosome markers in lysates from HCT116 cells cultured in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) medium for 24 h. Gel was loaded with equal amounts of protein (total cell lysate protein was reduced by 19 ± 4 % after glutamine depletion). The activity of mTORC1 was assessed via phosphorylation of S6 and 4E-BP1, using phospho-specific antibodies and a pan-4E-BP1 antibody, where the hyper-phosphorylated form of 4E-BP1 produced by mTORC1 gives the slowest migrating γ band. Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates.

(B-F) Western blot analyses of EV preparations. (B) EVs isolated by ultracentrifugation (UC) of medium from HCT116 cells cultured in glutamine-replete and -depleted conditions for 24 h, and loaded according to cell lysate protein levels to compare secretion on a per cell basis, as shown in bar chart from four biological replicates. (C) EV preparation isolated by size-exclusion chromatography (SEC) from glutamine-depleted HCT116 cells and then separated by high-resolution iodixanol-PBS density centrifugation. Note that exosomes (exos) and microvesicles (MVs) are found in only partially overlapping fractions and the pattern of Rab11a separation mirrors that of exosome markers (primarily 1.064 - 1.085 g/ml). (D) EVs isolated by UC under glutamine-depleted conditions as in (B) and then subjected to Proteinase K (Prot K) digestion in the presence or absence of Triton® X-100 (Triton-X). Only membrane-associated CD81 is digested in the absence of Triton® X-100, while CD63 is resistant to digestion, even in the presence of Triton® X-100. SEC-isolated EVs also behave similarly (Appendix Fig S6C). (E) EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD63 antibodies coupled to magnetic beads. This method only pulls down a fraction of CD63, so the protein from pull-down of eight times the input is loaded for comparison. Approximately the same low levels of Rab11a are captured by control IgG and anti-CD63 beads. (F) EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD81 antibodies coupled to magnetic beads. All or most CD63 and CD81 appears to be pulled down, but less than 10% of Rab11a and Cav-1 is captured. NTA analysis suggests about 10% of all particles are pulled down with this approach.

Data information: Bar charts derived from at least three independent experiments and analysed by the Kruskal-Wallis test: *P < 0.05; n.s. = not significant. Bars and error bars denote mean +/- SD. Significantly decreased levels are in blue and increased levels are in red.
Glutamine deprivation alters the origin and function of cancer cell exosomes (2020)
Figure 5

Figure 5. Reduction in extracellular glutamine or Akt/mTORC1 signalling induces a switch to Rab11a-positive exosome secretion in three different cancer cell lines

Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein

(A) EVs isolated by UC of medium from HeLa cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24 h (see Appendix Fig S7A'' for analysis of SEC-isolated EVs).

(B) EVs isolated by size-exclusion chromatography (SEC; fractions two to seven) from LNCaP cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24 h.

(C) EVs isolated by SEC from LNCaP cells cultured in the presence or absence of 120 nM Torin 1 for 24 h.

(D) EVs isolated by UC from HCT116 cells cultured for 24 h following three days of raptor or non-targeting (NT) shRNA knockdown.

(E) EVs isolated by SEC from HCT116 cells cultured in the presence or absence of 3 µM Akt inhibitor AZD5363 for 24 h.

(F) EVs isolated by SEC from HCT116 cells cultured under glutamine-replete conditions in the presence or absence of 10 µM KRAS inhibitor BI-2852 (or control compound Bl2853) for 24 h.

(G) Table summarising the EV protein analyses in Figs 4 and 5 (normalised to cell lysate protein levels). Data analysed by the Kruskal-Wallis test: *P < 0.05. Significantly decreased levels are in blue and increased levels are in red.

Data information: Bar charts derived from at least three independent experiments and analysed by the Kruskal-Wallis test: *P < 0.05; n.s. = not significant. Bars and error bars denote mean +/- SD. Significantly decreased levels are in blue and increased levels are in red.
Glutamine deprivation alters the origin and function of cancer cell exosomes (2020)
Figure 7

Figure 7. EVs Induced by glutamine depletion promote amphiregulin- and ERK-dependent growth in HCT116 recipient cells

(A) Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete or -depleted HCT116 cells or vehicle (PBS). Bar chart shows ratio of p-ERK to ERK levels from triplicate independent experiments.

