Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury
2013; Springer Nature; Volume: 32; Issue: 17 Linguagem: Inglês
10.1038/emboj.2013.171
ISSN1460-2075
AutoresIkuko Maejima, Atsushi Takahashi, Hiroko Omori, Tomonori Kimura, Yoshitsugu Takabatake, Tatsuya Saitoh, Akitsugu Yamamoto, Maho Hamasaki, Takeshi Noda, Yoshitaka Isaka, Tamotsu Yoshimori,
Tópico(s)Autophagy in Disease and Therapy
ResumoArticle6 August 2013free access Source Data Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury Ikuko Maejima Ikuko Maejima Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Japan Science and Technology Agency CREST, Tokyo, Japan Search for more papers by this author Atsushi Takahashi Atsushi Takahashi Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Hiroko Omori Hiroko Omori Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Tomonori Kimura Tomonori Kimura Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Yoshitsugu Takabatake Yoshitsugu Takabatake Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Tatsuya Saitoh Tatsuya Saitoh Laboratory of Host Defense, WPI Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan Search for more papers by this author Akitsugu Yamamoto Akitsugu Yamamoto Faculty of Bioscience, Nagahama Institute of BioScience and Technology, Shiga, Japan Search for more papers by this author Maho Hamasaki Maho Hamasaki Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Takeshi Noda Takeshi Noda Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan Search for more papers by this author Yoshitaka Isaka Yoshitaka Isaka Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Tamotsu Yoshimori Corresponding Author Tamotsu Yoshimori Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Japan Science and Technology Agency CREST, Tokyo, Japan Search for more papers by this author Ikuko Maejima Ikuko Maejima Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Japan Science and Technology Agency CREST, Tokyo, Japan Search for more papers by this author Atsushi Takahashi Atsushi Takahashi Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Hiroko Omori Hiroko Omori Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Tomonori Kimura Tomonori Kimura Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Yoshitsugu Takabatake Yoshitsugu Takabatake Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Tatsuya Saitoh Tatsuya Saitoh Laboratory of Host Defense, WPI Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan Search for more papers by this author Akitsugu Yamamoto Akitsugu Yamamoto Faculty of Bioscience, Nagahama Institute of BioScience and Technology, Shiga, Japan Search for more papers by this author Maho Hamasaki Maho Hamasaki Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Takeshi Noda Takeshi Noda Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan Search for more papers by this author Yoshitaka Isaka Yoshitaka Isaka Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Tamotsu Yoshimori Corresponding Author Tamotsu Yoshimori Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Japan Science and Technology Agency CREST, Tokyo, Japan Search for more papers by this author Author Information Ikuko Maejima1,8, Atsushi Takahashi2, Hiroko Omori3, Tomonori Kimura2, Yoshitsugu Takabatake2, Tatsuya Saitoh4, Akitsugu Yamamoto5, Maho Hamasaki1, Takeshi Noda6, Yoshitaka Isaka2 and Tamotsu Yoshimori 1,7,8 1Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan 2Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan 3Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 4Laboratory of Host Defense, WPI Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan 5Faculty of Bioscience, Nagahama Institute of BioScience and Technology, Shiga, Japan 6Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan 7Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan 8Japan Science and Technology Agency CREST, Tokyo, Japan *Corresponding author. Department of Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871 Osaka, Japan. Tel:+81 6 6879 3580; Fax:+81 6 6879 3589; E-mail: [email protected] The EMBO Journal (2013)32:2336-2347https://doi.org/10.1038/emboj.2013.171 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 Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis. Introduction The lysosome, a single-membrane