EMBO/soda-nel huggingface dataset:
Lysosome-mediated processing of chromatin in senescence. (2013)
Figure 5.

Senescent cells in vitro and in vivo contain reduced histone content. (A) Immunofluorescent confocal images of histone H3 in control or OIS cells (11 d after activation of ER-RASG12V). Yellow arrows indicate cells with most pronounced SAHF. (B) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells. Representative of two independent experiments. (C) Progressive loss of core histones in senescent IMR90 cells in replicative senescence (RS). Lysates were normalized by cell number and an equal number of cells was loaded per each lane. Lamin A/C was used as a confirmatory loading control. See Materials and methods for details of time course. (D) Immunohistochemistry for histone H3 in human dermal nevus (n) and adjacent epidermis (e). Right-hand panels are expanded from boxed areas on the left. Note decreased nevus staining for H3 (right bottom) as compared with epidermis (right top). Bars: (left) 100 µm; (right) 50 µm. (E) Immunofluorescence of histone H3 (red) and S100 (melanocytes [green]) in human benign nevus and adjacent epidermis. Bars, 20 µm. (F) Higher magnification of boxed regions in E shows reduced staining for H3 in nevus (bottom panels), compared with epidermal melanocytes (top panels). Bars, 20 µm. (G) Quantitative immunofluorescence of H3 in epidermal and nevus melanocytes. Images were obtained in blue (DAPI), green (S100), and red (H3) channels and then H3 intensity was measured in either S100+ epidermal cells, strictly adjacent to the basal membrane, or in S100+ nevus melanocytes. H3 intensity histograms represent fluorescence intensity distribution, combined from three individual nevi. At least 150 epidermal (50 per nevus) and 300 nevus (100 per nevus) melanocytes were assessed.
Lysosome-mediated processing of chromatin in senescence. (2013)
Figure 6.

Senescence-associated loss of histones is V-ATPase dependent. (A) Bafilomycin A1 (BafA1) blocks the loss of nuclear histone H3 content in cells undergoing OIS. BafA1 was added to cells at 50 nM on day 5 after RASG12V induction. Cells were harvested 24 h later, and stained for histone H3. Bars, 10 µm. (B) Quantitative histone H3 immunofluorescence in cells from A. Single representative experiment out of three repeats. (C) RASG12V-induced OIS cells were treated with BafA1 as described in A and then assessed for histones by Western blot. LC3 I/II has been used as a control for BafA1 activity. Lysates from 10,000 cells were loaded per well. (D) BafA1 blocks the accumulation of H3cs.1 in cells undergoing OIS. (E) BafA1 and concanamycin A (Con A) block the accumulation of H3cs.1 in RS cells.
Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains (2022)
Figure 1

Figure 1 - PolyUb chains with varied lengths and linkages differentially affect UBQLN2 LLPS.

A Fluorescence microscopy showing UBQLN2 (Alexa Fluor 647) in solutions contained 50 µM FL UBQLN2 with increasing amounts of K48- and K63-linked chains of different lengths in 20 mM NaPhosphate and 200 mM NaCl (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub monomers and UBQLN2 molecules. The same image is used for the 0:1 condition. Scale bar, 5 µm. Yellow cartoons illustrate one major solution conformation of each chain.

B Representative results from spectrophotometric turbidity assay comparing the effects of varying amounts of K48- and K63-linked chains of different lengths on UBQLN2 LLPS at 30 μM UBQLN2.

C Temperature−composition phase diagrams showing changes in cloud point temperatures as a function of Ub:UBQLN2. The black dashed lines denote biologically relevant temperature 37 ºC, at which 12.5 µM UBQLN2 only undergoes LLPS with K63-Ub4.
Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains (2022)
Figure 2

Figure 2 - K48- and K63-Ub4 bind differently to UBQLN2 450-624.

A CSPs for UBQLN2 450-624 residues at 1:1 ratio with Ub4. The highlighted regions in the domain map are important for the homotypic interactions that drive UBQLN2 LLPS (Dao et al, 2018).

B Amide peak intensities between the unbound and Ub4-bound forms of UBQLN2 450-624 with the 555-570 region highlighted in gray.

