ER –plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI 3P synthesis
2017; Springer Nature; Volume: 36; Issue: 14 Linguagem: Inglês
10.15252/embj.201797006
ISSN1460-2075
AutoresAnna Chiara Nascimbeni, Francesca Giordano, Nicolas Dupont, Daniel Grasso, María I. Vaccaro, Patrice Codogno, Étienne Morel,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle26 May 2017free access Transparent process ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis Anna Chiara Nascimbeni Anna Chiara Nascimbeni Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Francesca Giordano Francesca Giordano orcid.org/0000-0002-5942-1753 Institut Jacques Monod, CNRS UMR 7592, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Daniel Grasso Daniel Grasso Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Maria I Vaccaro Maria I Vaccaro Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Patrice Codogno Patrice Codogno Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Etienne Morel Corresponding Author Etienne Morel [email protected] orcid.org/0000-0002-4763-4954 Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Anna Chiara Nascimbeni Anna Chiara Nascimbeni Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Francesca Giordano Francesca Giordano orcid.org/0000-0002-5942-1753 Institut Jacques Monod, CNRS UMR 7592, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Daniel Grasso Daniel Grasso Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Maria I Vaccaro Maria I Vaccaro Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Patrice Codogno Patrice Codogno Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Etienne Morel Corresponding Author Etienne Morel [email protected] orcid.org/0000-0002-4763-4954 Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Author Information Anna Chiara Nascimbeni1,2, Francesca Giordano3,4, Nicolas Dupont1,2, Daniel Grasso5, Maria I Vaccaro5, Patrice Codogno1,2 and Etienne Morel *,1,2 1Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France 2Université Paris Descartes-Sorbonne Paris Cité, Paris, France 3Institut Jacques Monod, CNRS UMR 7592, Paris, France 4Université Paris Diderot-Sorbonne Paris Cité, Paris, France 5Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina *Corresponding author. Tel: +33 172606474; Fax: +33 172606399; E-mail: [email protected] The EMBO Journal (2017)36:2018-2033https://doi.org/10.15252/embj.201797006 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 The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy. Synopsis Early autophagic markers are recruited to endoplasmic reticulum-plasma membrane (ER-PM) contact sites established by tethering factors extended synaptotagmins, allowing for local phosphatidylinositol-3-phosphate synthesis and autophagosome biogenesis. Autophagy induction is accompanied by ER-PM contact site mobilization. E-Syt2, a major tethering protein of ER-PM contact sites, forms a complex with VMP1 and Beclin1, two regulators of PI3KC3 complex activity. Local autophagosome biogenesis is initiated by local PI3P synthesis via the targeting of PI3KC3 complex at ER-PM contact sites. Introduction Macro-autophagy (hereafter referred to as autophagy) is a highly regulated intracellular degradation pathway necessary for cellular homeostasis (Boya et al, 2013). Autophagy is initiated by the formation of a specific double-membrane organelle called the autophagosome. The biogenesis of autophagosome is orchestrated by multiple signalling pathways and complexes that regulate membrane dynamics that contain autophagy-related (ATG) proteins. Autophagy initiates with biogenesis of a pre-autophagosomal double-membrane structure, termed the phagophore, which emanates from the omegasome, a subdomain of the endoplasmic reticulum (ER) membrane positive for PI3P (phosphatidyl-inositol-3-phosphate) and PI3P-binding proteins (Axe et al, 2008). The PI3P pool engaged in autophagosome biogenesis is synthesized by the class 3 PI3kinase complex (PI3KC3), comprised of VPS34, VPS15, ATG14L, Beclin1, and regulating adaptors, such as VMP1, NRBF2 and Ambra1, and is dependent on ULK1 complex signalling (Nascimbeni et al, 2017). The phagophore then elongates and is close to form a mature autophagosome that will latter fuse with the lysosome. Although the ER membrane requirement is well established, other membrane sources, like