ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival

Abstract

Although lysosomes perform a number of essential cellular functions, damaged lysosomes represent a potential hazard to the cell. Such lysosomes are therefore engulfed by autophagic membranes in the process known as lysophagy, which is initiated by recognition of luminal glycoprotein domains by cytosolic lectins such as Galectin-3. Here, we show that, under various conditions that cause injury to the lysosome membrane, components of the endosomal sorting complex required for transport (ESCRT)-I, ESCRT-II, and ESCRT-III are recruited. This recruitment occurs before that of Galectin-3 and the lysophagy machinery. Subunits of the ESCRT-III complex show a particularly prominent recruitment, which depends on the ESCRT-I component TSG101 and the TSG101- and ESCRT-III-binding protein ALIX Interference with ESCRT recruitment abolishes lysosome repair and causes otherwise reversible lysosome damage to become cell lethal. Vacuoles containing the intracellular pathogen Coxiella burnetii show reversible ESCRT recruitment, and interference with this recruitment reduces intravacuolar bacterial replication. We conclude that the cell is equipped with an endogenous mechanism for lysosome repair which protects against lysosomal damage-induced cell death but which also provides a potential advantage for intracellular pathogens.

Keywords: autophagy; endosome; lysophagy; lysosome; membrane repair.

Figure 1. The ESCRT‐III subunit CHMP4B is recruited to Galectin‐3‐positive damaged endolysosomal membranes

1. Representative fluorescence micrographs of HeLa cells treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO (Ctrl) for 1 h before fixation and immunostained with Hoechst (blue), anti‐CHMP4B (green), anti‐GAL3 (red), and anti‐LAMP1 (white) are presented. Cells treated with LLOMe show increased recruitment of ESCRT‐III protein to lysosomes when compared to Ctrl cells. Number of foci per cell was quantified and is indicated as mean ± SD. Data are quantified from >86 cells per condition from three independent experiments. CHMP4B, GAL3, LAMP1 foci per cell (Ctrl versus LLOMe treatment): P = 0.0398, P = 0.0033, P = 0.2400 (Student's t‐test), respectively.

2. HeLa cells stably expressing CHMP4B‐eGFP and mCherry‐Galectin‐3 treated as in (A) and stained with CD63 antibody are shown. Number of foci per cell was quantified and is indicated as mean ± SD. Data are quantified from >87 cells per condition from three independent experiments. CHMP4B, GAL3, CD63 foci per cell (Ctrl versus LLOMe treatment): P = 0.0111, P = 0.0050, P = 0.3793 (Student's t‐test), respectively.

Data information: Scale bars: 10 μm and 1 μm (inset).

Figure EV1. CHMP4B is recruited to Galectin‐3‐positive damaged endolysosomal membranes in various cell lines

1. Representative fluorescence images of a HeLa‐mCherry‐Galectin‐3 stable cell line treated with 250 μM lysosomotropic drug LLOMe for 1 h, fixed and labeled with CD63 antibody. To determine the statistical significance, the number of CHMP4B, GAL3, and CD63 foci per cell observed after LLOMe treatment was compared to Ctrl using Student's t‐test. P‐values for CHMP4B, GAL3, and CD63 are as follows: P = 0.000001, P = 0.0017, P = 0.4160, respectively.

2. Experimental setup as in (A) where RPE‐1 cells are labeled with CHMP4B, Galectin‐3, and LAMP1 antibodies. P‐values for CHMP4B, GAL3, and LAMP1 foci per cell (Ctrl versus LLOMe treatment): P = 0.00001, P = 0.00018, P = 0.1368 (Student's t‐test), respectively.

3. Representative images of fixed H‐460 cells after 1‐h treatment with 250 μM LLOMe. Cells were stained for endogenous CHMP4B, GAL3, and LAMP1. CHMP4B, GAL3, and LAMP1 foci per cell (Ctrl versus LLOMe treatment): P = 0.0192, P = 0.0493, P = 0.6333 (Student's t‐test), respectively.

Data information: For all conditions, the number of foci per cell was quantified from >70 cells per condition from three independent experiments. Data are presented as mean ± SD. Scale bars: 10 and 1 μm (inset).

Figure EV2. CHMP4B is recruited to damaged endolysosomal membranes after treatment with different agents

1. HeLa cells stably expressing CHMP4B‐eGFP and mCherry‐Galectin‐3 were treated with 250 μM lysosomotropic drug GPN for 1 h, fixed and imaged. Representative images showing recruitment of CHMP4B to damaged endolysosomal membranes are presented. Number of foci per cell (>90 cells per condition from four independent experiments) was quantified and is indicated as mean ± SD. P‐values for comparisons CHMP4B, GAL3, and LAMP1 foci per cell (Ctrl versus GPN treatment) are as follows: P = 0.0002, P = 0.0008, P = 0.1113 (Student's t‐test), respectively.