C CSPs for residues in UBQLN2 487-624 at 1:1 ratio with Ub4.

D CSPs for residues in UBQLN2 450-624 lacking residues 551-569 (red/gray region) at 1:1 ratio with Ub4.

E Normalized titration curves of 11 amide resonances for UBQLN2 450-624.

F Comparison of binding affinity (Kd) measurements obtained from NMR and fluorescence anisotropy. Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis. For visual comparison, gray dashed line is Kd of Ub binding to UBQLN2 450-624 as determined from NMR and FA experiments (green bars).

G Fluorescence anisotropy values for fluorescently-labeled UBQLN2 450-624 in the presence of increasing K48- or K63-Ub4. Error bars represent the standard deviation over three biological trials.

H Fluorescence anisotropy values for fluorescently-labeled K48- or K63-Ub4 in the presence of increasing UBQLN2 450-624. Error bars represent the standard deviation over three biological trials.
Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains (2022)
Figure 3

Figure 3 - The Ub-Ub interface in K48-Ub4 opens to bind to UBQLN2.

A Representative structures of K48-Ub4 (PDB ID 2O6V) and K63-Ub4 (PDB ID 3HM3). The cyan proximal Ub unit was selectively 15N-labeled for NMR binding experiments. Yellow spheres represent hydrophobic patch residues L8, I44, and V70. Red sticks indicate K48 or K63 linkage. CSPs > 0.05 ppm for proximal Ub resonances vs. monoUb positions (in the absence of UBQLN2) are color-coded orange.

B Proximal Ub resonances in Ub4 were compared to monoUb, as noted in schematic. CSPs > 0.05 ppm (orange dashed line) are mapped onto the proximal Ub unit of Ub4 in panel A.

C 15N-1H SOFAST-HMQC NMR spectra of proximal Ub of K48-Ub4 or K63-Ub4 at 50 µM in the absence and presence of UBQLN2 450-624 at a 2:1 UBQLN2:Ub4 ratio. Unbound monoUb spectrum (black) is overlaid for comparison. NMR spectra collected and processed under identical conditions.

D CSPs of the same proximal Ub resonances in K63-Ub4 vs K48-Ub4. Gray bars indicate K48-Ub4 resonances that were too weak to be detected.
Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains (2022)
Figure 4

Figure 4 - Compact K11 and K48-Ub4 bind UBQLN2 differently than extended K63-Ub4, M1-Ub4 and HOTag6-G10-Ub.

A Cartoons depicting a possible solution conformation for each of the Ub4 chains studied here. B Normalized pair distance distribution function P(r) from SAXS data for four Ub4 chain types.

C CSPs at 1:1 Ub4:UBQLN2 for all chain types, but clustered into two categories for clarity.

D Peak intensity comparison in the absence and presence of Ub4 at 1:1 Ub4:UBQLN2 with residues 555-570 highlighted in gray.

E TROSY-HSQC spectra of UBQLN2 450-624 in the absence and presence of different Ub4 chains. Some of the peaks in the 555-570 region are more perturbed in the presence of K11-Ub4 and K48-Ub4 but not the other chains, whereas the UBA peaks (580-624) are equally perturbed in the presence of any of the chains.

F Normalized titration curves of UBA resonances showing more pronounced sigmoidal behaviors upon titration of K48-Ub4 into UBQLN2 450-624 than upon titrations of K11-Ub4, K63-Ub4, M1-Ub4 and HOTag6-Ub.

G Fluorescence anisotropy values for fluorescently-labeled UBQLN2 450-624 in the presence of increasing amounts of different Ub4 chains. Error bars represent the standard deviation over three biological trials.

H Comparison of Kd values obtained from NMR and fluorescence anisotropy. Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis. For visual comparison, gray dashed line is Kd of Ub binding to UBQLN2 450-624 as determined from NMR and FA experiments (green bars).
Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains (2022)
Figure 5

Figure 5 - The effects of polyUb chains on UBQLN2 LLPS are dependent on chain flexibility / accessibility to Ub binding patch.

