Lipid and protein content profiling of isolated native autophagic vesicles
2022; Springer Nature; Volume: 23; Issue: 12 Linguagem: Inglês
10.15252/embr.202153065
ISSN1469-3178
AutoresDaniel Schmitt, Süleyman Bozkurt, Pascale Henning‐Domres, Heike Huesmann, Stefan Eimer, Laura Bîndilă, Christian Behrends, Emily Boyle, Florian Wilfling, Georg Tascher, Christian Münch, Christian Behl, Andreas Kern,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoResource10 October 2022Open Access Transparent process Lipid and protein content profiling of isolated native autophagic vesicles Daniel Schmitt Daniel Schmitt The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Writing - review & editing Search for more papers by this author Süleyman Bozkurt Süleyman Bozkurt orcid.org/0000-0003-4965-3195 Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Pascale Henning-Domres Pascale Henning-Domres The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Data curation, Investigation, Writing - review & editing Search for more papers by this author Heike Huesmann Heike Huesmann The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Investigation Search for more papers by this author Stefan Eimer Stefan Eimer Department of Structural Cell Biology, Institute for Cell Biology and Neuroscience, Goethe University, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Laura Bindila Laura Bindila Clinical Lipidomics Unit, Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Christian Behrends Christian Behrends orcid.org/0000-0002-9184-7607 Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University, Munich, Germany Contribution: Resources, Data curation, Writing - review & editing Search for more papers by this author Emily Boyle Emily Boyle orcid.org/0000-0001-8803-3939 Mechanisms of Cellular Quality Control, Max Planck Institute of Biophysics, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Florian Wilfling Florian Wilfling Mechanisms of Cellular Quality Control, Max Planck Institute of Biophysics, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Georg Tascher Georg Tascher Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Christian Münch Christian Münch orcid.org/0000-0003-3832-090X Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Christian Behl Christian Behl The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Data curation, Supervision, Writing - review & editing Search for more papers by this author Andreas Kern Corresponding Author Andreas Kern [email protected] orcid.org/0000-0003-0993-9818 The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Writing - original draft, Writing - review & editing Search for more papers by this author Daniel Schmitt Daniel Schmitt The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Writing - review & editing Search for more papers by this author Süleyman Bozkurt Süleyman Bozkurt orcid.org/0000-0003-4965-3195 Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Pascale Henning-Domres Pascale Henning-Domres The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Data curation, Investigation, Writing - review & editing Search for more papers by this author Heike Huesmann Heike Huesmann The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Investigation Search for more papers by this author Stefan Eimer Stefan Eimer Department of Structural Cell Biology, Institute for Cell Biology and Neuroscience, Goethe University, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Laura Bindila Laura Bindila Clinical Lipidomics Unit, Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Christian Behrends Christian Behrends orcid.org/0000-0002-9184-7607 Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University, Munich, Germany Contribution: Resources, Data curation, Writing - review & editing Search for more papers by this author Emily Boyle Emily Boyle orcid.org/0000-0001-8803-3939 Mechanisms of Cellular Quality Control, Max Planck Institute of Biophysics, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Florian Wilfling Florian Wilfling Mechanisms of Cellular Quality Control, Max Planck Institute of Biophysics, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Georg Tascher Georg Tascher Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Christian Münch Christian Münch orcid.org/0000-0003-3832-090X Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Contribution: Data curation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Christian Behl Christian Behl The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Data curation, Supervision, Writing - review & editing Search for more papers by this author Andreas Kern Corresponding Author Andreas Kern [email protected] orcid.org/0000-0003-0993-9818 The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Daniel Schmitt1, Süleyman Bozkurt2, Pascale Henning-Domres1, Heike Huesmann1, Stefan Eimer3, Laura Bindila4, Christian Behrends5, Emily Boyle6, Florian Wilfling6, Georg Tascher2, Christian Münch2, Christian Behl1 and Andreas Kern *,1 1The Autophagy Lab, Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany 2Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany 3Department of Structural Cell Biology, Institute for Cell Biology and Neuroscience, Goethe University, Frankfurt am Main, Germany 4Clinical Lipidomics Unit, Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany 5Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University, Munich, Germany 6Mechanisms of Cellular Quality Control, Max Planck Institute of Biophysics, Frankfurt am Main, Germany *Corresponding author. Tel: +49 6131 3923185; E-mail: [email protected] EMBO Reports (2022)23:e53065https://doi.org/10.15252/embr.202153065 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. 