EMBO/soda-nel huggingface dataset:
The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage (2019)
Figure 1

Figure 1 - Microscopy-based siRNA screen of human E2 ubiquitin-conjugating enzymes identifies UBE2QL1 to be required for ubiquitination of damaged lysosomes.

A Robust immunofluorescence detection of ubiquitination on LAMP1-positive compartments with pan-ubiquitin FK2 or K48 chain-specific antibodies. HeLa cells were treated with 250 µM LLOMe or EtOH (untreated) for 3 h, fixed and stained with indicated antibodies. Confocal microscopy was followed by automated image analysis. Graphs represent data from three independent experiments with ≥ 50 cells per condition (mean ± SD). Scale bar: 20 µm.

B Screen overview.

C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes. HeLa cells were seeded in 384-well plates, transfected with a siRNA library in quadruplicates for 72 h. Cells were LLOMe-treated and processed for imaging as in (A). Plates were imaged automatically with a spinning disk confocal microscope. Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined. Robust z-scores were calculated for LLOMe-treated samples for each depletion against the values of the whole plate. Samples exceeding the threshold of -2 of robust z-score (highlighted in red) were considered for the secondary screen.

D Selected candidates were re-screened with the same work pipeline as in (C) but with the four single siRNAs of pools of each candidate from the primary screen. Candidates exceeding the threshold in both stainings are highlighted in red.

E Damage-induced translocation of UBE2QL1 to lysosomes. The three best candidates (from D) were transiently expressed with N-terminal HA-tag in HeLa cells. Cells were treated with LLOMe or EtOH (untreated) for 3 h, fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy. Scale bar: 10 µm.

F Automated quantification of (E). Shown are Pearson correlation coefficients (P.C.C) of HA and LAMP1 signals. Graph represents data from three independent experiments with ≥ 30 cells per condition (mean ± SD). ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).

G Western blot detection of endogenous UBE2QL1 and depletion efficiency. Cells were transfected with control siRNA (Ctrl), two UBE2QL1 siRNAs from the screen (#2 or #4) or an additional siRNA (#5), and lysates probed with an antibody to UBE2QL1 as indicated. GAPDH was probed as loading control.
The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage (2019)
Figure 2

Figure 2 - UBE2QL1 depletion affects ubiquitination of damaged lysosomes.

A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h, and LLOMe or control-treated (untreated) for 3 h. Cells were fixed and immuno-stained for LAMP1 along with antibodies specific for K63 or K48-linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope. Note the strong reduction of damage-induced K48 ubiquitination and decreased intensity of the K63 signal. Scale bar: 20 µm.

B, C Automated image quantification of (A). Shown are percentages of K48 or K63-positive LAMP1 vesicles per cell. Graphs represent data from three independent experiments with ≥ 70 cells per condition (mean ± SD). **P<0.01; ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).
The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage (2019)
Figure 3

Figure 3 - UBE2QL1 recruitment coincides with the delayed K48-linked ubiquitination response.

A HeLa cells expressing UBE2QL1-HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy. Cells were stained with antibodies specific for LAMP1 (not shown), and combinations of HA with K48 chains, K63 chains or p62 as indicated. Note the delayed co-emergence of K48 chains and UBE2QL1 vesicles, compared to the earlier appearance of K63 chains and p62. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 10 µm, 2 µm for inlays.

B Automated quantification of (A). Shown are the Pearson correlation coefficients (P.C.C.) representing colocalization of UBE2QL1-HA, K48 chains, K63 chains or p62 with LAMP1-positive vesicles. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD).

C Automated quantification of (A). P.C.C.'s representing colocalization of K48 chains, K63 chains or p62 with UBE2QL1-HA in cells treated with LLOMe or vehicle alone (untreated) for 180 min. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD).
The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage (2019)
Figure 4

Figure 4 - UBE2QL1 translocates in the vicinity of damage sensors, ubiquitination targets and ubiquitin effectors at permeabilized lysosomes.