(B) Bar chart shows HCT116 recipient cell growth over 120 h, after pre-treatment with EV preparations isolated as in (A) or with PBS, and then incubation for the first 24 h of culture in the presence or absence of the ERK inhibitor SCH772984 (1.00 µM). Growth curves are shown in Appendix Fig S8C.

(C) Western blot analysis showing levels of the EGFR ligand, Amphiregulin (AREG) in EVs isolated by SEC of medium from HCT116 cells cultured in glutamine-replete (2.00 mM) or -depleted (0.15 mM) conditions for 24 h. Gel loading was normalised to cell lysate protein levels. AREG's molecular weight (approximately 26-28 kDa) suggests it is in its membrane-associated form (see Appendix Fig S8D). In the bar chart, the levels of putative exosome proteins were normalised to cell lysate protein levels. Significantly decreased levels are in blue and increased levels are in red.(C') Growth curves in low (1%) serum are for HCT116 recipient cells pre-treated with the EV preparations [isolated as in (C)], which had themselves been pre-treated with and without anti-AREG neutralising antibody or with a control IgG. Solid red line shows growth-promoting effect of EVs isolated under glutamine depletion, which is blocked by anti-AREG antibody (red dashed line). ***P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and anti-AREG-treated cells (red). (C'') Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete (2.00 mM) or -depleted (0.15 mM) HCT116 cells or vehicle (PBS), which were pre-treated with anti-AREG (AREGab) or a control immunoglobulin (Ig). Note increase in ERK phosphorylation using EVs from glutamine-depleted cells, which is blunted by the addition of the anti-AREG antibody.

Data information; Growth curves were reproduced in three independent experiments and analysed by two-way ANOVA. Bar charts derived from three independent experiments and analysed by ANOVA or the Kruskal-Wallis test: ***P < 0.001, **P < 0.01, *P < 0.05. Bars and error bars denote mean +/- SD.
Glutamine deprivation alters the origin and function of cancer cell exosomes (2020)
Figure 8

Figure 8. EVs from glutamine-depleted HCT116 cells increase tumour cell turnover and vessel growth in mouse HCT116 xenografts

HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System.

(A) Sections immunostained for CD31, which labels endothelial cells and blood vessels, with blood vessel number (upper) and total area (lower) represented in bar charts.

(B) Sections immunostained for Ki67, which stains proliferative cells. Proportion of tumour cells with Ki69 staining is represented in bar chart.

(C) Sections stained with haematoxylin and eosin, which highlights necrotic regions (pale staining). Proportion of tumour area that is necrotic is represented in bar chart.

(D) Sections immunostained for CA9, which is expressed in hypoxic regions. Proportion of tumour cells with CA9 staining is represented in bar chart.

(E) Schematic model showing how in cancer cells, regulation of endosomal trafficking by depletion of exogenous glutamine or reduced Akt/mTORC1 signalling can induce a change in the balance of exosome production. Relative levels of a mixed population of exosomes from the established Rab7-late endosomal multivesicular bodies (MVBs), termed 'CD63-exosomes', are reduced relative to the mixed exosome population from Rab11a-labelled recycling endosomal MVBs, termed 'Rab11a-exosomes', a subset of which appear to be marked with Rab11a. Although CD63 does not appear to be trafficked through these compartments, the 'classical' exosome marker CD81 appears to be present at undefined levels and marks some Rab11a-containing exosomes. The resulting vesicles can increase ERK signalling and cell growth in recipient tumour cells, and enhance growth and stability of network formation in endothelial cells. The growth effects are Rab11a-dependent and can be blocked by anti-AREG antibodies. Thickness of arrows indicates relative levels of membrane flux through the two exosome-generating endosomal routes.

Data information: Scale bar is 250 µm, except for (C), which is 500 µm. Data were analysed by one-way ANOVA; n ≥ 4; * P < 0.05, ** P < 0.01, *** P < 0.001. Bars and error bars denote mean +/- SD.

Expanded View Figure Legends
SRD-1 in AWA neurons is the receptor for female volatile sex pheromones in C. elegans males (2019)
Figure 2

Figure 2. AWA chemosensory neurons are responsible for pheromone detection.