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 Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin–proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses. Synopsis This report introduces an antibody-based FACS-mediated method for isolation of native autophagic vesicles in large quantities, enabling subsequent phospholipid and cargo analyses. The antibody-based FACS-mediated isolation method enriches intact, unmanipulated autophagic vesicles at large quantities. Besides phosphoglycerides, lipidomic analysis identifies sphingomyelin as a component of autophagic vesicles. Proteomic analysis of isolated autophagic vesicles identifies autophagy substrates and reveals treatment-specific alterations in cargo profiles. Introduction Macroautophagy (hereafter autophagy) is a eukaryotic catabolic pathway responsible for the removal and recycling of an extensive portfolio of cytosolic cargoes. Numerous proteins, aggregates, organelles, cellular compartments, or pathogens have been characterized as autophagy substrates, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation (Mizushima & Komatsu, 2011). Autophagosome formation starts with a cup-shaped membrane, the phagophore, which expands around the degradable material until it surrounds it and seals (Lamb et al, 2013). This de novo vesicle synthesis is initiated by activation of the ULK1/2 kinase and the phosphoinositide 3-kinase (PI3-kinase) complex that facilitates the recruitment of proteins and phospholipids (PLs) essential for phagophore generation (Nascimbeni et al, 2017; Mercer et al, 2018). The exact lipid sources are elusive so far; however, recent studies suggest a direct PL transfer from the ER via ATG2A/B (Osawa et al, 2019; Valverde et al, 2019) and from post-Golgi compartments via ATG9A (Gomez-Sanchez et al, 2021). The elongation of the phagophore is linked to two ubiquitin-like conjugation reactions (Mizushima, 2020). ATG12 is conjugated to ATG5, which facilitates the conjugation of ATG8 proteins to phosphatidylethanolamine (PE) (Kabeya et al, 2004; Noda & Inagaki, 2015). In humans, six Atg8 family members have been identified: MAP1LC3A, MAP1LC3B, MAP1LC3C (shortly LC3A-C), as well as GABARAP, GABARAPL1, and GABARAPL2 (Slobodkin & Elazar, 2013). Lipidated ATG8 proteins (referred to as ATG8-II) are inserted into both sides of the growing phagophore membrane and stay attached to mature autophagosomes, facilitating phagophore elongation and closure as well as autophagosome-lysosome fusion (Weidberg et al, 2010; Nguyen et al, 2016; Tsuboyama et al, 2016). Besides their prominent roles in autophagy, distinct lipidated ATG8 proteins have additionally been associated with nonautophagic vesicles, modulating their maturation, trafficking, or degradation (Florey & Overholtzer, 2012; Heckmann et al, 2017; Nieto-Torres et al, 2021). Moreover, ATG8 proteins provide binding sites for cargo receptors. Autophagy was initially described as a nonselective process that degrades random material of the cytosol to recycle building blocks responding to changing metabolic requirements. The identification of cargo receptors, though, established a selective part, resulting in the degradation of specific substrates (Stolz et al, 2014; Khaminets et al, 2016). Via its LC3 interacting domain and its ubiquitin-binding domain, the cargo receptor SQSTM1/p62, for example, binds to ATG8 proteins and directs selective cargoes into autophagosomes (Pankiv et al, 2007; Kirkin et al, 2009; Gatica et al, 2018). Autophagy is highly dynamic and rapidly adapts to changing cellular conditions. Unstressed cells are characterized by a basal autophagy rate that constantly degrades and recycles cellular material at (comparably) low levels. Various stress situations alter autophagy, resulting in a substantially enhanced cargo degradation (He & Klionsky, 2009). However, the impact of different autophagy conditions on the exact PL and cargo profiles of autophagosomes is not defined in detail yet. Several studies have isolated autophagosomes using elaborate cellular fractionation methods (Gao et al, 2010; Dengjel et al, 2012; Mancias et al, 2014) or have performed cargo analyses employing proximity labeling in combination with quantitative proteomics (Le Guerroue et al, 2017; Zellner