A Fluorescence microscopy showing enhancement or inhibition of UBQLN2 droplet formation at increasing amounts of chains at 50 µM UBQLN2 in 20 mM NaPhosphate and 200 mM NaCl (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub monomers and UBQLN2 molecules. Solution is spiked with Alexa Fluor 647-labeled UBQLN2. The same image is used for the 0:1 condition. For visual comparison, images for K48-Ub4 and K63-Ub4 are identical to those shown in Figure 1A. Scale bar, 5 µm.

B Representative results from spectrophotometric turbidity assay as a function of temperature at 30 μM UBQLN2.

C Temperature−component phase diagrams showing the changes in LCST phase transition cloud-point temperatures as a function of Ub:UBQLN2 for different Ub4 at 30 µM UBQLN2.

D Phase diagrams showing the dense and dilute phase concentrations of UBQLN2 (top) and Ub chains (bottom). Error bars represent the standard deviation over three biological trials. Green dashed line (top) is the fixed total UBQLN2 concentration used (80 µM). For visual comparison, cyan dashed line (bottom) represents UBQLN2 concentration in the dense phase (> 2 mM) as determined from top panel.

E Fluorescence microscopy showing the distribution of Ub4 (spiked with Dylight 488-labeled chains) inside and outside droplets containing 50 µM UBQLN2 (spiked with Alexa Fluor 647-labeled UBQLN2). Scale bar, 5 µm.
Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains (2022)
Figure 6

Figure 6 - LLPS of UBQLN2 is induced during in vitro K63-selective ubiquitination assays.

A SDS-PAGE gels monitoring the formation of K63-linked polyUb chains and possibly ubiquitinated GST-Ubc13 over time, as indicated by asterisks and curly brackets. K63-Ub3 migrates on the gel similarly as His-Mms2, right below 20 kDa. Experimental conditions: ± 50 µM Ub and UBQLN2, 30 nM Dylight 650-labeled UBQLN2, 1 µM mE1, 2 µM His-Mms2, 4 µM GST-Ubc13, 10 mM ATP and MgCl2, 3 mM TCEP, 50 mM Tris pH8, 37 ºC. The K63-linked polyUb chain reactions were done in the absence (top) and presence (bottom) of UBQLN2. Since UBQLN2 competes with E1 and E2 to bind to Ub, the presence of UBQLN2 slowed down chain formation (indicated by comparing disappearance rates of Ub). The band intensity for GST-Ubc13 decreased with time. The time-dependent appearance of species between 50-85 kDa were obstructed in the presence of UBQLN2. These species are most likely ubiquitinated GST-Ubc13, and not free polyUb chains, since these bands were observed with BioRad Stain-free gels (right) that detect proteins with tryptophan residues unlike Ub or polyUb chains.

B Time-lapse fluorescence microscopy monitoring UBQLN2 droplet formation in the presence of K63-selective ubiquitination machinery and with or without Ub. Experimental conditions were the same as in (A). Imaging was done in solution above the coverslip. Scale bar, 5 µm.

C Time-lapse fluorescence microscopy monitoring formation of UBQLN2- and GST-Ubc13-containing droplets in the presence of Ub and K63-selective ubiquitination machinery. Experimental conditions were the same as in (A) with addition of 30 nM Alexa Fluor 488-labeled GST-Ubc13. Imaging was done at the coverslip surface. Scale bar, 5 µm.
Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites (2018)
Figure 1

Figure 1: Identification of MOSPD2, a new FFAT motif-binding protein

A: Sequence of the two peptides used for the pull-down assay. The peptides are composed of an amino-terminal biotin, a linker sequence and a random sequence (control peptide) or the FFAT sequence of ORP1 (FFAT peptide). The FFAT sequence of ORP1 is in bold and corresponds to residues 469-483 (Accession number Q9BXW6-1). Aromatic and acidic residues are in blue and red, respectively.

B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows.

C: Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction. Score: protein score based on the sum of the ion scores of all peptides identified; Cov.; percentage of the protein sequence covered by identified peptides; Pep.: number of unique peptide sequences identified.

D: Schematic representation of MOSPD2, VAP-A, and VAP-B. Numbers correspond to the predicted positions of the beginning and the end of each domain. Calculated molecular weights (MW) of the proteins are shown.