the Golgi apparatus, endosomes, the mitochondria and the plasma membrane (PM), have been proposed to participate, directly, indirectly or partially, in autophagosome biogenesis (Molino et al, 2017), from phagophore generation to growth of the organelle (Ravikumar et al, 2010a,b; Rubinsztein et al, 2012). The ER is a dynamic and complex membranous network that extends throughout the cell impacting a multiplicity of cellular functions (Friedman & Voeltz, 2011). There is growing evidence that close appositions between the ER and the membranes of virtually all other organelles play major roles in cell physiology (Helle et al, 2013). Notably, ER–mitochondria contact sites actively participate in autophagosome biogenesis via the regulation of PI3KC3 complex (Hamasaki et al, 2013). ER-PM contact sites are important for lipid metabolism and transport, notably of phosphoinositides, and these domains have the potential to affect membrane trafficking and signalling events that occur at the PM (Stefan et al, 2013). In higher eukaryotes, three ER-localized proteins, the extended synaptotagmins (E-Syts 1, 2 and 3), play crucial roles in tethering the ER to the PM and are thus considered as key regulators, as well as precise markers, of ER-PM tethering zones (Giordano et al, 2013). Because both ER and PM have been directly associated with autophagy regulation and because ER tethering could be important for membrane remodelling, we hypothesized that the ER cooperates with plasma membrane during the very first steps of the autophagosome biogenesis via the establishment of ER-PM specialized contact sites. Indeed, we show here that stress situations that induce autophagy lead as well to ER-PM contact site mobilization, highlighting a connection between ER-PM tethering and the autophagy machinery. We observed local recruitment of autophagic and pre-autophagic markers at E-Syts domains of the cortical ER during autophagy initiation. Further, autophagy was enhanced in E-Syt-overexpressing cells, whereas inhibition of E-Syts expression reduced autophagosome biogenesis. Finally, we demonstrated that ER-PM contact sites are required for local PI3P synthesis by the PIK3C3 complex. We found that the PIK3C3 complex at ER-PM contact sites is mobilized at the ER membrane via the binding of VMP1 (Molejon et al, 2013a), the ER partner of Beclin1, the major regulator of autophagy-associated PI3P synthesis. Results We first studied the behaviour of ER-PM contact sites in conditions that promote autophagy using a HRP-myc-KDEL reporter that allows indirect visualization of ER lumen by electron microscopy (EM; Giordano et al, 2013). We analysed and quantified ER-PM contact zones in control and in HeLa cells starved to induce autophagy. We observed a massive increase in the number of ER-PM contact sites compared to control situation (Fig 1A and B), and this increase correlated with autophagy induction (Fig 1C). Levels of E-Syt2 and 3 proteins increased after 1 and 4 h of starvation, whereas levels of calnexin (an ER marker), syntaxin 17 [STX17, previously identified as a marker of an autophagy-related ER–mitochondria contact sites (Hamasaki et al, 2013)] or PTPIP51 [an ER–mitochondria tethering protein recently shown to participate in autophagy regulation (Gomez-Suaga et al, 2017)] were not affected (Fig 1D and E). The increase in E-Syt2 was also induced by mechanical stress in KEC cells (Fig EV1A), a condition that promotes autophagy (Orhon et al, 2016), and by serum starvation or mTOR chemical inhibition (Fig EV1B). Figure 1. Starvation increases ER-PM contact sites density A. Electron micrographs of HeLa cells grown under complete medium conditions (control, ctrl) or starved for 1 h (1 h STV). HeLa cells were transfected with the ER luminal marker ssHRP-myc-KDEL (which enables ER identification via an electron-dense (dark) HRP reaction) to allow detection of ER-PM contact sites (black arrows). Scale bar, 2 μm. Representative images from one of three independent experiments are shown. B. Quantification of ER-PM contact sites visualized in electron micrographs with 20 cells analysed per condition in each of three experiments. Means ± s.e.m. are plotted. ***P < 0.001, unpaired two-tailed t-test. C–E. HeLa cells were grown under control and starvation conditions for 1 and 4 h. Representative Western blots of lysates for (C) p62 and lipidated LC3 and (D) E-Syts, calnexin (CLNX), STX17, and PTPIP51 are shown. Actin was used as a loading control. (E) Quantification of Western blots from three independent experiments. Means ± s.e.m. are plotted. