EMBO/soda-nel huggingface dataset:
OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination (2016)
Figure 7

Figure 7. OTUB1 up-regulation in wt KRAS lung adenocarcinomas correlates with increased MAPK signaling and poorer patient survival. A, Representative examples of OTUB1 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Materials and Methods. Scale bar, 100 micron. B, OTUB1 expression in lung non-small cell carcinomas with different KRAS mutation status. OTUB1 expression was assessed in TMA samples by IHC. C, OTUB1 up-regulation is an early event in lung adenocarcinoma development. Lung adenocarcinoma patients expressing different levels of OTUB1 were stratified by tumor stages (T1-T3). Total number of patients, n. D, Representative examples of phosphorylated-ERK1/2 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Material and Methods. Scale bar, 100 micron. E, OTUB1 levels correlates with increased levels of phosphorylated-ERK1/2 in wt KRAS lung adenocarcinomas. Association between OTUB1 and pERK1/2 expression levels was assessed in the same tumor samples by IHC. F, Ki67 expression in lung adenocarcinomas expressing different levels of OTUB1. Ki67 expression was assessed by IHC. The percentage of Ki67-positive cells was counted, and the samples were scored as negative/low, <10%; medium, between 10 and 40%; high, > 40%. See Figure EV4 for representative images of Ki67 staining. G, Wt KRAS lung adenocarcinoma TCGA patients with medium OTUB1 expression have poorer overall survival. H, OTUB1 expression levels are not associated with survival rate of KRAS-mutant lung adenocarcinoma TCGA patients. G,H, Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Overall survival of lung adenocarcinoma patients expressing different levels of OTUB1 mRNA as measured by Kaplan–Meier curves. p-values were determined by Log Rank Test.
Clustering of Tau Fibrils Impairs the Synaptic Composition of α3-Na+/K+-ATPase and AMPA receptors (2018)
Figure 1

Figure 1. Rapid uptake and redistribution of preformed Fib-Tau following injection in the CA1 region of the hippocampus

(A-B) Coronal brain section, bregma: - 2.88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus (A). 8 h post-injection, majority of the injected Fib-Tau-1N3R-ATTO550 (red) is taken up by the corpus callosum of the injected side (B). Images are shown at low (left, 10X) and high (right, 63X) magnification.

(C-H) Representative images of the distribution of Fib-Tau (red) 8 h (C, E) and 24 h (D, F) after injection. Dendrites are immunolabeled using MAP2 antibody (green). 8 h after injection, both diffused and clustered distribution of exogenous Fib-Tau is observed in stratum oriens and CA1 cell body region (C, E, Appendix Fig S3). No exogenous Fib-Tau is detected in the adjoining cortex, stratum radiatum and entrorhinal cortex (see Appendix Fig S3). 24 h after injection, clustered distribution is primarily restricted to the pyramidal (CA) cell body layer (D). Uptake of Fib-Tau is seen in some cells in both stratum oriens and pyramidal cell body layer (D). The number of Fib-Tau clusters per µm2 within stratum oriens decreases between 8 h and 24 h post-injection (G). A similar decrease in the total fluorescence (clusters + diffused) is observed indicating the diffusion and/or clearance of exogenous Fib-Tau (H). Paired-t-test performed between '8/24h-right' and '8/24h-left'; Unpaired t-test performed between '8h-left' and '24h-left' columns (n is number of animals: 6 for 8h and 5 for 24h); ***p<0.001, **p<0.01, *p<0.05, ns= not significant. Note: The images presented in panels C and D were independently acquired images under non-identical exposure setting at low magnification for display purpose; Panels (E and F) are shown for same exposure and are used for quantification.