acidic organelle, is present in all eukaryotic cells. Lysosomes act as a cellular ‘digestive apparatus’ that degrades materials delivered either from outside via the endocytic pathway or from inside via the autophagic pathway. These compartments provide cells with nutrients, including amino acids and lipids, by degrading proteins and other macromolecules, and they also function in plasma membrane repair, defense against pathogens, antigen presentation, and cell death (Saftig and Klumperman, 2009). Lysosomal rupture results in leakage of contents, including Cathepsins, from the lysosomal lumen into the cytosol; in the worst cases, this can cause apoptotic or necrotic cell death (Boya and Kroemer, 2008). Even when the damage is not lethal, lysosomal rupture provokes oxidative stress due to the release of H+ from the lysosomal lumen into the cytosol, DNA damage, and reduction in the catabolic capacity of the lysosome, which might in turn affect the cellular functions in which lysosomes are involved (Boya and Kroemer, 2008; Hornung et al, 2008; Johansson et al, 2010). Emerging evidence indicates that lysosomal rupture activates the NLRP3 inflammasome, which induces the secretion of proinflammatory cytokines including IL-1β, promoting inflammation and enhancing pathogenesis (Hornung et al, 2008; Salminen et al, 2012). Therefore, lysosomal rupture is a potentially harmful and stressful event for cells. Diverse substances, including mineral crystals such as silica and monosodium urate (MSU), bacterial or viral toxins, lipids, β-Amyloid, lysosomotropic compounds, and cell death effectors can impair lysosomal membranes in vivo. These substances can cause pathologies, including neurodegenerative disorders such as Parkinson's disease, inflammation, and the development of hyperuricemic nephropathy (Emmerson et al, 1990; Kroemer and Jäättelä, 2005; Boya and Kroemer, 2008; Dehay et al, 2010; Salminen et al, 2012). Autophagy is an intracellular bulk degradation system that is drastically induced under several cellular stress conditions such as nutrient starvation, and plays diverse physiological and pathological roles as a prosurvival mechanism of cells through maintaining cellular and tissue homeostasis (Mizushima and Levine, 2010). Autophagy is initiated by de novo generation of the isolation membrane in the cytoplasm. This membrane elongates to engulf cytoplasmic macromolecules and organelles, and encloses these cargos to form the autophagosome. The autophagosome itself does not have the ability to degrade its contents. Fusion with the lysosome provides an acidic environment and hydrolases, enabling degradation of autophagosomal contents. LC3, a mammalian Atg8 homologue, is a ubiquitin-like protein that is essential for autophagy. The cysteine proteinase Atg4 processes the C-terminal 22 residues of newly synthesized precursor LC3, producing a soluble form of LC3 (LC3-I) that exposes the C-terminal glycine residue (Kabeya et al, 2000). The exposed C-terminus of LC3 is conjugated to the head group amine of phosphatidylethanolamine (PE) by a ubiquitin-like system composed of Atg7 (E1 enzyme), Atg3 (E2 enzyme), and the Atg12–Atg5–Atg16L complex (E3 enzyme) (Ichimura et al, 2000; Hanada et al, 2007; Fujita et al, 2008b). Lipidated LC3 (LC3-II), a widely used autophagic marker, is anchored to the forming autophagosome membrane. Upon membrane closure, LC3-II is cleaved by Atg4, and LC3 dissocites from PE (Kirisako et al, 2000; Kim et al, 2001). Thus, Atg4 is involved in both lipidation and delipidation of LC3. Furthermore, overexpression of Atg4BC74A, an inactive mutant of Atg4B (a mammalian Atg4 homologue), strongly inhibits lipidation of LC3 by sequestering LC3 orthologues including LC3A, LC3B, GABARAP, GATE16, and Atg8L prior to lipidation (Fujita et al, 2008a). Although autophagy is considered to be a non-selective degradation system, growing evidence has revealed autophagic pathways that selectively degrade aggregation-prone proteins, invading pathogens, and damaged or superfluous organelles such as mitochondria, peroxisomes, and endoplasmic reticulum (ER) in order to maintain cellular homeostasis (Komatsu and Ichimura, 2010; Tanida, 2011). In mammalian cells, ubiquitination plays a crucial role in determining the