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Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling

2019; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês

10.15252/embr.201947734

1469-3178

Jane Fraser, Joanne Simpson, Rosa Fontana, Chieko Kishi‐Itakura, Nicholas T. Ktistakis, Noor Gammoh,

Calcium signaling and nucleotide metabolism

Article26 August 2019Open Access Transparent process Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling Jane Fraser Jane Fraser Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Joanne Simpson Joanne Simpson Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Rosa Fontana Rosa Fontana Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Chieko Kishi-Itakura Chieko Kishi-Itakura Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Nicholas T Ktistakis Nicholas T Ktistakis Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Noor Gammoh Corresponding Author Noor Gammoh [email protected] orcid.org/0000-0001-9402-9581 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Jane Fraser Jane Fraser Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Joanne Simpson Joanne Simpson Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Rosa Fontana Rosa Fontana Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Chieko Kishi-Itakura Chieko Kishi-Itakura Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Nicholas T Ktistakis Nicholas T Ktistakis Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Noor Gammoh Corresponding Author Noor Gammoh [email protected] orcid.org/0000-0001-9402-9581 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Jane Fraser1, Joanne Simpson1, Rosa Fontana1, Chieko Kishi-Itakura2, Nicholas T Ktistakis2 and Noor Gammoh *,1 1Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 2Signalling Programme, Babraham Institute, Cambridge, UK *Corresponding author. Tel: +44 1316 518526; E-mail: [email protected] EMBO Reports (2019)20:e47734https://doi.org/10.15252/embr.201947734 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 Despite recently uncovered connections between autophagy and the endocytic pathway, the role of autophagy in regulating endosomal function remains incompletely understood. Here, we find that the ablation of autophagy-essential players disrupts EGF-induced endocytic trafficking of EGFR. Cells lacking ATG7 or ATG16L1 exhibit increased levels of phosphatidylinositol-3-phosphate (PI(3)P), a key determinant of early endosome maturation. Increased PI(3)P levels are associated with an accumulation of EEA1-positive endosomes where EGFR trafficking is stalled. Aberrant early endosomes are recognised by the autophagy machinery in a TBK1- and Gal8-dependent manner and are delivered to LAMP2-positive lysosomes. Preventing this homeostatic regulation of early endosomes by autophagy reduces EGFR recycling to the plasma membrane and compromises downstream signalling and cell survival. Our findings uncover a novel role for the autophagy machinery in maintaining early endosome function and growth factor sensing. Synopsis Targeting of damaged organelles by autophagy is crucial for cell homeostasis. This study identifies a novel function of the autophagy machinery in recognising damaged early endosomes, the absence of which compromises EGFR trafficking and signalling. Aberrant early endosomes accumulate in the absence of autophagy. Lysosomes are recruited to damaged early endosomes requiring the activities of TBK1, Gal8, and the autophagy machinery. The consequence of unresolved early endosome damage is the disruption of EGFR trafficking and compromised EGF-mediated signalling and survival. Introduction Vesicular trafficking processes are important in controlling various aspects of cell fate decisions and homeostasis. Of these processes, both endocytosis and autophagy are known to deliver cellular cargoes to lysosomes for degradation and are involved in relaying cues from the extracellular milieu. Recent studies have begun to uncover mechanisms through which the endocytic and autophagic compartments can act in concert to facilitate their optimal activities. Endocytosis begins with the internalisation of plasma membrane cargoes into early, or "sorting", endosomes marked by specific adaptor proteins and small Rab GTPases 1-3. These vesicles can then mature into endosomes destined for various cellular compartments including the plasma membrane (recycling endosomes) or lysosomes (late endosomes), thereby regulating the activities of endocytic cargoes such as receptor tyrosine kinases (RTKs) 4. Efficient signalling from a subset of endocytosed RTKs has been shown to utilise autophagy-related membranes as signalling platforms 5-7, suggesting that autophagy proteins can regulate pro-growth signalling independent of their degradative function. This is of particular interest given that the perturbed activities of many RTKs, including the epidermal growth factor receptor (EGFR), have been associated with various cancers and developmental defects 8-11. Autophagy-mediated degradation of various cellular components is critical to maintain cellular homeostasis 12. Recently, autophagy has been shown to target damaged lysosomal membranes and bacteria-containing vesicles, suggesting that it can mediate the degradation of these vesicles 13-15. Such endomembrane damage is recognised by the Galectin family of proteins that bind glycans on the luminal face of the vesicles 15, 16. Tank-binding kinase 1 (TBK1) is then required to recruit autophagy players, including WIPI2 and ATG16L1, to the damage site where they facilitate the ubiquitin-like conjugation of ATG8 paralogues, such as LC3, to phosphatidylethanolamine and subsequently autophagosome biogenesis 16, 17. While the role of LC3 in cargo recognition by binding to receptor proteins has been well studied, increasing evidence suggests that upstream autophagy