Data information: Scale bar, 100µm in the left panel in (B), 10µm everywhere else.
Clustering of Tau Fibrils Impairs the Synaptic Composition of α3-Na+/K+-ATPase and AMPA receptors (2018)
Figure 2

Figure 2. Time-, Concentration- and Lateral Diffusion-dependent clustering of Fib-Tau on neuronal plasma membrane

(A-C) Time and concentration dependent clustering of Fib-Tau in primary neurons. Representative images are shown for certain conditions to illustrate Fib-Tau clustering time dependence (A, top-row) and concentration dependence (A, bottom-row). Quantification of the number of Fib-Tau clusters per µm2 (B) or fluorescence intensity of clusters (C, indicating size, refer to Material and Methods). At low concentrations (up to 0.72nM) the density of clusters increased with time (between 10 and 60 min) but the increase in intensity was small. At high concentrations of Fib-Tau (≥1.8nM), both density and size increased with increasing time. Box-plot represents: median, interquartile range and 10-90% distribution; One-way ANOVA with Dunnett post-hoc test, number of images analysed from 3 cultures (from left to right: 25, 25, 25, 25, 70, 45, 45, 45, 45, 45, 30, 40, 40, 40, 40 and 40 images).

(D-F) Single Particle Tracking using Quantum Dots (SPT-QD) of biotin tagged Fib-Tau. Representative single molecule trajectories of Fib-Tau following 10 or 60 min exposure are shown (D). Note after 60min exposure (0.36nM), single molecules are more confined suggesting they are trapped and clustered. Quantification of diffusion coefficient (E) and explored area (F, extracted from mean squared displacement (MSD), see methods) shows that both these parameters decrease after 60min exposure to Fib-Tau. Unpaired t-test, n is averaged value per cells imaged in 3 experiments (10 min: 22, 60 min: 23).

(G) Neurons were exposed for 60 min to Fib-Tau (0.36nM) labeled both with biotin and ATTO488 (red). Cell surface exposed biotin was labelled using streptavidin-550 (green) followed by live imaging. Note that most of the clusters of ATTO488 (red) are co-labelled with Streptavidin 550 (green) indicating that the clusters are at the cell surface.

(H-J) Clearance of Tau clusters from neurons. Neurons were exposed (0.36nM) to ATTO 550 labeled Fib-Tau for 10 min and the unbound fibrils were washed. Cells were fixed immediately (10min) or allowed to recover in culture medium for 60 min. Two representative images (H) and quantifications (I, J) show that following 60 min recovery most of the Tau clusters disappear/dissociate as indicated by a decrease in their density. Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test, n is number of images analyzed from 3 cultures (49 images).

Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 5µm in (G), 2µm everywhere else.
Clustering of Tau Fibrils Impairs the Synaptic Composition of α3-Na+/K+-ATPase and AMPA receptors (2018)
Figure 3

Figure 3. Equilibrium between clustered and free Fib-Tau on neuronal plasma membrane

(A) Super-resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib-Tau (0.36nM). Primary neurons were fixed 10, 60, 120 and 240 min after exposure to ATTO647N labeled Fib-Tau. Images rendered with a pixel size of 5nm show both clustered and diffused (non-clustered) distribution of exogenous Fib-Tau molecules. Scale bar, 1µm.

(B-F) Quantification of various parameters from the super-resolution images. Averaged values per image is plotted. Increased "total detections per µm2" (B) and "cluster-density" (C) of Fib-Tau bound to neurons with increasing exposure time is observed. No change in the "size of cluster" (D) or "number of molecules within a cluster" (E) is seen with increased exposure time. The proportion of single molecule events detected within clusters remained nearly constant (~ 50%) in all conditions (F) indicating an equilibrium between clustered and non-clustered Fib-Tau single molecules. Unpaired t-test to compare difference with 10 min, n is number of images (8 images). *p<0.05; **p<0.01; ***p<0.001; ns= not significant.
Clustering of Tau Fibrils Impairs the Synaptic Composition of α3-Na+/K+-ATPase and AMPA receptors (2018)
Figure 5

Figure 5. Clustering of Fib-Tau at excitatory synapses redistributes α3-NKA and AMPA but not NMDA receptors

(A, B) Primary neurons were exposed to Fib-Tau-ATTO550 (0.36nM, red) for 10 (top row) or 60 min (bottom row) and excitatory (Homer, green) and inhibitory synapses (Gephyrin, blue) were immuno-labelled. Fib-Tau clusters co-localized and/or apposed to Homer (arrowheads) but not Gephyrin (A). Quantitative analysis shows that 10 to 20% of Fib-Tau clusters are localized at Homer containing synapses (B). Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test to compare difference between 10 and 60 min, n is number of images analyzed from 3-experiments (10 min: 49; 60 min: 50). Scale bar, 5µm.