target of selective autophagy, working in conjunction with adaptor proteins such as p62/SQSTM1, which enable interaction between ubiquitin and LC3 paralogues. Following interaction with adaptors, ubiquitinated substrates are engulfed by autophagosomes via binding between adaptors and LC3. Here, we examined the possibility that cells induce autophagy in order to avoid the associated risks that is caused by lysosomal rupture. We found that lysosomes damaged by lysosomotropic reagents are selectively isolated by autophagy. Furthermore, we suggest a possibility that this selective isolation plays critical roles in lysosomal biogenesis in cells and in suppression of acute kidney injury in vivo. Results Lysosomal rupture induces autopahgy To disrupt the lysosomal membrane, we used either the lysosomotropic compound L-Leucyl-L-leucine methyl ester (LLOMe) or crystalline silica. LLOMe accumulates in lysosomes and is converted into its membranolytic form (Leu-Leu)n-OMe (n>3) by a lysosomal thiol protease, dipeptidyl peptidase I (DPPI) (Thiele and Lipsky, 1990; Uchimoto et al, 1999). Crystalline silica, also known as silicon dioxide (SiO2), exists in nature as sand or quartz. Although silica itself is usually harmless, crystalline silica has been shown to disrupt the lysosomal membrane in alveolar macrophages and activate lung inflammation in vivo (Mossman and Churg, 1998; Hornung et al, 2008). To test whether autophagy is induced by lysosomal rupture, we measured lipidation of endogenous LC3 by immunoblotting in the murine macrophage cell line J774 cells and mouse embryonic fibroblasts (MEFs). LLOMe treatment stimulated cytosolic release of Cathepsin D in a dose-dependent manner (Supplementary Figure S1A), and the level of lipidated LC3 dose dependently increased upon either LLOMe or silica treatment (Supplementary Figure S1B and C). These results suggest that lysosomal rupture induces autophagy. Then, we found the colocalization of LC3 and Lamp1 in LLOMe- or silica-treated J774 cells (Supplementary Figure S1D) using confocal microscopy. In particular, silica-treated cells exhibited obvious recruitment of LC3 to silica particles surrounded by Lamp1. These results raise the possibility that autophagy selectively targets damaged lysosomes. Autophagic machinery is selectively recruited to damaged lysosomes To determine whether LC3 is specifically recruited only to damaged lysosomes, we used Galectin-3 (Gal3), a recently established marker of damaged endomembranes (Paz et al, 2010). Galectin-3/Mac-2 is a member of the Galectins, a lectin protein family defined by conserved sequence and affinity for β-galactosides. Gal3 is distributed throughout the cytoplasm and nucleus, whereas β-galactose-containing glycoconjugates are present only on the cell surface and in the lumens of endocytic compartments, the Golgi apparatus, and post-Golgi secretory compartments (Houzelstein et al, 2004). Therefore, these proteins normally do not interact with each other; however, endosomal membrane rupture allows Gal3 to access the lumenal glycoproteins of these compartments (Paz et al, 2010). We generated Atg7-deficient MEFs, in which Atg5–Atg12 conjugation is impaired, stably expressing GFP-Gal3 to investigate the recruitment of LC3 to membrane-damaged lysosomes. Under untreated conditions, GFP-Gal3 and LC3 were diffusely distributed throughout the cytosol and did not colocalize with Lamp1 (Figure 1A). However, upon LLOMe or silica treatment, several GFP-Gal3 puncta appeared in the cytosol. The number of these punctate structures increased in an LLOMe dose-dependent manner (Supplementary Figure S2A) and Gal3 puncta extensively colocalized with Lamp1 in LLOMe- or silica-treated Atg7+/+ and Atg7−/− MEFs; conversely, we could not observe Lamp1-negative Gal3 puncta. GFP-Gal3-positive Lamp1 puncta were not stained with Lysotracker (Supplementary Figure S2B). Video microscopy observations showed that GFP-Gal3 puncta were never stained with Lysotracker throughout its recruitment in the presence of LLOMe (Supplementary Figure S3A; Supplementary Movie1), indicating that Gal3 is recruited to lysosomes that have lost their acidic interior environment following membrane damage. Gal3 was also recruited to Lamp1 and colocalized with LC3 under oxidative