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Multiple autophagy stimuli induce E-Syt2 expression Mechanical stress: Western blots and protein quantifications of protein lysates from KEC cells under basal (ctrl) and mechanical stress (flow) conditions for 4–72 h (n = 3). Plotted are mean ± s.d. mTOR inhibition and serum starvation: Western blots and protein quantifications (compared to ctrl) of protein lysates from HeLa cells grown under basal (ctrl) and autophagy-inducing conditions. Cells were cultured with 1.5 μM Torin1, an mTOR inhibitor, for 2 h or in a medium without serum for 1 h, respectively (n = 3). Plotted are mean ± s.d. Download figure Download PowerPoint Since our data suggested that autophagy and formation of ER-PM contact sites are stimulated in the same time frame, we next investigated whether autophagosomal markers could be detected at the cortical ER, near the PM. Indeed, in HeLa cells, we detected the phagophore, autophagosome and autolysosome marker LC3 near the cell boundary as early as 15 min post-starvation, a time at which most of LC3 was associated with the ER marker Sec61β (Fig 2A). The presence of LC3 in the vicinity of the plasma membrane was further confirmed by total internal reflection fluorescence (TIRF) microscopy (Fig 2B). The number of LC3 puncta in the TIRF zone increased with time under starvation conditions (Fig 2B and C), showing that LC3 autophagic structures appear at the immediate vicinity of the PM during starvation-induced autophagy. A similar LC3 pattern was observed in MDCK cells under mechanical stress (Orhon et al, 2016; Fig 2D). These results indicated that in different cell types treated with different autophagy inducers, LC3 staining was detected very close to the PM, suggesting that these autophagic structures might be associated with ER-PM contact sites. Figure 2. Early autophagic structures are detected in the vicinity of the plasma membrane HeLa cells were transfected with RFP-Sec61β (an ER marker) and immunostained for the autophagosome marker LC3. The two markers co-distribute in the vicinity of the plasma membrane (empty arrowheads) after 15 min of starvation (STV 15 min) as shown by confocal microscopy. TIRF analysis after 0 (control), 15 (STV 15 min) and 60 (STV 60 min) min of starvation. Quantification of LC3 puncta per TIRF section (n = 3; 20 cells analysed per condition). **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Means ± s.e.m. are plotted. Representative confocal microscopy images and 3D reconstructions of MDCK cells immunostained for LC3, Na/K-ATPase and DAPI under mechanical stress conditions. Arrowheads indicate LC3 puncta at vicinity of plasma membrane. Data information: Scale bars, 10 and 4 μm (magnified area in A). Download figure Download PowerPoint To analyse further whether ER-PM contact zones are sites of autophagosome formation, we used tagged E-Syt2 and E-Syt3 proteins as markers of ER-PM contact sites: while E-Syt1 can be detected on perinuclear as well as cortical ER structures, E-Syt2 and E-Syt3 localizations are restricted only to cortical ER engaged in ER-PM contact sites (Appendix Fig S1A and Fernández-Busnadiego et al (2015); Giordano et al (2013)). In HeLa cells starved for 15 min, we observed co-distribution of LC3 with the ER markers Sec61βRFP and E-Syt2GFP by confocal microscopy (Fig EV2A and C) at the basal level of the cells and by super-resolution two-colour stimulated emission depletion (STED) microscopy (Fig 3A). 