et al, 2021). Still, a potent method for the isolation of unmanipulated autophagic vesicles at large quantities that enables rapid and efficient lipid and cargo profiling is missing to date. Here, we now introduce an antibody-based FACS- (fluorescence-activated cell sorting-) mediated isolation approach to purify intact native autophagic vesicles. We characterized the quality of the isolates and performed quantitative lipidomics and proteomics analyses to identify PLs and cargo proteins of autophagic vesicles enriched upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. Results and Discussion Isolation of intact native autophagic vesicles in large quantities We established a protocol for the isolation of native autophagic vesicles, employing antibody-based fluorescence tagging of ATG8 proteins and subsequent sorting via FACS (Fig 1A, Appendix Fig S1A). Upon mild cell disruption, we incubated the cellular extract with a primary antibody directed against an ATG8 protein, followed by treatment with a secondary fluorophore-conjugated antibody. The selective and stable attachment of the fluorophore allowed the specific purification of the labeled granular structures using FACS. The sorting resulted in preparations of approx. 1,000 positive events per μl PBS and thus allowed the isolation of autophagic vesicles in large quantities. In order to investigate the successful enrichment of autophagic vesicles by the FACS-based approach, we analyzed the presence of specific autophagosomal proteins within the isolate fractions via Western blotting. Upon isolations using antibodies directed against LC3B or all GABARAP isoforms (Fig 1B), we observed the lipidated variants of both ATG8 proteins and SQSTM1/p62 within the isolate fractions, confirming the enrichment of autophagic vesicles. Unlipidated ATG8 proteins, which are not bound to autophagic vesicles, were hardly detectable. They were effectively depleted from the isolates by the centrifugation steps and the FACS-based sorting included in the isolation protocol (Appendix Fig S1B, Fig 1A). To emphasize the quality of the preparations, we investigated the presence of proteins specific for the cytosol (SOD1), cytoskeleton (Tubulin), ER (DFCP1), Golgi (FTCD), lipid droplets (PLIN2), or mitochondria (SIRT4). Importantly, all investigated proteins were quantitatively excluded from the isolate fractions, illustrating the quality of the FACS-based method (Fig 1B). To additionally stress the potency of the isolation approach, we used a cell line that expresses endogenously HA-tagged GABARAP (Appendix Fig S2). Employing an antibody directed against the HA-tag, we efficiently enriched HA-positive autophagic vesicles without accumulating additional cellular organelles or compartments (Fig EV1). Figure 1. FACS-mediated isolation of autophagic vesicles Schematic representation of the antibody-based FACS-mediated isolation method. TL, total lysate; P1-2, pellet fractions; S1-2, supernatants. Western blot analysis of purified autophagic vesicles. Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms, respectively, and are represented with total lysate (TL). Depicted are representative blots of 14 independent approaches. Quantification of fluorophore-labeled events in WT and ATG5 KO HeLa cells. Shown percentages represent the relative number of detected events in three independent experiments. Quantification of fluorophore-labeled events in WT and FIP200 KO MEFs. Shown percentages represent the relative number of detected events in three independent experiments. Co-localization of fluorescence signals linked to antibodies directed against LC3B and all GABARAP isoforms. Shown percentages represent the average distribution of three independent experiments, excluding double negative events. Data information: (C–E) Statistics are depicted as mean ± SD; t-test (C + D) or one-way ANOVA (E); *P ≤ 0.05; ***P ≤ 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Isolation of autophagic vesicles from HA-GABARAP-expressing HeLa cellsWestern blot analysis of purified HA-tagged autophagic vesicles. Isolations were based on an antibody directed against the HA-tag and are represented with total lysate (TL). Shown blots are representative for three independent experiments. Download figure Download PowerPoint For further quality control, we investigated whether total amounts of fluorophore-labeled events detected via FACS correlated with the numbers of ATG8-positive structures present within cells and employed ATG5 KO HeLa cells and FIP200 KO MEFs. We generated ATG5 KO HeLa cells and confirmed that the stable loss of ATG5 deteriorated LC3B lipidation and abrogated canonical autophagy (Appendix Fig S3A). FIP200 is functionally involved in ULK1-mediated autophagy induction and its knockout disturbs autophagosome formation (Hara et al, 2008). However, although at decreased levels, the lipidated form of LC3B is still present in FIP200 KO MEFs and is primarily associated with nonautophagic vesicles (Appendix Fig