A) Inspection of specific amphid-neuron-defective mutants revealed that AWA neurons are most likely responsible for pheromone perception. Here, an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant. The color code of edges represents the average C.I. of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities. The region colored green shows the range of wild-type sex pheromone response in the male. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.

B) AWA-laser-ablated males were not attracted by the sex pheromone in single-worm chemoattraction assays. Both AWA-defective mutant males (lin-11 and odr-7) and AWA-laser-ablated males failed to reach the sex pheromone within 30 min. Wild-type C. elegans males responded normally to the sex pheromone and wild-type C. elegans hermaphrodites were not attracted by same-sex pheromone. The single-worm chemoattraction assay was performed as described in Materials and Methods. Sample size of males in AWA-laser-ablation experiment: 12; all other experiments: 100 worms.
SRD-1 in AWA neurons is the receptor for female volatile sex pheromones in C. elegans males (2019)
Figure 3

Figure 3. GPCR signaling cascade components are required for pheromone perception.

A) If the sex pheromone receptor is a GPCR as hypothesized, it could elicit chemoattraction through the signal transduction pathways that control cGMP-gated channels and TRPV channels. Knocking out any key component in the two pathways should reduce or abolish chemoattraction behavior.

B) Mutant males of 4 GPCR signaling cascade components failed to respond to C. remanei sex pheromone, suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.

C) In males from both AWA-specific-rescue odr-3 mutant strain and odr-3 rescued mutant strain, the sex pheromone response was significantly restored in single male worm arrival assays. However, males from the CEM-specific-rescue odr-3 mutant strain failed to reach the sex pheromone within 30 min. In odr-3 and him-5 strain experiments, the sample sizes were 12 and 20 worms, respectively; in all other experiments, 40 worms were used.
SRD-1 in AWA neurons is the receptor for female volatile sex pheromones in C. elegans males (2019)
Figure 4

Figure 4. Characteristics of SRD-1 as the receptor required for sex pheromone perception and expression pattern of srd-1 in C. elegans.

A) srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1000 diacetyl and 10 mg/mL pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception.

B) Summary of srd-1 expression pattern. The transcriptional reporter Psrd-1::gfp was detected in both male and hermaphrodite ASI neurons in the head region, and only in male AWA and ADF neurons.

C) SRD-1::GFP fusion construct revealed predominant SRD-1 localization at the cilia of AWAs in C. elegans males. Scale bar = 5 μm.

D) Examination of srd-1 transcriptional reporter revealed that srd-1 is expressed in AWA neurons in males but not hermaphrodites. A transcriptional reporter for odr-7 indicated the locations of AWAs (arrowheads). Scale bar = 50 μm.

E) Schematic diagram of reprogramming of chemotaxis in C. elegans. Rescue-experiment results demonstrated the necessity of SRD-1 in sex pheromone perception; ectopic expression of SRD-1 in the chemorepulsion-responsible neuron, AWB, revealed that this SRD-1 expression is sufficient for altering sex pheromone preference in C. elegans.

F) AWA-specific rescue of srd-1 restored sex pheromone perception ability in both males and hermaphrodites.

G) Ectopic expression of srd-1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites.

Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.
SRD-1 in AWA neurons is the receptor for female volatile sex pheromones in C. elegans males (2019)
Figure 5

Figure 5. SRD-1 is necessary for sex pheromone to activate a calcium response in AWAs.

A) The GCaMP5 reporter was used to visualize activation of the SRD-1 receptor by the sex pheromone in AWA neurons. To facilitate real-time imaging of the process, the heads of the transgenic worms were immobilized on soft-agar pads permeable to sex pheromone.

B) Excitation of the GCaMP5 reporter in AWA neurons could be observed through both lateral and top views but the top view was clearer and more suitable for quantification. Arrowheads: AWA neurons. Scale bar = 50 μm.

C) Relative fluorescence-intensity change was quantified through calcium imaging. Wild-type males showed receptor excitation by sex pheromones from C. elegans hermaphrodites or C. remanei females. This response was lost in srd-1-mutant-strain males but was restored following AWA-specific rescue of srd-1. Red triangles: time points of sex pheromone addition. Three males were assayed from each strain.