ESCRT ‐mediated lysosome repair precedes lysophagy and promotes cell survival
2018; Springer Nature; Volume: 37; Issue: 21 Linguagem: Inglês
10.15252/embj.201899753
ISSN1460-2075
AutoresMaja Radulovic, Kay Oliver Schink, Eva M. Wenzel, Viola Nähse, Antonino Bongiovanni, Frank Lafont, Harald Stenmark,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle12 October 2018free access Source DataTransparent process ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival Maja Radulovic Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Kay O Schink Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Eva M Wenzel orcid.org/0000-0002-5561-3344 Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Viola Nähse Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Antonino Bongiovanni Center of Infection and Immunity of Lille: CNRS UMR8204, INSERM U1019, Institut Pasteur de Lille, Lille Regional University Hospital Centre, Lille University, Lille, France Search for more papers by this author Frank Lafont Center of Infection and Immunity of Lille: CNRS UMR8204, INSERM U1019, Institut Pasteur de Lille, Lille Regional University Hospital Centre, Lille University, Lille, France Search for more papers by this author Harald Stenmark Corresponding Author [email protected] orcid.org/0000-0002-1971-4252 Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Maja Radulovic Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Kay O Schink Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Eva M Wenzel orcid.org/0000-0002-5561-3344 Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Viola Nähse Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Antonino Bongiovanni Center of Infection and Immunity of Lille: CNRS UMR8204, INSERM U1019, Institut Pasteur de Lille, Lille Regional University Hospital Centre, Lille University, Lille, France Search for more papers by this author Frank Lafont Center of Infection and Immunity of Lille: CNRS UMR8204, INSERM U1019, Institut Pasteur de Lille, Lille Regional University Hospital Centre, Lille University, Lille, France Search for more papers by this author Harald Stenmark Corresponding Author [email protected] orcid.org/0000-0002-1971-4252 Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Author Information Maja Radulovic1,2, Kay O Schink1,2,‡, Eva M Wenzel1,2,‡, Viola Nähse1,2, Antonino Bongiovanni3, Frank Lafont3 and Harald Stenmark *,1,2 1Faculty of Medicine, Centre for Cancer Cell Reprogramming, University of Oslo, Oslo, Norway 2Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway 3Center of Infection and Immunity of Lille: CNRS UMR8204, INSERM U1019, Institut Pasteur de Lille, Lille Regional University Hospital Centre, Lille University, Lille, France ‡These authors contributed equally to this work *Corresponding author. Tel: +47 22781818; E-mail: [email protected] EMBO J (2018)37:e99753https://doi.org/10.15252/embj.201899753 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Damaged lysosomes are lethal to cells and cleared by lysophagy. While pathways exist to protect lysosomes from damage, repair mechanisms for injured organelles are unknown. Here ESCRT proteins, originally identified as regulators of protein sorting, are shown to promote the repair of lysosomal membrane and bacteria-containing vacuoles to protect cells against cell death and provide replicative advantage to pathogens, respectively. ESCRT-I, -II and -III proteins are recruited to damaged endolysosomal membranes. The ESCRT machinery mediates repair of damaged lysosomes. ESCRT recruitment to damaged lysosomes precedes lysophagy. ESCRT-mediated lysosome repair promotes cell viability after lysosome injury. ESCRT recruitment to Coxiella burnetii vacuoles promotes bacterial replication. Introduction Lysosomes are essential organelles that carry out numerous cellular functions, including degradation of macromolecules, pathogen killing, and signaling functions. On the other hand, because of their low intraluminal pH and high content of Ca2+ and enzymes that can potentially trigger cell death, lysosome damage caused by pathogens, sharp crystals, amphiphilic drugs, or other membrane-disrupting agents impose a serious threat to cell viability (Kroemer & Jaattela, 2005; Papadopoulos & Meyer, 2017). Previous work has uncovered a lysosome-protective function of heat-shock protein 70 (Kirkegaard et al, 2010) and the existence of an autophagic pathway that sequesters and degrades damaged lysosomes (Hung et al, 2013; Maejima et al, 2013). This pathway, termed lysophagy, is triggered by sensing of lysosomal membrane lesions by cytosolic lectins such as Galectin-3, which recognize exposed intraluminal carbohydrate chains of lysosomal glycoproteins (Maejima et al, 2013; Papadopoulos & Meyer, 2017). This is followed by ubiquitination of lysosomal membrane proteins, processing by the ATPase p97, and mobilization of LC3-containing autophagic membranes (Papadopoulos & Meyer, 2017). The existence of pathways that protect lysosomes from damage and incapacitate damaged lysosomes has begged the question whether repair mechanisms for damaged lysosomes also exist (Papadopoulos & Meyer, 2017). Here, we have tested the hypothesis that the endosomal sorting complex required for transport (ESCRT) machinery (Henne et al, 2013) might play a role in lysosome repair and thereby have a cytoprotective function. Originally identified for its function in protein sorting to the yeast lysosome equivalent, the vacuole (Katzmann et al, 2002), the ESCRT machinery has recently been shown to have a more general function in membrane involution and scission processes that occur in the direction "away" from cytosol, such as virus budding from the plasma membrane, daughter cell separation during cytokinesis, and sealing of the nuclear envelope during mitotic exit (Christ et al, 2017; Schoneberg et al, 2017). Interestingly, the ESCRT machinery has also been shown to mediate repair of both the plasma membrane and the nuclear envelope (Jimenez et al, 2014; Scheffer et al, 2014; Denais et al, 2016; Raab et al, 2016), raising the possibility that it might also function in repair of other membranes. Based on biochemical and genetic evidence, the ESCRT machinery can be divided into four subcomplexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, of which ESCRT-III is thought to mediate membrane sealing/scission through formation of membrane-active oligomeric filaments (Christ et al, 2017). Even though ESCRT-0 and ESCRT-II are important during endosomal protein sorting into intraluminal vesicles of endosomes (Katzmann et al, 2001, 2003; Babst et al, 2002b; Raiborg et al, 2002; Bache et al, 2003), these subcomplexes appear to be dispensable for most known ESCRT functions (Christ et al, 2017). On the other hand, ESCRT-I is frequently involved in recruitment of ESCRT-III, and so is ALIX, a Bro1 domain-containing protein which can potentially form a physical link between ESCRT-I and ESCRT-III (Bissig & Gruenberg, 2014; Christ et al, 2017). Here, we show that ESCRT-III is indeed recruited to damaged lysosomes and that this requires ESCRT-I and ALIX. Interference with this mechanism abolishes the cell's ability to repair damaged lysosomes and causes otherwise reversible lysosome damage to become cell lethal. Surprisingly, we also find that ESCRTs are not only recruited to vacuoles containing the replicating form of the intracellular bacterium Coxiella burnetii, but they also provide the bacterium with an advantage by maintaining a niche for its intracellular replication. Results The ESCRT-III subunit CHMP4B is recruited to damaged lysosomes In order to achieve permeabilization of lysosomes in an acute manner, we used L-leucyl-L-leucine methyl ester (LLOMe), which is converted into a membranolytic polymeric form in the lysosome lumen by lysosomal hydrolases (Thiele & Lipsky, 1990). Using an antibody against Galectin-3 as marker for lysosome permeabilization (Paz et al, 2010), we observed by fluorescence microscopy that Galectin-3 translocated to vesicular structures in HeLa cells treated with LLOMe, and its co-occurrence with the lysosome marker LAMP1 confirmed that these structures are lysosomes (Fig 1A). We generated a stable HeLa line expressing low levels of mCherry-tagged Galectin-3 and the major ESCRT-III subunit CHMP4B tagged with eGFP and monitored the effect of LLOMe on CHMP4B distribution. Interestingly, whereas both mCherry-Galectin-3 and CHMP4B-eGFP displayed mainly cytosolic staining in untreated cells, treatment with a low (250 μM) concentration of LLOMe for 1 h caused a strong redistribution of both molecules to vesicles positive for the late endosome/lysosome marker CD63 (Fig 1B). Likewise, antibodies against CHMP4B stained CD63-, Galectin-3-, and LAMP1-positive lysosomes after LLOMe incubation of HeLa, RPE-1, and H-460 cells, in contrast to untreated cells (Fig EV1A–C). We conclude that LLOMe-induced membrane damage causes recruitment of CHMP4B to lysosomes in various cell types. Figure 1. The ESCRT-III subunit CHMP4B is recruited to Galectin-3-positive damaged endolysosomal membranes 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. 