EMBO/soda-nel huggingface dataset:
APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting (2018)
Figure 3

Figure 3: RAB21 interacts and colocalizes with the WASH and retromer complexes. Strumpellin and VPS35 are in close proximity to RAB5 and RAB21. (A) Anti-biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35. Lysates correspond to 1% of input, n=3 independent experiments. RAB21 can be seen interacting with various WASH and retromer complexes subunits. GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and endogenous (B) VPS26 immunoblot and (C) FAM21, Strumpellin and VPS35 immunoblots. Lysates correspond to 5% of input, n≥ 3 independent experiments. RAB21 actively pull-downs WASH and retromer complex subunits (D) Bacterially-purified and GTP-loaded GST:RAB21 pull-down of HeLa cell lysates followed by Strumpellin, FAM21, CAPZα and VPS35 immunoblots. Ponceau staining reveals the purity of the GST and GST:RAB21 used for the pull-downs. n=3 independent experiments. (E) RAB21 colocalizes with the WASH and retromer complexes. Transiently expressing GFP:RAB21 HeLa cells were fixed and stained for endogenous EEA1, FAM21, WASH and VPS26. Boxed region is magnified and single channels are depicted. Scale bars: 10µm or 5µm in enlarged view. (F) Per cell Pearson Correlation between RAB21 and the various markers. Error bars are SEM, n=2 independent experiments.
APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting (2018)
Figure 4

Figure 4: Characterization of RAB21 knockout HeLa cells. (A) Low RAB21 expression levels in two independent RAB21 KO cell populations. Immunoblot analysis of endogenous RAB21, RAB5, RAB7 and GAPDH. (B) Endo-lysosomal compartments are unaffected by the loss of RAB21. Immunofluorescences of APPL1, TfR, RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations. Scale bars :10µm, n=3 independent experiments.
APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting (2018)
Figure 5

Figure 5: RAB21 modulates WASH and retromer endosomal recruitment and WASH activity. (A) The stability of the retromer cargo sorting complex is affected in RAB21 KO cells. Immunoblot analysis of VPS35, VPS26, VPS29, GAPDH and RAB21 in parental and the two RAB21 KO cell populations. Ratio of VPS35, VPS26 and VPS29 integrated densities to GAPDH for four independent experiments; SEM. * represents p <0.05 (B) WASH complex proteins are not affected by RAB21 deletion. Immunoblot of Strumpellin, WASH, FAM21, CAPZα, GAPDH and RAB21. Ratio of WASH, FAM21, Strumpellin and CAPZα integrated densities to GAPDH for four independent experiments; SEM. (C-E) RAB21 knockout decreases VPS35 localization at endosomes. (C) Immunofluorescence of endogenous VPS35 and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (D) Per cell Pearson Correlation between VPS35 and EEA1; SEM, n=3 independent experiments. (E) Average number of VPS35 puncta per cell; SEM, n=3 independent experiments. (F-H) RAB21 loss impairs endosomal recruitment of the WASH complex. (F) Immunofluorescence of endogenous WASH and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (G) Per cell Pearson Correlation between VPS35 and EEA1 and (H) FAM21 and EEA1; SEM, n=2 independent experiments. (I-J) RAB21 knockout reduces endosomal F-actin levels. (I) Immunofluorescence of endogenous F-actin using Alexa-488 conjugated phalloidin and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (J) Per cell Pearson Correlation between internal F-actin and EEA1; SEM, n=3 independent experiments.

Data information: In (C, F, I) scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: (A and B) - One sample t-tests, (D) - Unpaired t-tests, (E and J) - Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.
APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting (2018)
Figure 6

Figure 6: RAB21 is required for endosomal sorting of specific cargo types. (A-C) RAB21 is not required for CI-MPR or GLUT1 trafficking. (A) Immunofluorescence of endogenous CI-MPR and TGN46 or GLUT1 with LAMP1 in RAB21-knockout cells. Boxed region is magnified. (B) Per cell Pearson Correlation between CI-MPR and TGN46 or (C) GLUT1 and LAMP1; SEM, n=3 independent experiments. MCT1 trafficking requires RAB21. (D) Immunofluorescence of endogenous MCT1 in wild type or RAB21-depleted cells. (E) Per cell integrated MCT1 intensity (in Relative Fluorescence Unit); SEM, n=2 independent experiments. (F) RAB21 interacts with SLC3A2. FLAG immunoprecipitation of FLAG:RAB21 and immunoblot of co-expressed SLC3A2:HA. Lysates correspond to 5% input, n=3 independent experiments. (G-H) RAB21 is required for appropriate SLC3A2, Basigin and CD44 trafficking. (G) Antibody-uptake assays in parental or knockout RAB21 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Note that non-internalized antibodies were removed by an acid wash, which was not fully efficient for CD147 and CD44, explaining plasma membrane labeling. (H) Percentage of cells with tubules in parental or RAB21 knockout cells; SEM, n=3 independent experiments.