A, B, C Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (A) and (B) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 and K48. (A) p62 (10 nm gold particles) localizes as a cap (arrowheads) to the outer limiting membrane of lysosomes (L). (B) K48 ubiquitin chains (15 nm gold particles, arrows) are found in the lumen of lysosomes, that also contain p62 (10 nm gold particles, arrowheads) at their outer membrane. (C) Overexpressed UBE2QL1-GFP (10 nm gold particles) is found in late endosomes (LE, Inset) and lysosomes. M = mitochondrion. Scale bars: 200 nm.

D, E, F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram (D) and displayed high correlation coefficients (E). (F) Results from the four experiments are summarized in a volcano plot. Horizontal dotted line represents the significance threshold (p > 0.05). The vertical lines indicate the fold change cutoff (log2 (H/L) ≥ 1.5). Red dots indicate proteins above the significance and fold-change thresholds. Protein names are color-coded in blue (endosome/lysosome-associated), green (endolysosomal damage/autophagy-associated) and orange (VCP/p97 and its cofactors).
The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage (2019)
Figure 5

Figure 5 - UBE2QL1 abrogates recruitment of VCP/p97, reduces accumulation of p62 and compromises association of LC3 with damaged lysosomes.

A HeLa cells stably expressing p97-GFP were control or UBE2QL1-depleted for 48 h and expression of p97-GFP was induced for the last 24 h. Cells were LLOMe or control treated for 1 h, fixed after a chase of 2 h, and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated. Note the reduction of the K48 and p97-GFP signals on damaged lysosomes in UBE2QL1-depleted cells. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 10 µm for inlays. See Fig EV4A for images of cells treated with an alternative siRNA.

B Automated quantification of (A) and Fig EV4A. Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p97-GFP and LAMP1 signals, normalized to untreated siCtrl. Data are from four independent experiments with ≥ 30 cells per condition.

C HeLa cells were control or UBE2QL1-depleted for 60 h, fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays. See Fig EV4B for images of cells treated with an alternative siRNA and control cells.

D Quantification of (C) and Fig EV4B. Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p62 on LAMP1 signals, normalized to untreated siCtrl. Graph represents data from three independent experiments with ≥ 30 cells per condition.

E HeLa cells were treated as in (C), but fixed after additional 2 h of chase. Samples were stained for Galectin-3 (Gal3) to detect damaged lysosomes, and LC3B as marker for autophagosomes. Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.

F Quantification of (E). Shown are fold increases of Pearson correlation coefficients (P.C.C.) of LC3B and Gal3 signals, normalized to untreated siCtrl, representing LC3B colocalization with Gal3. Graph represents data from three independent experiments with ≥ 50 cells per condition.

G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h, treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A/B. GAPDH was probed as loading control.

Data information: In (B, D and F) data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).
The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage (2019)
Figure 6

Figure 6 - UBE2QL1 is essential for efficient lysophagy and cell survival after lysosomal damage.

A Gal3 puncta assay for damaged lysosomes. HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone (untreated) for 1 h. Cells were fixed at indicated times after washout, stained with DAPI and Gal3 and LAMP1 antibodies, and processed for confocal microscopy. Note increased number of Gal3 puncta in untreated UBE2QL1-depleted cells and their persistence 10 h after LLOMe-induced damage. Scale bars: 10 µm, 2 µm for inlays. Arrows indicate Gal3 and LAMP1 colocalizing vesicles.

B Automated quantification of (A). The percentage of cells with more than three Gal3 puncta is shown. Graph represents data from three independent experiments with ≥ 50 cells per condition (mean ± SD). *P<0.05; **P<0.01 (One-way-ANOVA with Dunnett's multiple comparison test).

C Survival assay. Control or UBE2QL1-depleted HeLa cells (48 h) were treated with increasing concentrations of LLOMe as indicated. Cell viability was measured with the MTS assay. Graph represents data from one experiment including three replicates (mean ± SD). *P<0.05; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).
The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage (2019)
Figure 7

Figure 7 - UBE2QL1 and its homologue UBC-25 in C. elegans are essential for maintenance of lysosomal integrity.