S3B). Importantly, due to autophagy dysfunction, both KO lines accumulated SQSTM1/p62 aggregates that co-localized with LC3B, confirming previous studies (Pankiv et al, 2007; Kishi-Itakura et al, 2014) (Appendix Fig S3). Upon starvation-mediated autophagy induction, we purified ATG8-positive structures and detected reduced quantities of fluorophore-labeled events in both KO lines compared with appropriate wild-type (WT) cells (Fig 1C and D, Appendix Fig S4). ATG5 KO cells showed a strong decline in total numbers, and FIP200 KO MEFs displayed reduced amounts. Thus, levels of isolatable material and eventually isolated structures indeed correlated, which emphasized the specificity of the isolation approach. We analyzed the proteins present within the isolate fractions of both KO lines via Western blotting and detected the unlipidated form of LC3B for ATG5 KO cells and lipidated LC3B for FIP200 KO MEFs (Fig EV2). The cargo receptor SQSTM1/p62 was present in both fractions. Thus, besides autophagic vesicles, the employed LC3B antibody sufficiently labeled additional targets and enabled their purification. This emphasized the potential of the isolation approach to enrich every epitope-offering structure of granular appearance. These granular structures might include SQSTM1/p62-positive aggregate particles that co-localize with unlipidated and lipidated LC3B (Runwal et al, 2019) as well as LC3B-positive nonautophagic vesicles. Click here to expand this figure. Figure EV2. Western blot analysis of isolates from ATG5 KO HeLa cells and FIP200 KO MEFs A, B. Western blot analysis of purified structures from ATG5 KO HeLa cells (A) and FIP200 KO MEFs (B). Isolations were performed with an antibody directed against LC3B and are represented with total lysate (TL). Shown blots are representative for three independent experiments. Download figure Download PowerPoint To gain deeper insights into antibody specificity and identity of the isolated structures in WT HeLa cells, we determined the co-localization of antibodies directed against LC3B and all GABARAP isoforms using FACS (Fig 1E, Appendix Fig S5). Importantly, both antibodies co-localized on approx. 93% of positive events. Concurrently, only a minor fraction was decorated exclusively by one ATG8 protein, which was possibly due to insufficient antibody binding and/or the presence of nonautophagic vesicles, such as LC3-associated phagosomes (Sanjuan et al, 2007). However, we treated cells with bafilomycin A1 prior to isolation, which resulted in the substantial accumulation of autophagic vesicles within cells and, moreover, reduced the recruitment of LC3 to nonautophagic lipidation processes (Florey et al, 2015; Stempels et al, 2022). This substantiated the clear predominance of autophagic vesicles within isolate fractions of WT HeLa cells. To directly visualize and characterize the purified structures, we performed microscopy. Differential interference contrast microscopy showed vesicular structures of different sizes without evidence of cellular debris or accumulations of other cellular material (Fig 2A). This was validated by negative stain electron microscopy, which identified intact vesicles (Fig 2B), whose inner core often showed a granular appearance and was filled with darkly stained materials most likely resembling proteinous cargoes. Figure 2. Isolated autophagic vesicles are sealed Differential interference contrast microscopy images of purified autophagic vesicles at high (I) or low (II) dilution. Images are representative of three independent approaches. Scale bar = 10 μm. Negative stain electron microscopy images of isolated vesicles. Scale bar = 500 nm. Size evaluation of isolated vesicles. The diameters of approx. 60 individual vesicles were determined using EM images. Statistics are depicted as mean ± SD. Western blot analysis of isolated autophagic vesicles upon proteinase K digestion. Mechanically opened vesicles served as positive control. For negative control, isolates were incubated with BSA instead of proteinase K. Depicted are two different blots that are representative for five independent experiments. Download figure Download PowerPoint Size evaluation of isolated vesicles demonstrated that their diameters ranged from 340 to 1,150 nm (Fig 2C), fully consistent with the size described for autophagosomes (Mizushima et al, 2002). Interestingly, the enriched vesicles could be clustered into different size groups: the group with the smallest diameters covered 426 nm on average, and the largest grouped with a mean diameter of 1,034 nm. Covering 42%, the most abundant vesicles showed a mean size of 651 nm in diameter. Thus, microscopical visualization confirmed the purification of sealed vesicles that showed typical size characteristics of autophagic vesicles. To further investigate whether the isolated autophagic vesicles were intact, we used proteinase K digestion and analyzed its impact on SQSTM1/p62 (Fig 2D). In closed vesicles, the cargo receptor is inaccessible