(C, D) Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Arrows indicate excitatory synapses where Fib-Tau and α3-NKA/AMPA/NMDA are co-localized (C). Quantification of the fluorescence intensity (indicating size of clusters, see Material and Methods) of synaptic α3-NKA/AMPA/NMDA spots (obtained after thresholding) following exposure to Fib-Tau showed a reduction in the size of α3-NKA and increase in the size of GluA2-subunit containing AMPA receptors (D). Mann-Whitney-test, n is number of images analyzed from 3-4 experiments (α3-NKA: 75; GluA1: 95; GluA2: 50; GluN1/N2B: 45). Box-plot represents: median, interquartile range and 10-90% distribution, **p<0.01; ns= not significant. Scale bar, 5µm.
Clustering of Tau Fibrils Impairs the Synaptic Composition of α3-Na+/K+-ATPase and AMPA receptors (2018)
Figure 6

Figure 6. Diffusion properties of GluA2-AMPA receptors and co-clustering with fibrillar Tau

(A-C) SPT-QD of pHluorin tagged AMPA (GluA1 and GluA2) and NMDA (GluN1 and GluN2B) receptor subunits using GFP-antibody pre-coupled to QD655. Primary neurons were exposed or not to Fib-Tau (0.36nM, 60 min). Cumulative frequency of diffusion coefficient of all trajectories (extracted from MSD plot, inset, A) show a slow-down in diffusion of GluA2-subunit but not GluA1, GluN1 and GluN2B subunits (A). To compute statistical differences, the diffusion coefficient of all trajectories per movie (from 3 independent experiments) were averaged (B). To measure statistical differences in MSD, the explored area (area under curve during 600-900ms of MSD plot) per movie (from 3 independent experiments) were averaged (C). Decreased diffusion coefficient and area explored for GluA2-AMPA was observed indicating increased confinement concomitant with increased clustering (Fig 5C) following exposure to Fib-Tau. Mann-Whitney-test, n is number of movies (left to right): 14, 15, 14, 13, 15, 15, 15, and 15. **p<0.01; ns= not significant.
Clustering of Tau Fibrils Impairs the Synaptic Composition of α3-Na+/K+-ATPase and AMPA receptors (2018)
Figure 7

Figure 7. Exogenous Fib-Tau destabilize α3-NKA and affect their turnover

(A) Single Particle Tracking using PALM (SPT-PALM) was performed on α3-NKA-Dendra expressing primary neurons. A representative image shows an epifluorescence image and trajectories (strands of different colors) of minimum 5 points in length.

(B, C) Extraction of diffusion coefficient using mean squared displacement (MSD) approach using minimum 5-point trajectory length. Spine and shaft were visually identified. An upward shift of MSD curve, indicating reduced confinement was observed following exposure to Fib-Tau (0.36nM, 60 min) (B). As expected, an increased diffusion coefficient (i.e. increased mobility) (C) of α3-NKA is observed in dendritic shaft following exposure to Fib-Tau. Mann-Whitney-test, n=21 cells, obtained from 4 independent experiments; Each dot represents average diffusion coefficient of all trajectories value per cell.

Mann-Whitney-test, in panel (B), n=21 cells, is the averaged value of diffusion coefficient per cell from 4 experiments.