A Control or UBE2QL1-depleted (for 60 h) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy. Note nuclear translocation of TFEB upon loss of UBE2QL1. Scale bar: 20µm. See Fig EV5A for images of cells treated with an alternative siRNA.

B Automated quantification of (A). The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions. Graph represents data from four independent experiments with ≥ 100 cells per condition (mean ± SD). ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).

C Cells as in (A) were stained for LAMP1 and mTOR. Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells. Scale bar: 20 µm. See Fig EV7B for images of cells treated with an alternative siRNA.

D Automated quantification of (C). The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients (P.C.C.) of the signals. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD). *P<0.05 (One-way-ANOVA with Bonferroni's multiple comparison test).

E Automated quantification of (C). Number of LAMP1-positive vesicles per cell is shown. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD).

F Western blot analysis of lysates of cells treated as in (C). As a positive control, cells were treated with the mTOR-inhibitor Torin1 (1 µM) for 2 h. GAPDH was probed as loading control.

G C.elegans wild-type (N2) and mutant strains for ubc-25(ok1732), for scav-3(qx193) or the ubc-25 and scav-3 double mutant (ok1732;qx193) carrying a EGFP-Gal3 transgene were imaged in adult worms (L4 + 24 h). 3D reconstructions of the hypodermis. Small and big units represent 1 and 5 µm, respectively.

H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified. At least 20 animals were scored in each strain. *P<0.05; **P<0.01; ****P<0.0001 (One-way-ANOVA with Dunnett's multiple comparison test or Student's two-tailed unpaired t test).

I Populations of worms with different extent of lysosome permeabilization were analyzed. Graph represents percentage of worms with less than 5, 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length.

Expanded view - figure legends
Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1 (2021)
Figure 1

Figure 1. Interaction of Rabaptin5 with FIP200 as identified by two-hybrid analysis in yeast and by co-immunoprecipitation in HeLa and HEK293A cells.

A Schematic representation of the sequence of Rabaptin5. Coiled-coil (CC) segments are shown in yellow. Colored backgrounds highlight the segments shown to interact with Rab4, Rab5, Rabex5, and the GAE and GAT domains of GGAs (Golgi-localizing, γ-adaptin ear homology domain, ARF-binding proteins). Below, the segments used to test yeast two-hybrid interaction with residues 257-444 of FIP200 are shown with their number (#) and the observed interaction (+ or -).

B Yeast two-hybrid analysis for interaction between the above-shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257-444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica-plated on medium with His or without His, but containing 3-amino-1,2,4-triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination.

C Schematic representation of the sequence of FIP200 with its coiled coil segments in yellow. Residues 281-439 (gray) indicates the minimal sequence identified to interact with Rabaptin5 in the yeast two-hybrid screen.

D FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti-GAPDH antibody.

E-H Lysates of HeLa cells transiently transfected with full-length FIP200-mCherry (FIP200-mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (∆280-440) were immuprecipitated with anti-mCherry (IP FIP200-mCh) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G). Co-immunoprecipitation of Rabaptin5, ATG13, and ULK1 with FIP200∆280-440 (FIP∆) was quantified in comparison to that with wild-type FIP200 (H; signals normalized to that of the immunoprecipitated protein; mean ± S.D. of three independent experiments each).
Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1 (2021)
Figure 2

Figure 2. Autophagy proteins are targeted to Rabaptin5-positive endosomes upon chloroquine treatment.

A-I To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK+Rbpt5) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared to untreated cells (-CQ, A'). Rabaptin5 levels were anayzed by immunoblotting in comparison to parental HEK293A cells (panel B). HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-galectin3 (mCh-Gal3), mRuby3-galectin8 (mRuby-Gal8), mCherry-FIP200, or -ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry-galectin3 or mRuby3-galectin8 (C), ubiquitin (Ub; D), p62 (E), mCherry-FIP200 (F), WIPI2 (G), mCherry-ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged, ring-like early endosomes. Rabaptin5-positive enlarged endosomes positive for mCherry-galectin3 (Gal3) or mRuby3-galectin8 (Gal8) were quantified (C'; mean ± S.D. and individual values of three independent experiments counting >45 structures each).