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). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. CHMP4B is recruited to Galectin-3-positive damaged endolysosomal membranes in various cell lines 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. 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. 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). Download figure Download PowerPoint We next investigated whether other agents that cause lysosomal membrane damage also induce CHMP4B recruitment. Glycyl-L-phenylalanine-beta-naphthylamide (GPN) is known to cause lysosomal membrane permeabilization via osmotic swelling (Berg et al, 1994), and we indeed observed that this compound caused a profound recruitment of both mCherry-Galectin-3 and CHMP4B-eGFP to LAMP1-containing lysosomes (Fig EV2A). Amphiphilic antihistamines have been shown to induce permeabilization of the lysosome membrane (Ellegaard et al, 2016), and the antihistamine terfenadine induced recruitment of Galectin-3 and CHMP4B to LAMP1-positive lysosomes in RPE-1 cells, as detected with antibodies against the endogenous proteins (Fig EV2B). Collectively, these results show that CHMP4B, like Galectin-3, is recruited to lysosomes upon various types of membrane damage. Click here to expand this figure. Figure EV2. CHMP4B is recruited to damaged endolysosomal membranes after treatment with different agents 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. 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). Download figure Download PowerPoint ESCRT-I, ESCRT-II, ESCRT-III, ALIX, and VPS4A are recruited to damaged lysosomes The observed recruitment of CHMP4B to damaged lysosomes begged the question of which other ESCRT subunits are recruited. Specifically, because ESCRT-0, ESCRT-I, and ESCRT-II are upstream of ESCRT-III in endosomal sorting (Babst et al, 2002a; Bache et al, 2003), we wondered if this might be the case with recruitment to damaged lysosomes as well. In HeLa cells treated with LLOMe for 30 min, we found no evidence for recruitment of the ESCRT-0 component HRS. In contrast, the ESCRT-I subunit TSG101 and the ESCRT-II subunit EAP30 were clearly recruited, as was VPS4A (Schoneberg et al, 2017), an ATPase that controls ESCRT-III dynamics (Fig 2). As expected, not only CHMP4B, but also another ESCRT-III subunit, CHMP2A was found to be recruited, as was the ESCRT-III-related protein IST1 (Figs 2 and EV3). Interestingly, ALIX, a Bro1 domain-containing protein that can bridge ESCRT-I with ESCRT-III (Bissig & Gruenberg, 2014), was also recruited, whereas we were unable to detect recruitment of another Bro1 domain protein, HD-PTP (Fig 2). We were also unable to detect recruitment of the VPS4 isoform VPS4B (Fig EV3). From these studies, we conclude that ESCRT-I, ESCRT-II, ESCRT-III, ALIX, and VPS4A are recruited to damaged lysosomes. Figure 2. ESCRT-I, ESCRT-II, ESCRT-III, ALIX, and VPS4A are recruited to endolysosomes upon LLOMe treatmentTo 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). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Screening for additional ESCRT proteins that are involved in the lysosomal repair processESCRT-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). Download figure Download PowerPoint TSG101 depletion inhibits CHMP4B recruitment on damaged lysosomes, whereas CHMP2A knockdown stabilizes it The above results suggested that TSG101 and ALIX might have a role in ESCRT-III recruitment, and we tested this further using siRNA to deplete various ESCRT proteins in HeLa cells expressing CHMP4B-eGFP and monitoring CHMP4B-eGFP recruitment by live-cell microscopy. Knockdown of the ESCRT-0 subunit HRS was without effect on CHMP4B-eGFP recruitment to lysosomes upon LLOMe treatment of the cells (Fig 3A, Movie EV1), consistent with our finding that this ESCRT subunit is not recruited itself. On the other hand, depletion of TSG101 caused a strong delay in CHMP4B-eGFP recruitment (Fig 3B, Movie EV2). Conversely, depletion of CHMP2A caused increased accumulation of CHMP4B-eGFP on LLOMe-damaged lysosomes (Fig 3C, Movie EV3), in agreement with previous studies on ESCRT recruitment to other membranes, suggesting that CHMP2A limits the extent