(D-F) Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model (see Material and Methods). Two diffusive states could be derived for α3-NKA with confidence (termed Bound / slow-diffusing or Free / fast-diffusing) (D) between which α3-NKA can transition. The characteristic parameters for neurons exposed or not to Fib-Tau- are shown (D). The proportion of single molecules transitioning to bound state decreased (from 71.4% to 57.1%) following neuron exposure to Fib-Tau. The occupancy time of α3-NKA in bound-state also decreased whereas that in free-state increased upon neuron exposure to Fib-Tau (E). Dwell-time of α3-NKA is also reduced in bound state indicating destabilization of native α3-NKA-complexes following interaction with Fib-Tau (F). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells).

(G-H) Cell-surface biotinylation on primary neurons exposed or not to Fib-Tau (1.8nM, 60 min). The amount of α-NKA at cell surface decreased upon exposure of neurons to Fib-Tau (G). Quantification was performed by normalizing the western blot band intensity with input. Paired-t-test, 3 independent experiments.

(I) Paraformaldehyde fixation α3-NKA-pHluorin (green, top row) reveals endocytic spots otherwise not visible in live neurons (green, bottom row). Many Fib-Tau-ATTO647 (red) clusters overlapped (arrow) with α3-NKA-pHluorin spots (exposure, 0.36nM, 60 min).

(J) The majority of the overlapping Fib-Tau-ATTO647 clusters (red) and α3-NKA-pHluorin spots (green) localized on top of Clathrin-mRFP (blue) puncta.

Data information: *p<0.05; ns= not significant. Scale bars: 1 µm in panel (A) and 2µm in panels (I and J).
Clustering of Tau Fibrils Impairs the Synaptic Composition of α3-Na+/K+-ATPase and AMPA receptors (2018)
Figure 8

Figure 8. α-synuclein but not amyloid-β fibrils enhance fibrillar Tau clustering within neuron membranes

(A-F) DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn (A) or Aβ (B) fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn or Aβ (shown here in green) or unlabeled α-Syn or Aβ fibrils]. A concentration-dependent increase in the binding and clustering of both α-Syn fibrils (A, C) and Aβ fibrils (B, D) is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 27, 25, 25, 26 and 28 in (C) and 18, 50, 50, 50, 25 in (D), from left to right. The data originate from 3 to 4 independent experiments. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils (A, B, E). The quantification in (E) includes all 5-experiments (n = number of images analyzed, left to right: 58, 54, 50, 62, 63, 65, 43, 35, 60, 60, 60, 35; One-Way ANOVA with Dunnette's post-hoc test was performed to compare the size of Fib-Tau clusters). A perfect co-localization between Fib-Tau and α-Syn clusters is also observed. The proportion of Fib-Tau clusters co-localizing with α-Syn fibrils increased with an increasing concentration of α-Syn fibrils reaching 60-70% overlap (A, F). A weaker, concentration independent, overlap (10-20%) was observed with Aβ fibrils (B, F). The quantification in (F) is derived from 3 to 4 independent experiments. The number of images analyzed is 27, 25, 25, 26, 28 for α-Syn and 18, 50, 50, 50, 25 for Aβ from left to right. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the co-localization differences from 100nM concentration. Box-plot represents: median, interquartile range and 10-90% distribution.

(G-I) Primary neurons were co-exposed to Fib-Tau (0.36nM, ATTO550 labelled) and Fib-α-Syn (50nM, monomer-equivalent, unlabeled) for 60min and immunolabelled for homer (G) or α3-NKA (H). Quantification of Fib-Tau clusters co-localized with homer or α3-NKA (I). Note an increased co-localization of Fib-Tau with homer in presence of Fib-α-Syn.

Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.

Expanded View Figure Legends
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 1.

Rag GTPases are required for recruitment of TFEB to lysosomes. (A and B) ARPE-19 cells were transfected with siRNA duplexes to raptor, RagA+B, p18, or nontarget. 60 h after transfection, cells were infected with adenovirus expressing either TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG (used to detect TFEB) and mTOR. Bars, 10 µm. (C) Quantification of A. (D) Quantification of B. Values are means ± SD of three independent experiments. ***, P < 0.001.
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 2.