Data information: In (A), scale bars represent 20µm or 5µm in enlarged views for CI-MPR, while they represent 10µm in enlarged views for GLUT1. (D and G) scale bars represent 20µm or 5µm in enlarged views. Statistical tests used: (B) - Unpaired t-tests, (C and H) - Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats
APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting (2018)
Figure 7

Figure 7: WASH and retromer complexes are required for SLC3A2 and Basigin sorting, and for full RAB21 activation and endosomal localization. (A) Validation of WASH and retromer knockouts and SLC3A2 protein levels. Immunoblots of parental or FAM21, VARP and VPS29 knockout cell populations. (B) Antibody-uptake assays in parental or knockout FAM21, VARP and VPS29 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. (C) Percentage of cells with tubules in parental or in various knockout cells; SEM, n=3 independent experiments. (D) Ratio of SLC3A2 integrated densities to GAPDH; SEM, n=3 independent experiments. (E-G) Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (E) EEA1 or (F) VPS26 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. (G) Per cell Pearson Correlation between GFP:RAB21 and EEA1 or VPS26; SEM, n=3 independent experiments.

Data information: (B) scale bars represent 20µm, (E and F) scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: (C) - Unpaired t-tests, (D) One sample T-tests (G) - Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.
Redox-regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress (2016)
Figure 1

Fig. 1: While ROS susceptibility of SUMO targets vary between cells, SUMO E1~E2 redox regulation is conserved.

A: Multiple pathways alter SUMOylation in response to oxidative stress.

B: Consequences of ROS on the SUMO proteome vary amongst cell lines. HeLa, MCF7, U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N-Ethylmaleimide (NEM). Samples were analyzed by immunoblotting with SUMO1 antibodies.

C: H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells. HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min. Cells were lysed with 1% SDS in PBS buffer and 20 mM NEM. Upon immunoprecipitation of the SUMO1 proteome, samples were analyzed by immunoblotting with TRIM28 antibodies.

D: Induction of the Uba2~Ubc9 disulfide is a conserved mechanism. HeLa, MCF7, U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min, lysed in non reducing buffer supplemented with 20 mM NEM, and analyzed by immunoblotting with Uba2 antibodies.
Redox-regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress (2016)
Figure 2

Fig. 2: A random mutagenesis screen for the identification of H2O2-resistant Ubc9.

A: Upper panel: Strategy for the identification of oxidation resistant Ubc9 variants. Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations. Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2. For this purpose, we used a FRET-based in vitro SUMOylation assay with recombinant SUMO E1, YFP-SUMO and CFP-RanGAPtail and ATP. Lower panel: Ubc9 W103R is H2O2 resistant. Bacterial lysates containing Ubc9 wt (left panel) or Ubc9 W103R (right panel) were tested as described.

B: Ubc9 W103 mutants are H2O2 resistant but not fully active. Left panel: Recombinant Ubc9 W103R, W103A and W103F were purified (embedded panel). Resistance against oxidation was tested under conditions of limiting E1 enzyme: 21 nM Aos1/Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP-SUMO1 and CFP-RanGAPtail. Right panel: To compare specific activities of wt and mutants in the absence of H2O2, sumoylation assays were carried out using limiting Ubc9 concentration. Reactions contained 35 nM Aos1/Uba2, 11 nM Ubc9, 85 nM each of YFP-SUMO1 and CFP-RanGAPtail and 1 mM ATP.
Redox-regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress (2016)
Figure 3

Fig. 3: Ubc9 D100A remains active in presence of H2O2.

A: Ubc9 possesses a specific insertion of two amino acids between the catalytic cysteine and tryptophane W103. Sequences of human UbcH5B (GI: 1145689), human E2-25k (GI: 1381164), human UbcH6 (GI: 1064914), human Ubc12 (GI:4507791), human Ubc9 (GI:4507785), and S. cerevisae Ubc9 (GI:1431070) were aligned using Clustalw with default parameters. The catalytic cysteine is shown in bold. Identical residues are indicated by an asterisk under the aligned sequences. The model of Ubc9 (PDB: 1A3S) was generated using Deepview and rendered using POV-Ray.

B: In FRET-based assay, Ubc9 D100A is active upon H2O2 treatment. Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig. 2B.

C: Ubc9 wt and D100A are equally active in CFP-RanGAPtail SUMOylation. Assays were with 35 nM Aos1/Uba2 and 15 nM Ubc9.

D: Ubc9 D100A is fully competent to form a thioester with SUMO. Single turnover reactions were performed using 630 nM E1, 3,3 µM SUMO1 and 600 nM Ubc9. Reactions were stopped at different time points by addition of non-reducing buffer..