3D reconstructions showed that LC3 was often directly connected to the ER membrane via E-Syt2-positive ER domains (Fig 3B) and sometimes appeared within a membranous niche positive for Sec61βRFP and E-Syt2GFP (Fig EV2A). Similar results were obtained when we used an antibody to ATG16L1 (Fig EV2B and C), a regulator of autophagosome biogenesis known to participate in the early events of LC3 recruitment to omegasome/phagophore structures (Wilson et al, 2014). The LC3-positive structures that were in the vicinity of the PM were negative for Rubicon (Appendix Fig S2), excluding the possibility of a non-autophagy-related LC3-associated phagocytosis (Levine et al, 2015). Using immunogold EM, we clearly observed E-Syt2myc and LC3GFP co-distribution on ER-PM contact sites in HeLa cells starved for 60 min (Fig EV2D); these are likely the same autophagic structures that we observed directly by electron microscopy in the immediate vicinity of cortical ER and PM in the same conditions (Fig 3C). Click here to expand this figure. Figure EV2. LC3 and ATG16L1 reside at ER-PM contact sites under starvation conditions A, B. Confocal microscope images and 3D reconstructions of HeLa cells co-transfected with GFP-E-Syt2 and RFP-Sec61β and immunostained for LC3 or ATG16L1 and DAPI. Arrowheads denote LC3 or ATG16L1 puncta near the E-Syt2-positive niche of the ER. Scale bars, 5 and 2.5 μm (magnified areas). C. Quantification of LC3 and ATG16L1 co-distribution with E-Syt2 at basal plan of the cell n = 80 cells. Mean ± s.e.m. shown. D. Immunogold electron micrographs of starved HeLa cells, showing co-distribution of the myc-E-Syt2 and anti-LC3 antibody at ER (empty arrowheads) and PM (black arrowheads) juxtaposition sites. Scale bar, 250 nm. Download figure Download PowerPoint Figure 3. Autophagosomes can form at ER-PM contact sites STED images of the basal plane of a HeLa cell co-transfected with vectors for expression of the ER marker Sec61βGFP and the ER-PM contact marker mCherry-E-Syt3 and immunostained for the autophagosome marker LC3. Scale bars, 5 μm. Arrowheads indicate autophagic structures (LC3) arising from ER niches (Sec61β-positive) at ER-PM contact sites (E-Syt3-positive). Three-dimensional reconstruction from confocal microscope images of HeLa cells co-transfected with vectors for expression of Sec61βRFP and E-Syt2GFP and immunostained for LC3. Scale bar, 2.5 μm. The arrowheads indicate the LC3-positive structures. Electron micrographs of HeLa cells starved 1 h and transfected with a vector for expression of the ER luminal marker ssHRP-myc-KDEL, showing early autophagic structures in the proximity of the PM. Scale bar, 400 nm. Download figure Download PowerPoint We next analysed distribution of the omegasome-marker (Axe et al, 2008) DCFP1GFP in HeLa cells after a short time of starvation, to maximize detection of autophagosome biogenesis-related events. We clearly observed co-distribution of DFCP1 with membranes positive for E-Syts, LC3 and Sec61βRFP by confocal microscopy (Fig 4A and B) and by time-lapse microscopy (Fig 4C). These results strongly suggest that at least some autophagosome biogenesis occurs at ER-PM contact sites. We then quantified the E-Syt2-positive omegasome structures and the omegasome structures at ER-mitochondria contact sites identified by mitochondrial protein TOM20 (Hamasaki et al, 2013). Our results indicate that, within 15 min of autophagy induction, approximately 30% of DFCP1-positive structures were associated with E-Syt2-positive domains (Fig EV3A and C), a ratio very close to the one we observed for DFCP1-positive ER–mitochondria contact sites (Fig EV3B and C). These results were further confirmed by electron microscopy analyses (Fig EV3D). Together, these data demonstrate that autophagosome assembly at ER-PM contact sites domains accounts for approximately 30% of total autophagosomes observed after a 15-min starvation of HeLa cells. Figure 4. LC3 and DFCP1 are present at ER-PM contact sites under starvation conditions Representative confocal microscopy images taken in the basal plane of a HeLa cell expressing myc-E-Syt2, RFP-Sec61β and GFP-DFCP1 and immunostained for LC3. Scale bars, 10 and 3 μm (magnified areas). 3D reconstructions of representative HeLa cell expressing myc-E-Syt2, RFP-Sec61β and GFP-DFCP1 and immunostained for LC3. Arrowheads denote DFCP1 and LC3 puncta connected with E-Syt2-positive niches of the ER. Scale bar, 5 μm. Time-lapse confocal images of HeLa cells expressing mCherry-E-Syt3 and GFP-DFCP1 after cells were starved for 15 min. Two channels were observed simultaneously using two cameras. Arrowheads denote DFCP1 puncta in E-Syt2-positive niches of the ER. Scale bar, 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Early autophagic structures form at ER-PM and ER–mitochondria contact sites A, B. Confocal microscope images of HeLa cells co-transfected with GFP-DFCP1 (an omegasome marker), RFP-Sec61β (an ER marker) and myc-E-Syt2 (an ER-PM contact sites marker) or immunostained for TOM20 (a mitochondria marker) under starved conditions (15 min). Arrowheads denote omegasome formation at (A) ER-PM or (B) ER–mitochondria contact sites. Scale bars, 10 μm. C. Quantification of percent DFCP1-positive structures at ER-PM (Sec61β/E-Syt2 interface) and ER–mitochondria (Sec61β/TOM20 interface) contact sites. Plotted are mean ± s.e.m., n = 80. D. Electron microscopy images of HeLa cells transfected with the ER luminal marker ssHRP-myc-KDEL and starved for 1 h showing autophagic structures adjacent (within 1 μm) to ER-PM contact sites (ER-PM), ER-mitochondria contact sites (ER-mito) and to neither organelle (cytoplasm). The quantification of these autophagic structures is shown as well. Plotted are means ± s.e.m., n = 80 cells. Download figure Download PowerPoint As previously reported (Giordano et al, 2013), overexpression of E-Syt2 or E-Syt3 stabilized and increased the density of ER-PM contact sites (Appendix Fig S2). In cells overexpressing E-Syts, the lipidation of LC3 was increased and more LC3-positive structures were observed both in fed and starved conditions compared to mock-transfected cells (Fig 5A and B). Interestingly, LC3 puncta were significantly increased in the vicinity of the PM in E-Syt3-overexpressing cells (Fig 5C). Electron microscopy analyses showed twice as many autophagic structures in HeLa cells overexpressing E-Syt2 as in control cells (Fig EV4A). In functional tests monitoring long-lived protein degradation, which depends on autophagy, we observed a significant increase of protein degradation in E-Syt2- and E-Syt3-overexpressing cells, as compared to control cells (Fig EV4B). Thus, the observed autophagic structures originating from the ER-PM contact zones appear to be functional. Figure 5. Overexpression of E-Syt2 and E-Syt3 induces autophagosome formation Western blot analysis of the autophagic flux in cell lysates from control (mock) and GFP-E-Syt2 or GFP-E-Syt3-expressing HeLa cells, under complete medium and starvation (1 h EBSS) conditions, without or with Bafilomycin A1 (+BAF. A1). HeLa cells expressing GFP-E-Syt2 or GFP-E-Syt3 were immunostained for LC3. Compared to control (mock), transfected cells showed a dramatic increase in LC3 puncta, in both basal (complete medium) and starved (1 h) conditions, as evidenced by counting of LC3 puncta (n = 3; 20 cells per condition). The increase in LC3 puncta observed in cells overexpressing E-Syt3GFP (similar results were obtained with E-Syt2GFP, data not shown) involves mainly peripheral rather than perinuclear cellular regions (n = 3). Arrowheads indicate peripheral LC3 puncta (n = 3; 20–70 cells per condition). Data information: Means ± s.e.m. are plotted. NS, non-significant, ***P < 0.001, unpaired two-tailed t-test. Scale bars, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Overexpression of E-Syt2 and E-Syt3 enhances autophagy An electron micrograph of GFP-E-Syt2-expressing HeLa cells starved for 1 h, showing an increased number of autophagic structures (arrowheads) compared to starved control cells. Scale bar, 1 μm. Wortmannin (wort, 100 nM) was used as a negative control, and autophagic structures were counted in 15 μm2 areas (n = 10 cells). Proteolysis analysis showing an increased protein degradation rate in GFP-E-Syt2- and GFP-E-Syt3-expressing cells (n = 3). 3-methyladenine (3-MA) was used at 10 mM. Data information: Means ± s.e.m. are plotted. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Download figure Download PowerPoint We then sought to test how impairing ER-PM contact sites formation would influence autophagy. To do this, we inhibited expression of the three E-Syt proteins (E-Syt1, E-Syt2 and E-Syt3) simultaneously using siRNAs targeting the mRNAs encoding each of these proteins (Fig 6A and B). Interestingly, in the E-Syt-deficient cells the total number of LC3GFP structures was decreased compared to control cells (Fig 6C and D). The difference was even more striking when we quantified the peripheral to perinuclear ratio of LC3 puncta in cells treated with Bafilomycin A1 (a V-ATPase inhibitor preventing fusion between autophagosome and lysosome) to maximize the number of autophagic structures (Fig 6C and D). The decrease observed in siE-Syt-treated cells was