TFEB interacts with active Rag heterodimers. (A) ARPE-19 cells were nucleofected with the indicated Rag-expressing plasmids. 6 h after nucleofection, cells were infected with adenovirus expressing TFEB-FLAG-WT. 12 h later, cells were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG (used to detect TFEB-WT). (B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-GFP–expressing plasmids. 18 h later, cells were lysed and subjected to immunoprecipitation with the anti-HA antibody (used to immunoprecipitate Rag proteins). The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP (used to detect Rag proteins and TFEB-WT, respectively). (C) ARPE-19 coexpressing TFEB and the indicated Rag-expressing plasmids were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG (used to detect Rag proteins and TFEB-WT, respectively). (D) Immunoblotting analysis of coimmunoprecipitated TFEB-FLAG-S211A with Rag heterodimers. IP, immunoprecipitation.
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 3.

Regulation of endogenous TFEB by mTOR and Rags. (A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin. (B) HeLa cells were treated as indicated in A, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB), Lamp1, or Histone H3. (C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A. (D) HeLa cells were starved in serum- and amino acid–free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB. (E) HeLa cells were incubated in normal medium (control) and serum- and amino acid–free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets. (F) HeLa cells were nucleofected with the indicated Rag-expressing plasmids. 18 h later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione–Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST (used to detect endogenous TFEB and Rag proteins, respectively). Bars, 10 µm.
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 4.

Inactivation of endogenous Rags by starvation prevents lysosomal localization of TFEB. (A) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-WT. 16 h later, cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid–free medium for 3 h. Cells were then fixed, permeabilized with 0.2% saponin, and double stained with antibodies against TFEB (used to detect recombinant TFEB) and Lamp1. (B) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-S211A. 12 h later, cells were starved in serum- and amino acid–free medium for 4 h (starvation) or kept in complete medium (control). Cells were then fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB-S211A). (C) HeLa cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid–free medium for 4 h with the addition of Torin-1 during the last hour of starvation. Cells were then washed, fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against TFEB (used to detect endogenous TFEB) and Lamp1. Regions within the dotted boxes are magnified in the insets. Bars: (A [main images], B, and C) 10 µm; (A, insets) 5 µm.
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 5.

The activation state of Rags determines localization and activation of TFEB. (A and B) ARPE-19 cells were transfected with either active or inactive RagB/D heterodimers and infected with adenovirus expressing TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). After 12 h, cells were double stained with antibodies against FLAG and GST (used to detect TFEB or Rag proteins, respectively). (C) Quantification of TFEB-WT nuclear localization from A. (D) Quantification of TFEB-S211A lysosomal localization from B. (E and F) ARPE-19 cells were cotransfected with plasmids expressing TFEB-GFP together with inactive (E) or active Rag heterodimers (F). Cells were then incubated with 250 nM Torin-1 for 1 h (E) or starved in serum- and amino acid–free medium for 3 h (F). Anti-GST antibodies were used to detect Rag proteins. Arrows point to cells that do not express Rag heterodimers. Values are means ± SD of three independent experiments. ***, P < 0.001; *, P < 0.05. Bars, 10 µm.
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 6.

The N-terminal region of TFEB is necessary for interaction with Rag heterodimers and lysosomal localization. (A) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE-19 cells treated with either DMSO or Torin-1. (B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-expressing plasmids. After 12 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST (used to detect TFEB and Rag proteins, respectively). (C) FLAG-tagged TFEB-WT or TFEB deletion mutants were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG, 14-3-3 binding motif, or 14-3-3. (D) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-WT and TFEB-S3A/R4A mutant upon incubation with DMSO (vehicle) or 250 nM Torin-1 for 1 h. Cells were fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB). (E) ARPE-19 cells expressing either TFEB-S211A or TFEB-S211A-Δ30 were double stained with antibodies against TFEB and Lamp1. Regions within the dotted boxes are magnified in the insets. IP, immunoprecipitation. Bars, 10 µm.