stress conditions, which is also known to cause lysosomal membrane damage (Supplementary Figure S2C) (Boya and Kroemer, 2008), suggesting that Gal3 can be used as a general marker of damaged lysosomes. LC3 was specifically recruited to GFP-Gal3-positive Lamp1 puncta upon LLOMe or silica treatment in Atg7+/+ MEFs, but not in Atg7−/− MEFs (Figure 1A). Since LC3 was recruited to Gal3 puncta, even when only a few lysosomes were damaged at the low concentration of LLOMe (Supplementary Figure S2D), autophagy is highly sensitive to lysosomal damage. Figure 1.The recruitment of ubiquitin and LC3 to GFP-Gal3-positive damaged lysosomes. (A–C) Atg7+/+ and Atg7–/– MEFs stably expressing GFP-Gal3 (A, C) or GFP-Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti-LC3 and anti-Lamp1 (A), anti-p62 and anti-Lamp1 (B), or anti-FK2 and anti-p62 (C). Bars: 10 μm. Download figure Download PowerPoint Gal3-positive damaged membranes were ubiquitinated and colocalized with p62 in both MEFs (Figure 1B and C). These results suggest that ubiquitination and the recruitment of p62 occurs in a manner similar to that observed for selective autophagy against invading bacteria. We also confirmed the colocalization of other GFP-tagged upstream Atg proteins (ULK1, Atg9L1, Atg14L, WIPI1, and Atg5) and Gal3-positive lysosomes upon either LLOMe or silica treatment (Supplementary Figure S3C). Futhermore, to clarify the cause–effect relationship between lysosomal rupture and autophagy induction, we observed puncta formation of GFP-Atg5, a marker of the isolation membrane, and mStrawberry-Gal3 using video microscopy (Supplementary Figure S3B; Supplementary Movie 2). Upon LLOMe treatment, the recruitment of GFP-Atg5 always followed the formation of mStrawberry-Gal3 puncta, suggesting that autophagy is induced after lysosomal rupture. Taken together, these results indicate that the core autophagy machinery is selectively recruited to damaged lysosomes. GFP-Gal3-positive lysosomes decrease in an autophagy-dependent manner What happens in damaged lysosomes after specific recruitment of Atg proteins? We exposed cells to 1000 μM LLOMe for 1 h, and after washing out the reagent cultured the cells for an additional 24 h in the absence of LLOMe. Then, we evaluated the percentage of GFP-Gal3-positive Lamp1 puncta at the indicated time points (Figure 2A and B; Supplementary Figure S4A and B). Three hours after LLOMe washout, almost 30% of Lamp1 puncta colocalized with GFP-Gal3 (Figure 2B). These Gal3-positive Lamp1 puncta dramatically decreased within 10 h, and completely disappeared until 24 h after LLOMe washout. In contrast to control cells, in cells stably expressing an inactive Atg4B mutant, Atg4BC74A, that sequesters LC3 paralogues prior to lipidation and strongly blocks autophagy (Fujita et al, 2008a), GFP-Gal3-positive Lamp1 puncta slightly decreased but were then maintained at a high level even 24 h after LLOMe washout (Figure 2B). These results suggest that disappearance of GFP-Gal3 puncta is due to autophagy. The total number of Lamp1 puncta remained stable in both control and Atg4B-mutant cells (Supplementary Figure S4A). Similar results were obtained in Atg7-deficient MEFs (Supplementary Figure S5A–C). Lysotracker staining showed that there remained Lysotracker-positive Lamp1 puncta representing acidic intact lysosomes at 3 h after LLOMe washout (Supplementary Figure S5D). Figure 2.Decrease in the number of GFP-Gal3 puncta is dependent on time and autophagy. (A, B) NIH3T3 cells stably expressing GFP-Gal3 and either empty vector (control) or mStrawberry-Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP-Gal3 or Lamp1 puncta per cell was quantified using G-Count (see also Supplementary Figure S4A and B). Then, the percent of GFP-Gal3-positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page. Source Data for Figure 2b&s4ab [embj2013171-sup-0001-SourceData-S1.xls] Download figure Download PowerPoint We also tested continuous treatment of cells with LLOMe for longer periods of time. NIH3T3 cells were treated with 1000 μM LLOMe for the indicated time, and analysed the percentage of GFP-Gal3-positive Lamp1 puncta (Supplementary Figure S6A–C). Interestingly, even in the continuous presence of LLOMe, GFP-Gal3-positive Lamp1 puncta significantly