primarily due to decreases in numbers of peripheral puncta rather than to decreases in perinuclear LC3. Figure 6. Autophagosome biogenesis is reduced in E-Syt-deficient cells A, B. HeLa cells were treated with control siRNA (siCTRL) or with siRNAs targeting mRNAs encoding each of the E-Syts (siE-Syts). (A) Western blots of cell lysates. Actin is used as a loading control. (B) E-Syts mRNAs were quantified. Means ± s.d. are plotted (n = 3). C. HeLa cells stably transfected with GFP-LC3 and treated with siE-Syts or siCTRL were not treated (−BAF. A1) or were treated with Bafilomycin A1 (+BAF. A1). Representative images are shown. Empty arrowheads indicate peripheral LC3 puncta. Scale bars, 10 μm. D. Quantification of experiments shown in panel (C) (n = 3; 20 cells per condition). Means ± s.e.m. are plotted. E. HeLa cells treated with control siCTRL or with siE-Syts were grown in complete medium or were starved for 1 or 4 h, and cells lysates were subjected to Western blot for indicated proteins. F. Quantification of Western blot shown in panel (E), with 20 cells analysed per condition (n = 5). Data information: NS, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Download figure Download PowerPoint Western blot analyses performed following a time course of starvation-induced autophagy revealed a decrease in LC3 lipidation as well as decreases of the amounts of the autophagosome biogenesis regulators ATG16L1 and ATG5—ATG12 in E-Syt-deficient cells (Fig 6E and F). Moreover, using the LC3GFP-RFP tandem dye, which is widely used to measure autophagic flux (Klionsky et al, 2016), we observed that the GFP/RFP ratio was not modified in the E-Syt-deficient cells compared to control cells (Fig EV5). Together, these data suggest that, although the number of autophagic structures was diminished when ER-PM contact sites were reduced, the maturation and transport to lysosomes of the remaining autophagosomes were not altered. Click here to expand this figure. Figure EV5. Autophagosome maturation is not affected in E-Syt-deficient cells A, B. HeLa cells stably transfected with mRFP-GFP-LC3 and treated with siE-Syts have fewer autophagosomes, but autophagosomes have normal functionality, as evidenced by (A) microscopy and (B) by RFP+/GFP+ and RFP+/GFP− LC3-puncta counting. Cells treated with Bafilomycin A1 (+BAF. A1) and siCTRL were used as a positive functionality control and showed decreased autophagosome maturation (i.e. reduced RFP+/GFP− LC3-puncta compared to siCTRL-treated cells) as expected (n = 3). ***P < 0.001, unpaired two-tailed t-test. Scale bar, 10 μm. Plotted are mean ± s.e.m. Download figure Download PowerPoint One of the major molecular events responsible for autophagosome biogenesis is the synthesis of PI3P at the omegasome on the ER membrane (Axe et al, 2008; Lamb et al, 2013; Roberts & Ktistakis, 2013). PI3P is synthesized not only at the omegasome membrane but also on early endosomes as well (Di Paolo & De Camilli, 2006; Marat & Haucke, 2016). We observed that the omegasome-marker and PI3P binding protein DFCP1 co-distributed with E-Syt2 domains on the ER (Fig 4). Therefore, we looked directly for PI3P lipid in proximity to these ER-PM contact sites during short-term starvation. Interestingly, we observed PI3P-positive structures [detected by FYVEGST/fluorescent anti-GST antibody indirect staining (Khaldoun et al, 2014)] in the immediate vicinity of ER membrane regions positive for E-Syt3 and LC3 but not in regions stained by endosomal marker EEA1 after 15 min of starvation (Appendix Fig S3A and B). We obtained similar results using 2x-FYVEGFP dye to stain for PI3P (Appendix Fig S3C) and when cells were stained using ATG16L1 and VPS35 (Seaman et al, 1998) to mark early autophagic structures and early endosomes, respectively (Appendix Fig S3D). Our results suggest that E-Syts directly or indirectly participate in autophagosome biogenesis. Because we observed PI3P at E-Syts domains after autophagy induction, we speculated that these proteins are involved in regulation of PI3P synthesis at ER-PM contact site-associated autophagosome biogenesis. To assess this hypothesis, we quantified PI3P puncta in control cells and cells deficient in all three E-Syt proteins under both fed and starved conditions. We used wort
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