E: Western blot analysis of proteins pulled-down using the control (Ctrl) peptide (left) or the FFAT (FFAT) peptide (right). The Input, FT (flow-through), and Elution fractions correspond to HeLa cell total protein extract, unbound proteins and bound proteins, respectively. Representative illustration of at least two independent experiments.
Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites (2018)
Figure 5

Figure 5: MOSPD2 interacts with FFAT-containing proteins

A: List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif (see Appendix Table S1 for the full list). Proteins co-precipitated with GFP-MOSPD2 and GFP-MOSPD2 RD/LD proteins were identified by mass spectrometry; proteins were filtered for their ability to bind MOSPD2 and not the RD/LD mutant. The sub-cellular localization of the proteins is indicated [53,65-69].

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagged STARD3 (B), STARD3NL (C), ORP1L (D), STARD11 (E), and HA-tagged PTPIP51 (F) (WT and FFAT-deficient). Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E), anti-ORP1 (D), anti-HA (F), anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F) or anti-ORP1 (C), and anti-GFP (B-F) antibodies.

G: Immunoprecipitation (anti-Flag) experiment between Flag-tagged STARD3NL (WT and FFAT-deficient) and endogenous MOSPD2. Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti-MOSPD2, anti-STARD3NL and anti-actin antibodies.
Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites (2018)
Figure 8

Figure 8: Endogenous MOSPD2 is recruited to inter-organelle contact sites by FFAT-containing proteins

A: GFP-tagged MOSPD2 (green) expressing cells were labelled with an anti-MOSPD2 antibody (magenta). The superposition of the green and magenta signals indicates that the anti-MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence.

B: Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells (WT) and in HeLa cells expressing a control shRNA (shCtrl) and two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Quantification of MOSPD2 protein level is shown below. Means and error bars (SD) are shown. n: three independent experiments.

C: Endogenous MOSPD2 (green) was labelled in control HeLa cells (WT), in cells expressing a control shRNA (shCtrl), and in cells expressing two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Scale bar: 10 µm.

D: Endogenous MOSPD2 (green) staining in HeLa cells expressing the ER marker GFP-ER (magenta).

E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressing Flag-STARD3NL (E) or Flag-STARD3NL ∆FFAT (F) (anti-Flag; magenta).

G: Pearson correlation coefficients between endogenous MOSPD2 and Flag-STARD3NL (WT or ΔFFAT) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-Flag-STARD3NL: 27; MOSPD2-Flag-STARD3NL ΔFFAT: 26; from three independent experiments). Means and error bars (SD) are shown. Mann-Whitney test (two-tailed P value; ****: P< 0,0001).

Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay. Black rectangles indicate the positions of late endosomes (E). Scale bars: 10 µm.
Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites (2018)
Figure 9

Figure 9: MOSPD2 silencing affects inter-organelle contact sites organization

A: Western blot analysis of MOSPD2, VAP-A and VAP-B proteins in control HeLa cells (WT) and in HeLa cells transfected with a control siRNA (siCtrl), and with siRNA targeting MOSPD2 (siMOSPD2), VAP-A and VAP-B (siVAP-A/VAP-B), and MOSPD2, VAP-A and VAP-B (siMOSPD2/VAP-A/VAP-B).

B-F: TEM images of control HeLa cells (B: HeLa; C: HeLa/siCtrl) and MOSPD2 (D: siMOSPD2), VAP-A and VAP-B (E: siVAP-A/VAP-B), and MOSPD2, VAP-A and VAP-B (F: siVAP-A/VAP-B/MOSPD2) -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively.

Scale bars: 500 µm.

G-I: Quantification by stereology of ER-mitochondria (G), ER-endosome (H) and endosome-endosome (I) contacts. The percentages of mitochondria perimeter in contact with the ER (G), endosome perimeter in contact with the ER (H) and endosome perimeter in contact with an endosome (I) are shown as means and error bars (SEM). (G) 200 (HeLa), 208 (siCtrl), 199 (siMOSPD2), 202 (siVAP-A/VAP-B), 199 (siMOSPD2/VAP-A/VAP-B) mitochondria from 8, 10, 8, 12 and 12 cells, respectively, were analyzed. (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001).
Gadd45α modulates aversive learning through post-transcriptional regulation of memory-related mRNAs (2019)
Figure 1

Figure 1. Gadd45α controls hippocampus-dependent memories.