E: Ubc9 wt and D100A are equally active in PIAS2β - dependent SUMOylation. Assays were with 300 nM GST-p53, 170 nM E1, 120 nM Ubc9, 5 µM SUMO2 and 49 nM GST-Pias2β

F: Ubc9 wt or D100A are equally active in RanBP2 – dependent SUMOylation. Assays were with 650 nM YFP-SP100, 110 nM of E1, 25 Ubc9, 40 nM RanBP2ΔFG and 3,5 µM SUMO1 (Left panel) or SUMO2 (Right panel).
Redox-regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress (2016)
Figure 4

Fig. 4: Ubc9 D100A sensitizes the E1~E2 disulfide to reduction.

A: Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT. 21 nM Aos1/Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP-SUMO1 and CFP-RanGAPtail diluted in DTT-free buffer. The final DTT concentration in the reaction was 6 µM. Prior to addition of ATP, extra amount of DTT was added at indicated final concentration.

B: Both Uba2~Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A.

100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2. One hour after H2O2 addition, 500 µM reduced glutathione was added. Samples were taken at indicated time points and the Uba2~Ubc9 disulfide was monitored by immunoblotting against Uba2. Lower panel: Quantification of the ratio between crosslinked Uba2 vs total Uba2
Redox-regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress (2016)
Figure 5

Fig. 5: Ubc9 D100A compromises cell survival upon oxidative stress.

A: Generation of polyclonal populations of stable cells. Top panel: Replacement strategy: U2OS cells transfected with pIRES-hrGFP II-based vectors were selected via addition of G418. After 4 weeks, low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks. Bottom panel: Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9: U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected. Cells were lysed in Laemmli buffer and Ubc9 and α-tubulin as loading control were detected by immunoblotting.

B: Ubc9 D100A is severely impaired for disulfide bond formation with Uba2. Stable U2OS cells were transfected with siRNA against Ubc9 for 72h, treated with 250 µM H2O2 for different time points, from 2 min to 2 hours, lysed in non-reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide (upper panel) or free Uba2 as loading control (see also Appendix Fig. S5). Lower panel: relative intensities of Ubc9 bands were measured using imageJ. Highest intensities in both cell population were set to 1.

C: Ubc9 D100A impairs cell survival upon H2O2 treatment. Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h, transferred into 6 well plates (20 000 cells / well) and treated with increasing H2O2 concentration for 1h. Ten days later, cells were washed with PBS, fixed with 4% formaldehyde and stained with crystal violet. The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin. An arbitrary value of 1 was given to the area occupied by non-treated cells. The graph is showing the mean of 4 independent experiments. Error bars are S.E.M. *: p < 0.05

D: Constitutive expression of Ubc9 D100A affects cell growth. 103 cells were plated per 10 cm plate and cultivated for 10 days in 20% or 5% O2 incubators. Subsequently, cells were colored using crystal violet. The area of each clone was measured using ImageJ. Representative plates are shown in the upper panel. Lower left panel: Distribution of colony sizes, shown for one of the three biological replicates. Lower right panel: Comparison of the colonies size between “Ubc9 wt“and “Ubc9 DA” cells. The average size of “Ubc9 wt” was set to 1. Shown is the mean of 3 independent experiments, each of which was performed in technical triplicates. Error bars are S.E.M.
Redox-regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress (2016)
Figure 6

Fig. 6: Ubc9 D100A cells have a defective DNA damage response pathway.

A: Phosphorylation of ATM is impaired in Ubc9 D100A cells. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies. B: Phosphorylation of gamma-H2AX is impaired in Ubc9 D100A. Experiment was performed as in A. C: Phosphorylation of Chk2 is impaired in Ubc9 D100A. Experiment was performed as described in A. D: Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure. Stable U2OS cell populations were exposed to 2.5 mM hydroxyurea or 250 µM H2O2. Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies. E: Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells. Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2.5 mM hydroxyurea. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.
Redox-regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress (2016)
Figure 7

Fig. 7: Ubc9 D100A cells fail to maintain repair foci despite DNA damage.

A: Impaired γ-H2AX and 53BP1 accumulation in Ubc9 D100A cells. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence was performed. Left panel: representative immunofluorescence images. Right panel: quantification of cells with γ-H2AX and 53BP1 foci. Error bars represent S.E.M, n=3 independent experiments * p < 0.05. B: H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells. Cells were treated as described in A. An alkaline comet assay was performed to evaluate the DNA damage. Representative images are shown. Error intervals represent S.E.M, n=4 independent experiments.
Redox-regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress (2016)
Figure 8

Fig. 8: The E1~E2 thiol switch maintains pATM level upon DNA damages.