J, K HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry-ATG16L1. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 (J) or mCherry-ATG16L1 (K), and M2 showing the respective inverse (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01, ***P<0.001).
Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1 (2021)
Figure 3

Figure 3. Chloroquine-, but not Torin1- or LLOMe-induced autophagy depends on Rabaptin5.

A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (-) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control.

B WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01).

C HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm.

D WIPI2 or LC3B puncta per cell were quantified (mean ± S.D. of three independent experiments).
Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1 (2021)
Figure 4

Figure 4. Chloroquine-induced autophagy requires the FIP200-ULK1/2-ATG13 complex.

A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ATG13 (siATG13) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls.

B WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: **P<0.01).

C, C' HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ULK1 (siULK1), ULK2 (siULK2), or both (siULK1+2) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ULK1 knockdown was assayed by immunoblotting on the left and its effect on autophagy induced by starvation and Torin1 treatment in Appendix Fig S1. The efficiency of ULK2 knockdown was assayed by quantitative RT-PCR and the result is shown in panel C'.

D WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01, ***P<0.001).

E HEK293A cells were incubated without (-), with 60 µM chloroquine (+CQ), or with chloroquine and 5 µM MRT68921 (+CQ +MRT) for 150 min and fixed and stained for WIPI2 and LC3B. Scale bar, 10 µm.

F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B (mean ± S.D. of three independent experiments).
Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1 (2021)
Figure 5

Figure 5. Rabaptin5 binds to the WD domain of ATG16L1 via a conserved interaction motif. A HeLa cells transiently transfected with full-length mCherry-ATG16L1 were treated with 60 µM chloroquine (CQ) for 0, 30, or 120 min, lysed, and immunoprecipitated with anti-Rabaptin5 (IP: Rbpt5) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for Rabaptin5 and ATG16L1. Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that without (0 min) chloroquine treatment (mean ± S.D. of six independent experiments; two-tailed Student's t test: *P<0.05, **P<0.001). B Co-immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP-KO). Anti-HA antibodies were used as a control (IP HA). On the right, HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatment (mean ± S.D. of four independent experiments; two-tailed Student's t test: *P<0.05, **P<0.001). C-D Lysates of HEK293A or HeLa cells transiently transfected with full-length mCherry-ATG16L1 (wt) or a mutant lacking the WD domain (∆WD; residues 1-319 of ATG16L1, precisely deleting only the WD40 repeats residues 320-607) were immunoprecipitated with anti-Rabaptin5 or anti-FLAG antibodies, and immunoblotted for Rabaptin5 and mCherry-ATG16L1 (C and D), or immunoprecipitated with anti-FIP200 or anti-HA antibodies, and immunoblotted for FIP200 and mCherry-ATG16L1 (E). In panel D, cells were incubated with or without 60 µM chloroquine for 30 min before analysis. Co-immunoprecipitation of ATG16L1∆WD with Rabaptin5 (C) was reduced to 6.6±2.1% and 3.4±2.1% in HEK293A and HeLa cells, respectively, relative to that of full-length ATG16L1 (signals normalized to the immunoprecipitated protein; mean ± S.D. deviation of three independent experiments each). E The consensus sequence of the ATG16L1 interaction motifs of TMEM59, NOD2, and TLR2 (above; Boada-Romero et al. 2013) is shown together with the matching sequence in Rabaptin5 (below). The three point mutations to alanine to produce the AAA mutant of Rabaptin5 are indicated. F Lysates of HeLa cells transiently transfected with myc-tagged wild-type Rabaptin5 (wt) or triple-alanine mutant (AAA) were immunoprecipitated with anti-myc (IP myc) or anti-FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1. Co-immunoprecipitation of ATG16L1 with Rabaptin5-AAA triple mutant was reduced to 1.5±1.2% relative to that with wild-type Rabaptin5 (signals normalized to that of the immunoprecipitated protein; mean ± S.D. of three independent experiments).