decreased, although there remained some at 24 h. Presumably, autophagic sequestration of damaged lysosomes overcomes continuous damage of them. Acidity of damaged lysosomes recovers in an autophagy-dependent manner At least two phenomena could explain the disappearance of GFP-Gal3 puncta after LLOMe washout: release of GFP-Gal3 from the damaged membrane, and quenching of the GFP signal in an acidic environment such as that generated during autophagy. To distinguish between these two possibilities, we constructed mRFP- and GFP-tandem-tagged Gal3 (tandem fluorescent-tagged Galectin-3, tfGal3) (Figure 3A). GFP and mRFP are differentially sensitive to acidic environments (Kneen et al, 1998; Campbell et al, 2002). GFP fluorescence is rapidly quenched, and GFP is degraded by lysosomal hydrolases, whereas mRFP fluorescence remains relatively stable. Thus, as shown in Figure 3B, tfGal3 makes it possible to monitor the pH change in damaged lysosomes. The change in the surrounding environment from neutral to acidic pH causes attenuation of GFP puncta signals, while mRFP puncta stably persist. Therefore, if GFP signal is quenched after LLOMe washout, the number of mRFP+GFP+ puncta should decrease, whereas the number of mRFP+ puncta (i.e., regardless of the presence or absence of GFP signal) should not be changed. In contrast, if Gal3 is released from damaged lysosomes, then the number of both mRFP+GFP+ and mRFP+ puncta should decrease. Figure 3.tfGal3 GFP signal in puncta attenuates in an autophagy-dependent manner. (A) Diagram of the primary structure of tandem fluorescence-tagged Galectin-3 (tfGal3). (B) Schematic diagram of the fate of tfGal3 recruited to damaged lysosomes. (C–E) HeLa cells transfected with tfGal3 and either One-STrEP-FLAG-tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G-Count (D). Then, the percent of GFP+RFP+ tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm. (F–H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry-Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B-mutant (H) cells was quantified by G-Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3de [embj2013171-sup-0002-SourceData-S2.xls] Source Data for Figure 3gh&s5g [embj2013171-sup-0003-SourceData-S3.xls] Download figure Download PowerPoint We transfected HeLa cells with a plasmid encoding tfGal3, with or without a plasmid encoding Atg4BC74A, and then subjected the cells to the experimental procedure as in Figure 2. As we expected, upon LLOMe treatment, tfGal3 formed several mRFP+GFP+ puncta in both control and Atg4B-mutant cells (Figure 3C). In control cells, the GFP+ puncta were almost completely abolished 24 h after LLOMe washout, whereas the mRFP+ puncta could be observed at the same level as at time 0 (Figure 3D and E). The attenuation of GFP puncta signals was cancelled by Bafilomycin A1, a specific inhibitor of the vacuolar-type ATPase. By contrast, tfGal3 puncta did not lose GFP signal in Atg4B-mutant cells. The total number of mRFP+ puncta did not decrease even 24 h after LLOMe depletion in either cell types (Figure 3D). From these data, we conclude that GFP-Gal3 is not released from damaged membranes, and that the acidity of damaged lysosomes recovers in an autophagy-dependent manner. We also tried to corroborate our conclusion by other approaches than those using tagged Gal3. In Supplementary Figure S5D, Lysotracker-positive intact lysosomes seemed to increase while Lysotracker-negative lysosomes were colocalized with decreased GFP-Gal3. Therefore, we next measured % of Lysotracker-negative lysosomes in total lysosomes representing damaged lysosomes leaking protons (Supplementary Figure S5E). Lysotracker-negative lysosomes increased after 1.5 h from LLOMe washout but decreased after 6 h from LLOMe washout in control cells, suggesting recovery of low pH in damaged lysosomes. On the other hand, in autophagy-deficient Atg7−/− MEFs, the increase in Lysotracker-negative lysosomes was higher after 1.5 h from LLOMe washout but their decrease after 6 h from LLOMe washout was small compared to control. The result also supports that acidity is recovered by autophagy. Endogenous Gal3 on damaged lysosomes is degraded by autophagy Endogenous Gal3 puncta that appeared upon LLOMe treatment exhibited similar