A: Diamide is inducing the SUMO E1~E2 disulfide but not ATM phosphorylation. U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2. Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer. The lysates were resolved by SDS-PAGE. B: Left panel: The PP2A inhibitor okadaic acid rescues the γH2AX defect of the “Ubc9 DA” cells. “Ubc9 wt” or “Ubc9 DA” cells were treated with 0.25 µM okadaic acid for 30 min. After addition of 250 µM H2O2 in fresh medium for 3 hours, immunofluorescence was performed as described in Fig. 7A. Error bars represent S.E.M, n=3 independent experiments. Right panel: Representative immunofluorescence images. C: Left panel: Ubc9 D100A enhances sensitivity to Ara-C or etoposide. Cell survival assays were performed as described in Fig. 5C. Cells were exposed to Ara-C or etoposide (VP16) for 1 h and evaluated 10 days later. Right panel: N-acetylcysteine treatment rescues the higher toxicity of Ara-C or etoposide on “Ubc9 DA” cells. Cells were treated with 0.5 mM NAC 16 h prior to addition of etoposide or Ara-C. Graphs represent means of three to four independent experiments, error bars represent S.E.M. * p < 0.05.
Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium (2022)
Figure 3

Figure 3. Localization, Co-immunoprecipitation and mass spectrometry analysis

(A) Schematic drawing of the life cycle stages of Paramecium tetraurelia. MIC and parental MAC are represented in red, representing the DAPI signal, and developing MAC (dm) is represented in green until fully developed, representing the GFP signal. (B) ISWI1-tagged C-terminally with GFP localizes in the developing MAC as soon as developing new MACs (panel Early Development) become visible and remain there throughout late MAC development (panel Late Development). Red: DAPI, Green: ISWI1-GFP. Blue arrows identify developing MAC; scale bar 10μm.

(C) Western blot analysis using anti-HA antibody after coimmunoprecipitation of ISWI1-3XFlagHA fusion protein. Non-transformed cells (WT) of the same strain were used as the negative control. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 12% SDS gel.

(D) Volcano plot illustrating the distribution of proteins identified in label-free MS in WT Vs ISWI1-3XFlagHA. Significantly abundant proteins (fold change ≥ 4) are highlighted in orange.

(E) Western blot analysis using anti-HA and anti-GFP antibodies after coimmunoprecipitation of Ptiwi01-3XFlagHA fusion protein co-transformed with ISWI1-GFP. Non-transformed cells (WT) of the same strain and ISWI1-GFP fusion protein transformation were used as negative controls. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 10%SDS gel.

Source data are available online for this figure.
The high-energy transition state of the glutamate transporter homologue GltPh (2020)
Figure 1

Figure 1: Gain-of-function GltPh variants.

(A) A GltPh protomer in the OFS and IFS is shown in semi-transparent surface representation with the scaffold and transport domains colored wheat and blue, respectively. HP1 (yellow) and HP2 (red) are emphasized as cartoons. Bound l-Asp and Na+ ions are shown as green sticks and spheres, respectively. Select amino acids mutated in this study are shown as black spheres.

(B) Schematic representation of the GltPh transport cycle showing one protomer. Comparatively rapid and slow steps of the cycle are shaded pink and blue, respectively.

(C) The topology of a GltPh protomer showing tested mutation sites (circles) with filled circles corresponding to those shown in (A).

(D, E) Representative examples of dose-response curves and time courses. Lines through the dose-response curves are fits to the Michaelis-Menten equation; time courses are fitted to linear equations. Shown are means and standard errors over at least three independent repeats.

(F) Fold increase (positive values) or decrease (negative values) in the initial uptake rates of the variants relative to WT GltPh. Circles are averages of technical triplicates. Bars are means over independent repeats (as indicated). The dashed line marks a four-fold increase in the initial rate.

Data information: Color coding is the same in all panels. In (D to F), data are colored according to the structural elements where the mutations are located; combination mutants are colored purple. See also Appendix Figure S1 and Appendix Tables S1 and S2.
The high-energy transition state of the glutamate transporter homologue GltPh (2020)
Figure 2

Figure 2: Transport domain dynamics under apo (A) and transport (B) conditions.

(A, B) Schematic representations of the experimental conditions are shown on the far left. Data are shown for WT GltPh and select gain-of-function mutants, as indicated above the panels. Transition density plots (top of each panel) show the frequency of transitions between the EFRET values. The number of trajectories analyzed (N) and the population-wide mean frequency of transitions is shown on the panels. Representative 50 s sections of single-molecule EFRET trajectories (middle) with raw data in black and idealizations in brown. Scale bar and the conformational states corresponding to the low and intermediate EFRET values are shown to the left of the panels. Dwell-time distributions for the OFS in blue and the IFS in red (bottom). Mean dwell times are shown on the panels in corresponding colors. Black lines represent photobleaching survival plots normalized from 1 to 0. Shown are means and standard errors over at least three independent repeats. See also Appendix Figures S2 -S4.