of CHMP4B recruitment (Vietri et al, 2015). Whereas depletion of ALIX was without detectable effect on CHM4B-eGFP recruitment (Fig 3D, Movie EV4), co-depletion of TSG101 and ALIX led to an almost complete lack of CHMP4B-eGFP recruitment (Fig 3E, Movie EV5). These results confirm that TSG101 in ESCRT-I is upstream of ESCRT-III in recruitment to damaged lysosomes and also reveal a role for ALIX. Figure 3. Dynamics of CHMP4B accumulation at the damaged endolysosomal membranes depend on other ESCRT componentsHeLa 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. Depletion of HRS does not alter the dynamics of CHMP4B-eGFP recruitment as compared to siCtrl during treatment with LLOMe. 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. CHMP2A depletion accumulates and stabilizes CHMP4B-eGFP-positive foci. siRNA-mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment. 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. Source Data for Figure 3 [embj201899753-sup-0012-SDataFig3.pdf] Download figure Download PowerPoint ESCRT is required for repair of the lysosome membrane We next asked whether the ESCRT machinery is involved in repair of damaged lysosome membranes. As an assay for membrane permeability, we used the ability of lysosomes to retain Lysotracker, a weak base which accumulates in acidic lysosomes and is fluorescent at low pH (Chazotte, 2011). As expected, incubation of HeLa cells with LLOMe at 250 μM caused reduced Lysotracker staining of lysosomes (Fig 4A). Interestingly, however, at this relatively low LLOMe concentration, Lysotracker staining fully recovered within 30–60 min, indicating that the LLOMe-induced membrane damage was repaired (Fig 4A and B). Importantly, whereas Lysotracker staining recovered after LLOMe treatment of control cells, lysosomes in cells depleted for TSG101 and ALIX failed to recover Lysotracker fluorescence (Fig 4C). This indicates that the ESCRT machinery mediates repair of damaged lysosomes. Figure 4. ESCRTs are essential for the repair after endolysosomal damage 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. 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. 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. Download figure Download PowerPoint ESCRT-III shows much faster recruitment kinetics than Galectin-3 to damaged lysosomes In order to monitor the dynamics of ESCRT recruitment to damaged lysosomes, we used live microscopy to study HeLa cells stably expressing low levels of CHMP4B-eGFP and mCherry-Galectin-3. Upon LLOMe addition, mCherry-Galectin-3 accumulated slowly on the lysosomes as expected (Fig 5). Remarkably, CHMP4B-eGFP accumulated much faster, with detectable levels already after 1 min and reaching a peak level at about 10 min (Fig 5 and Movie EV6). This indicates that the ESCRT machinery detects a subtler membrane damage than the exposure of intraluminal β-galactosides detected by Galectin-3. Figure 5. CHMP4B is recruited before Galectin-3 to the damaged endolysosomal membranesRepresentative 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. Download figure Download PowerPoint ESCRT-III recruitment to damaged lysosomes precedes lysophagy Because Galectin-3 recruitment to lysosomes triggers lysophagy, we next compared the dynamics of CHMP4B recruitment with the dynamics of various molecules involved in lysophagy by confocal microscopy of HeLa cells fixed at various time points after LLOMe addition. Ubiquitination of lysosomal membrane proteins is known to occur early during lysophagy (Maejima et al, 2013), and we therefore monitored the kinetics of ubiquitin acquisition on lysosomes using an antibody against conjugated ubiquitin. Like with Galectin-3 recruitment, ubiquitin appeared on the damaged lysosomes much slower than CHMP4B (Figs 6A and EV4A). We also studied recruitment of the autophagy adaptor p62/SQSTM1, a protein known to connect ubiquitinated autophagic cargoes to autophagic membranes (Bjørkøy et al, 2005; Maejima et al, 2013), and a major constituent of the autophagy machinery, LC3 (Kabeya et al, 2000; Maejima et al, 2013). Both p62 and LC3 appeared on damaged lysosomes with much slower kinetics than CHMP4B (Figs 6B and C, and EV4B and C). The autophagy inhibitor SAR405 (Ronan et al, 2014) was without effect on CHMP4B-eGFP recruitment (Movie EV7). We conclude from these studies that the ESCRT machinery is recruited much prior to the autoph
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