and protected from degradation by the proteinase (Velikkakath et al, 2012). Indeed, SQSTM1/p62 was not prominently degraded, indicating that the majority of autophagic structures were sealed. For control, we opened vesicles mechanically, which resulted in the exhaustive digestion of the cargo receptor. Thus, the antibody-based FACS-mediated isolation approach efficiently enriched intact native autophagic vesicles at large quantities, which qualified the isolates for subsequent PL and cargo profiling. Phospholipid composition of native autophagic vesicles The autophagosome shows explicit requirements regarding membrane curvature and fusion ability, which is determined by a distinct PL composition (Schutter et al, 2020; Laczko-Dobos et al, 2021). However, the PLs of native human autophagosomes are elusive so far, and thus, we performed targeted multiplex quantitative MS to quantify distinct PL species and associated fatty acids of isolated autophagic vesicles. Importantly, we identified all analyzed PL classes within autophagic vesicles with diverse fatty acid combinations concerning chain length and saturation level (Fig 3A). Compared with HeLa total lysates, the PL composition was essentially altered. In particular, the short-chained form of phosphatidylcholine (PC) was significantly enriched and accounted for approx. 42% of all detected PLs in autophagic vesicles. Remarkably, phosphatidylinositol (PI) was exclusively represented with its short-chained variant, and phosphatidylglycerol (PG) was almost completely excluded. Excitingly, besides phosphoglycerides, we also observed sphingomyelin (SM) within the isolate fractions (Fig 3A). SM is the only PL that is not exclusively synthesized within the ER, but is generated from ceramide within the Golgi apparatus. The PL is commonly found at the Golgi network, the plasma membrane, in the endocytic system, and in lysosomes (Slotte, 2013). To confirm the presence of SM in autophagic vesicles, we analyzed the co-localization of SM and LC3B employing immunocytochemistry. Dye-conjugated SM was detected at the expected cellular compartments and, indeed, co-localized with LC3B-positive structures (Figs 3B and C, and EV3, Appendix Fig S6A). However, the isolation approach does not distinguish between autophagosomes and autolysosomes, the autophagosome-lysosome fusion product. Consequently, autolysosomes will also be present within the isolate fractions and could be the source of the detected SM. Thus, to further elucidate the identity of SM- and LC3B-positive vesicles, we analyzed their co-localization with the lysosome marker LAMP2. Importantly, we observed autolysosomes, positive for all three markers, but also vesicles solely positive for SM and LC3B (Fig 3D, Appendix Fig S6B), indicating that SM is a component of autophagic vesicles. Figure 3. Phospholipid profiles of isolated autophagic vesicles Phospholipids identified in isolated autophagic vesicles in comparison with HeLa total lysates. Relative amounts were calculated based on total levels of detected phospholipids. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; SM, sphingomyelin. Distribution of SM within HeLa cells. BODIPY FL C5-SM (green) was used to localize SM. Nuclei were stained by DAPI. Shown image is representative for 28 slices of three independent experiments. Scale bar = 20 μm. Immunocytochemical analysis of SM (green) and LC3B (red). DAPI was used to stain nuclei. Shown image is representative for 33 slices from three independent experiments. Pearson's correlation coefficient for co-localization: 0.44 ± 0.09. Single channels are presented in Appendix Fig S6A. Scale bars = 20 or 2 μm. Immunocytochemical analysis of SM (green), LC3B (red), and LAMP2 (blue). Shown image is representative for 21 slices of three independent experiments. Single channels are presented in Appendix Fig S6B. Scale bars = 20 or 2 μm. Phospholipids identified in autophagic vesicles isolated upon different conditions. Relative amounts were calculated based on total levels of detected phospholipids. Abbreviations are depicted in (A). Data information: (A, E) Statistics are depicted as mean ± SD of three independent samples for each condition; one-way ANOVA; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. No significant alterations were observed in (E). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Localization analysis of SM within HeLa cells SM is localized at the trans-Golgi network. Immunocytochemical analysis of SM (green) and TGN (red). DAPI was used to stain nuclei. Shown images are representative for 27 slices of three independent experiments. Pearson's correlation coefficient for co-localization: 0.57 ± 0.08. Scale bars: 20 and 2 μm. SM is localized at lysosomes. Immunocytochemical analysis of SM (green) and LAMP2 (red). Nuclei were stained by DAPI. Shown images are representative for 30 slices of three independent experiments. Pearson's correlation coeffic
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