2. Representative fluorescence images showing recruitment of CHMP4B to damaged membranes in HeLa cells stably expressing CHMP4B‐eGFP and mCherry‐Galectin‐3 and RPE‐1 cells after 2‐h treatment with 7 μm cationic amphiphilic drug terfenadine. In HeLa cells, there is a slight but not significant increase in CHMP4B‐positive foci. On the other hand, RPE‐1 cells labeled with CHMP4B, GAL3, and LAMP1 antibodies have elevated number of CHMP4 and GAL3 foci per cell. For HeLa‐CHMP4BeGFP‐mCherry‐Galectin‐3: CHMP4B, GAL3 foci per cell (Ctrl versus terfenadine treatment): P = 0.2679, P = 0.2266 (Student's t‐test), respectively. For RPE‐1 cell line: CHMP4B, GAL3 foci per cell (Ctrl versus terfenadine treatment): P = 0.0169, P = 0.1806 (Student's t‐test), respectively. Number of foci per cell was quantified from >75 cells per condition from two independent experiments and is indicated in the figure as mean ± SD.

Data information: Scale bars: 5 μm and 1 μm (inset).

Figure 2. ESCRT‐I, ESCRT‐II, ESCRT‐III, ALIX, and VPS4A are recruited to endolysosomes upon LLOMe treatment

To screen for ESCRT proteins that are involved in the endolysosomal repair process, cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence. TSG101, a component of the ESCRT‐I complex, and EAP30, an ESCRT‐II protein, are clearly recruited to the sites of damage compared to the DMSO control (Ctrl). As shown, the ESCRT‐III complex together with ALIX is recruited to damaged endolysosomes. In contrast, HRS, a component of the ESCRT‐0 complex, and HD‐PTP show no recruitment to damaged endomembranes. Number of foci per cell was quantified from >85 cells per condition from three independent experiments and are presented as mean ± SD. Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student's t‐test, the P‐values for which are as follows: HRS P = 0.9257, GAL3 P = 0.0050; TSG101 P = 0.0243, GAL3 P = 0.0489; EAP30 P = 0.0006, GAL3 P = 0.0307; CHMP2A P = 0.0284, GAL3 P = 0.0260; CHMP4B P = 0.0028, GAL3 P = 0.0414; VPS4A P = 0.0309, GAL3 P = 0.0461; ALIX P = 0.0249, GAL3 P = 0.0471; HD‐PTP P = 0.4898, GAL3 P = 0.0105. Scale bars: 5 μm and 1 μm (inset).

Figure EV3. Screening for additional ESCRT proteins that are involved in the lysosomal repair process

ESCRT‐0: To clarify the role of HRS, a cell line stably expressing eGFP‐HRS was incubated with 250 μm LLOMe for 30 min, pre‐permeabilized with PEM buffer (for details see Materials and Methods), and fixed. HRS shows no significant changes upon lysosomal damage when compared to untreated cells. ESCRT‐III: After treatment with 250 μm LLOMe for 30 min, HeLa cells stably expressing mCherry‐IST1 or eGFP‐VPS4B were fixed and imaged. As shown in the representative fluorescence images, IST1 is recruited to damaged lysosomes whereas VPS4B shows no change in localization upon endolysosomal membrane damage. Number of foci per cell was quantified and is indicated as mean ± SD. Data were obtained from >71 cells per condition from three independent experiments. Number of foci per cell (Ctrl versus LLOMe treatment): HRS P = 0.1241, GAL3 P = 0.0309 (Student's t‐test); IST1 P = 0.0038, GAL3 P = 0.0164 (Student's t‐test); VPS4B P = 0.1102, GAL3 P = 0.0387 (Student's t‐test). Scale bars: 5 μm and 1 μm (inset).

Figure 3. Dynamics of CHMP4B accumulation at the damaged endolysosomal membranes depend on other ESCRT components

HeLa cells stably expressing CHMP4B‐eGFP were transfected with siRNAs against the following: (A) HRS, (B) TSG101, (C) CHMP2A, (D) ALIX, and (E) both TSG101 and ALIX. Forty‐eight hours post‐transfection cells were used for live‐cell imaging experiments. LLOMe was added using perfusion system.

1. Depletion of HRS does not alter the dynamics of CHMP4B‐eGFP recruitment as compared to siCtrl during treatment with LLOMe.

2. Downregulation of TSG101, with two independent siRNAs, causes a delay in CHMP4B‐eGFP recruitment as compared to siCtrl, indicating an important role of the ESCRT‐I complex in recruiting the downstream components.

3. CHMP2A depletion accumulates and stabilizes CHMP4B‐eGFP‐positive foci.

4. siRNA‐mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment.

5. Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage.

Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live‐cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity).Source data are available online for this figure.

Figure 4. ESCRTs are essential for the repair after endolysosomal damage

1. HeLa cells stably expressing CHMP4B‐eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND‐99 and fixed at different time points as indicated. After 10 min of LLOMe treatment, CHMP4B is recruited whereas the number of Lysotracker‐positive foci is reduced. After 30 min, lysosomes gain back functionality (judging by the increased number of Lysotracker foci) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes. Representative confocal images for each time point are shown. Scale bars: 5 μm.

2. Quantification graph (>250 cells per condition from four independent experiments) showing CHMP4B and Lysotracker‐positive foci per cell at different time points. Error bars correspond to 95% confidence intervals.