decrease as exogenous-tagged Gal3 puncta in control and Atg4B-mutant cells (Figure 3F–H), and in Atg7+/+ and Atg7−/− MEFs (Supplementary Figure S5F and G). The number of Gal3 puncta was not affected in Atg4B-mutant cells by protease inhibitors, whereas Gal3 puncta accumulated in control cells in the presence of protease inhibitors (Figure 3G and H). Most likely, these results indicate that endogenous Gal3 on damaged lysosomes is degraded in an autophagy-dependent manner. We also measured the amount of endogenous Lamp1 in the presence of cycloheximide to inhibit synthesis of new proteins up to 10 h after LLOMe washout (Supplementary Figure S5H). There was no significant change observed. Presumably, Lamp1 on damaged lysosomes turns over very slowly due to its resistance to lysosomal proteases. Indeed, the half-life of Lamp1 was reported to be 1.6 days (Meikle et al, 1999). In addition, the increase in LC3 lipidation upon LLOMe treatment decreased after LLOMe washout, returning to the basal level within 10 h after LLOMe washout (Supplementary Figure S4C and D), consistent with the kinetics of change in the number of Gal3-positive lysosomes. This is not due to impaired autophagy flux, since we could observe autophagy flux by using mRFP-GFP-LC3, which is an established probe for autophagy flux (Kimura et al, 2007). In this assay, mRFP+GFP+ LC3 puncta represent forming autophagosomes or autophagosomes, and mRFP+-only puncta indicate autolysosomes. After LLOMe washout, the percentage of mRFP+GFP+ LC3 puncta decreased with increased mRFP+-only puncta in a time-dependent manner (Supplementary Figure S4E and F), indicating that autophagy flux (formation of autolysosome) is not significantly hindered in the experimental condition. Autophagosomes engulf damaged lysosomes To reveal how autophagy restores the acidic and proteolytic environment in damaged lysosomes, we observed the ultrastructure of GFP-LC3- and mStrawberry-Gal3-positive damaged lysosomes by correlation of light and electron microscopy (CLEM). HeLa cells expressing GFP-LC3 and mStrawberry-Gal3 were treated with LLOMe for 1 h, and immediately fixed (Figure 4A–F). CLEM revealed that LC3- and Gal3-positive puncta are double membranes, a typical autophagosome structure, tightly sequester swollen lysosomes (Figure 4C–F). The single-membrane vesicles were partially or completely sequestered in double-membrane structures (Figure 4G and H). We could not find such structures in cells that were not treated with LLOMe (Supplementary Figure S7A–D). In contrast to control cells, all of the obtained images in Atg4B-mutant-expressing cells treated with LLOMe showed that lysosomes were partially attached with flattened membranous sacs but were never enclosed in double-membrane structures (Figure 4I). These results suggest that damaged lysosomes are selectively engulfed by autophagosomes. Presumably, autophagosomes containing damaged lysosomes fuse with remaining intact lysosomes, resulting in the recovery of acidity and proteolysis activity. Figure 4.CLEM analysis of mSt-Gal3- and GFP-LC3-associated membranes. (A–F) HeLa cells stably expressing GFP-LC3 were transfected with mStrawberry-Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B–F). Green: LC3; magenta: Gal3; blue: DAPI. (G–I) NIH3T3 cells stably expressing CFP-Gal3 and YFP-LC3, and either empty vector (control) (G, H) or mStrawberry-Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane. Download figure Download PowerPoint Autophagy suppresses development of acute hyperuricemic nephropathy in mice Finally, we examined the pathophysiological importance of autophagic isolation of damaged lysosomes in vivo. Acute hyperuricemic nephropathy is a type of acute kidney injury observed in patients with tumour lysis syndrome, caused by chemotherapeutic treatment of haematopoietic malignancies (Ejaz et al, 2006). In this disease, oversaturation of uric acid (UA) in urine causes precipitation of UA and MSU in the renal tubule (Nickeleit and Mihatsch, 1997; Ejaz et al, 2006). A previous study showed that urate crystals cause the cytosolic release of lysosomal enzymes in MDCK cells, and that this lysosomal damage is involved in the development of hyperuricemic nephropa
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