A Experimental apparatus of the passive avoidance (PA) test to assess context-dependent associative learning. In this paradigm, the animal receives a foot shock immediately after entering the dark compartment. This leads to hippocampal-dependent aversive learning, which can be assessed by the latency of subjects to enter the dark compartment in subsequent phases of the experiment.

B Latency to enter dark compartment for Gadd45a-WT (white bars, n=11) and Gadd45a-KO (blue bars, n=11). A significant reduction was found in the Gadd45a-KO at 24 hours and 7 days after the training. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.

C In situ hybridization to detect Gadd45a mRNA in adult wild-type hippocampus (n=8 animals; antisense, left panel; sense, middle panel) and in Gadd45a-KO sections (n=6 animals; right panel). Scale bars: 500 µm (black bar).

D Experience-dependent changes in mRNA levels were evaluated under control conditions (home cage) and exposed to the PA (context, and context + shock groups; n=6 for all groups), respectively. Hippocampal Gadd45a mRNA levels remained unchanged, while Gadd45b was induced (context; t10=2.427, context + shock t10=2.319). Concomitantly, mRNA levels of activity-regulated cytoskeleton protein (Arc), as a proxy of neuronal activation, were increased in mice exposed to the PA context (t10=3.406) and context + shock (t10=3.141). Values shown are mean ± SEM; 1-Way ANOVA and unpaired t-test: * = p<0.05, *** = p<0.001.
Gadd45α modulates aversive learning through post-transcriptional regulation of memory-related mRNAs (2019)
Figure 2

Figure 2. Gadd45a-overexpression enhances memory consolidation.

A Diagram of the virally-mediated approach to induce Gadd45a-overexpression in hippocampal glutamatergic neurons of Nex-Cre(+/-) mice.

B Gadd45a mRNA level in the hippocampus of Gadd45a-overexpressing mice (red, n=7) was significantly increased (t10=4.189) as compared to Gadd45a-WT (n=5). Values shown are mean ± SEM; unpaired t-test: ** = p<0.01.

C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (Nex-Cre(-/-), n=5) and Gadd45a-overexpressing hippocampi (Nex-Cre(+/-), n=5) 8 weeks after AAV injection. Note that the Gadd45α-HA signal in Gadd45a-overexpressing mice is predominantly found in the stratum pyramidale (sp) but also in the stratum oriens and stratum radiatum (so and sr, respectively). Scale bar: 250 µm.

D Cellular distribution of Gadd45α was analyzed by HA-western blot on nuclear and cytoplasmic protein fractions of Gadd45a-WT and Gadd45a-overexpressing mice (n=2 for both groups). HA was found predominantly in the nuclear fraction of Gadd45a-overexpressing hippocampi, with a smaller but recognizable expression also in the cytoplasm.

E Latency to enter the dark compartment for Gadd45a-WT (white bars, n=10) and Gadd45a-overexpressing mice (red bars, n=12) in the PA test. A significant increase was found 24 hours and 7 days after the PA training in the Gadd45a-overexpresion group. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05.
Gadd45α modulates aversive learning through post-transcriptional regulation of memory-related mRNAs (2019)
Figure 3

Figure 3. Gadd45a regulates hippocampal synaptic plasticity.

A Nissl stained slice (left) and corresponding section from brain atlas (right). Left panel shows hippocampus and position of the stimulating (left) and recording (right) electrode. Scale bars: 500 µm (black bar).

B LTP protocol was applied to 16 slices of Gadd45a-WT mice (white dots, n=5 animals) and 11 slices of Gadd45a-KO mice (blue dots, n=5 animals), resulting in a significantly (p<0.05, Student t-test) lowered induction of LTP in Gadd45a-KO mice as compared with Gadd45a-WT. Values shown are mean ± SEM.

C Gadd45a-overexpressing mice (red dots, n=10 slices from 6 animals) presented a slightly increased LTP as compared with Gadd45a-WT (white dots, n=8 slices from 6 animals), which became more pronounced during the late phase of the experiment. Values shown are mean ± SEM.