3. Quantification graph showing dynamics of Lysotracker recovery in control (siCtrl) and both TSG101‐ and ALIX‐depleted cells. HeLa cells stably expressing CHMP4B‐eGFP were co‐transfected with siRNAs against TSG101 and ALIX. Forty‐eight hours post‐transfection, cells were pre‐treated for 20 min with 75 nM Lysotracker Deep Red, which was used as a read‐out. While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl, simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair. Data from >40 cells per condition from four independent live‐cell imaging experiments are shown. Graph is normalized to the area occupied by the cells. Error bars correspond to 95% confidence intervals.

Figure 5. CHMP4B is recruited before Galectin‐3 to the damaged endolysosomal membranes

Representative movie montage of a live‐cell imaging experiment illustrating the dynamics of CHMP4B‐eGFP and mCherry‐Galectin‐3 recruitment at different time points before and after LLOMe treatment. Using the perfusion system, 250 μM LLOMe was added to HeLa cells stably expressing CHMP4B‐eGFP and mCherry‐Galectin‐3 and images were acquired every 20 s for a period of 20 min. As shown in the representative movie montage and the quantification graph, CHMP4B recruitment precedes Galectin‐3, which is an established marker for detection of damaged endolysosomal membranes. The quantification graph is normalized to the area occupied by the cells. Data were obtained from >40 cells per condition from three independent experiments. Error bars correspond to 95% confidence intervals. Scale bars: 5 μm.

Figure 6. ESCRT‐III recruitment to damaged lysosomes precedes lysophagy

1. HeLa cells stably expressing CHMP4B‐eGFP and mCherry‐Galectin‐3 were incubated with 250 μM LLOMe, fixed at different time points as indicated and stained for ubiquitin. As presented, CHMP4B is recruited before ubiquitin and Galectin‐3 upon lysosomal membrane damage.

2. HeLa cells stably expressing CHMP4B‐eGFP were treated as in (A) and stained for p62. CHMP4B is recruited before p62 on the damaged lysosomes. The number of LAMP1‐positive foci appears stable independently of LLOMe treatment.

3. Experimental setup as in (A and B) where HeLa cells stably expressing CHMP4B‐eGFP and mCherry‐Galectin‐3 were stained for LC3. As shown, CHMP4B is recruited prior to LC3.

Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments.

Figure EV4. Representative confocal images corresponding to Fig 6

1. A

   Recruitment of CHMP4B as early as 5 min following LLOMe treatment whereas ubiquitin appears to be recruited later.

2. B, C

   Recruitment of CHMP4B to damaged endolysosomal membranes precedes p62 and LC3. In addition, p62 is recruited before LC3.

Data information: Scale bars: 5 and 1 μm (inset).

Figure 7. ESCRTs are essential for cell viability after endolysosomal damage

1. HeLa cells were transfected with different siRNAs as indicated. Forty‐eight hours post‐transfection, cells were treated with 250 μM LLOMe for 3, 6, and 10 h harvested and stained for Annexin V. Cells were immediately processed for flow cytometry. Already after 3 h of treatment with LLOMe, cells co‐transfected with TSG101+ALIX siRNA showed an increase in apoptotic cell death, whereas TSG101 siRNA‐transfected cells showed elevated Annexin V staining after 6 h of treatment.

2. Experimental set up as in (A) with additional propidium iodide (PI) staining of transfected HeLa cells.

Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one‐way ANOVA test. P‐values are presented where significant. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001.

Figure EV5. ESCRTs are essential for cell viability after endolysosomal damage

1. Cells were transfected with control (siCtrl), TSG101, ALIX, or both siRNAs. Forty‐eight hours after transfection, cells were harvested and profiled with flow cytometry. Dot plots and gating strategy of one out of three experiments are shown.

2. HeLa cells were transfected with control (siCtrl), ALIX, TSG101, or both siRNA. Efficient depletion was confirmed by Western blot analysis. β‐actin was used as loading control.

Source data are available online for this figure.

Figure 8. ESCRT‐III and Galectin‐3 are recruited to the Coxiella burnetii vacuole

1. HeLa cells stably expressing CHMP4B‐eGFP were transfected with the mCherry‐Galectin‐3 plasmid. Twenty‐four hours later, cells were infected with WT Coxiella burnetii. Live‐cell imaging started 24 h post‐infection, and several time points are indicated during the next 24 h of observation (time indicated in minutes, see Movie EV9). Arrows indicate Coxiella‐containing vacuole (DRAQ5 labeling) becoming positive for CHMP4B‐eGFP and mCherry‐Galectin‐3. Scale bars: 50 μm.

2. Bacterial viability and replication assay upon TSG101 KD. HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h. Then, cells were infected with mCherry‐Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells. Seventy‐two hours later, infected cells were fixed, DAPI stained, and processed for quantitative image analysis. Between 30 and 40 fields representing more than 9,000 cells per condition of three independent experiments were analyzed. Error bars represent SD. Statistical significance was determined using one‐way ANOVA test. **P ≤ 0.01 and ***P ≤ 0.001.