players can also play an important role in recruiting autophagosome substrates 18, 19. Autophagy and the endocytic pathway share multiple commonalities 20. For example, phosphoinositide-3-phosphate (PI(3)P) is essential for the proper function of both early endosomes and autophagosomes 3, 21-23. Its biogenesis from phosphoinositol is mediated by vacuolar protein sorting-associated protein 34 (VPS34), also known as phosphoinositide-3-kinase class III (PIK3C3) 24. VPS34 can generate PI(3)P on either endocytic or autophagic membranes by existing in two distinct complexes: the "autophagic" complex I (comprised of ATG14, Beclin-1 and VPS15) and the "endocytic" complex II (comprised of UVRAG, Beclin-1 and VPS15) 25-28. These complexes can be regulated by the availability of growth factors and nutrients 29-31. The endocytic pathway can also contribute to autophagosome biogenesis as a number of autophagy players (such as ATG16L1, ATG9, WIPI2 and ULK1) can localise to Rab11-positive (Rab11+) recycling endosomes 32-34. Furthermore, endocytic regulators (e.g. VAMP3, Rab7, CHMP2A and STX17) have been found to be required for autophagosome maturation, closure and their eventual fusion with lysosomes 35-38. While extensive studies highlight the contribution of the endocytic pathway to autophagosome formation 20, less is known on how autophagy is required for the optimal function of endosomes. Here, we uncover a novel role for autophagy in maintaining endosomal homeostasis by recognising and targeting perturbed early endosomes. A consequence of inhibiting this process is the disruption of EGFR endocytic sorting which impacts overall cellular response to growth factor signalling. Results Loss of autophagy perturbs EGFR endocytic trafficking Given the close interplay between endocytic and autophagic vesicular trafficking, we were interested to understand how the loss of autophagy can impact the endocytic pathway. To test this, we used EGFR as a model endocytic cargo as its regulation has been extensively studied 39. Glial cells transformed by Ink4a/Arf deletion as well as Tp53 and Nf-1 knockdown (shNf-1/shTp53) were used as a model system 40, 41, and CRISPR/Cas9-mediated gene editing of autophagy-related genes (denoted as sgAtg) was introduced to inhibit autophagy (Fig EV1). To observe the overall endocytic trafficking of EGFR in real time, Alexa 555-labelled EGF (555-EGF) was monitored in cells by live spinning disc confocal microscopy. Tracking the paths of these vesicles revealed that while EGF exhibited a dynamic trafficking pattern in control cells, it accumulated in the perinuclear region of Atg7 knockout cells (Fig 1A). Immunofluorescence staining of EGFR confirmed its perinuclear accumulation in the absence of ATG7 after 15 min of EGF treatment (Fig 1B and C). To further investigate this disrupted endocytic trafficking, we assessed changes in ligand–receptor colocalisation 42 and observed an increased percentage of EGFR vesicles that remained positive for 555-EGF in autophagy-deficient cells (Fig 1D and E). This elevated ligand–receptor binding is suggestive of a perturbed trafficking of EGFR at early endosomes. To test this, we assessed the colocalisation between EGFR and the early endosome marker Rab5. Figure 1F and G shows that at early time points (5 min), EGFR occupancy in Rab5+ endosomes was comparable between control and sgAtg7 cells, indicating that early endocytic uptake of EGFR from the plasma membrane is not affected by ATG7 loss. However, at a later time point (15 min), EGFR residency in early endosomes was strikingly increased in sgAtg7 cells compared to control cells. Overall, these data suggest that autophagy inhibition alters EGFR trafficking resulting in its accumulation at early endosomes. Click here to expand this figure. Figure EV1. Confirmation of CRISPR/Cas9-mediated gene editing and autophagy inhibition in cellsWestern blot analyses of glial cells infected with shRNA against Nf-1 and Tp53, and then with Cas9 and sgRNAs against Atg7. Download figure Download PowerPoint Figure 1. Loss of autophagy perturbs EGFR endocytic traffickingThe following experiments were performed in glial shNf-1/shTp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). Spinning disc confocal live cell imaging of Alexa 555-EGF (555-EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/ml 555-EGF for the indicated durations. Scale bar: 10 μm. Immunofluorescence staining of EGFR following 5- or 15-min stimulation with 20 ng/ml EGF. Scale bar: 20 μm. Quantification of EGFR vesicles in a perinuclear region (within 30 μm diameter of the centre of the nucleus) (in B). Cells were stimulated with 20 ng/ml 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm. Quantification of percentage of total EGFR vesicles that colocalise with 555-EGF (in D). Cells stably expressing mCherry-Rab5 were stimulated with 20 ng/ml EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm. Pearson's colocalisation coefficient between mCherry-Rab5 and EGFR (in F). Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two-tailed Student's t-test: NS P > 0.05, *P < 0.05, **P < 0.01. Download figure Download PowerPoint Autophagy inhibition increases PI(3)P-positive early endosomes To further investigate a potential defect in early endosomes upon autophagy deficiency, we examined the levels of PI(3)P, a key phosphoinositide determinant of autophagosome biogenesis and early endosome function 2, 43. To do this, we employed a post-fixation technique using an Alexa 488-conjugated recombinant FYVE-domain probe in order to avoid interference with endosomal trafficking as a result of PI(3)P-binding domain overexpression in cells 44. Interestingly, inhibition of autophagy in glial cells by ATG7 or ATG16L1 deletion (Fig EV2A) resulted in increased overall cellular levels of PI(3)P (Figs 2A and B, and EV2B). We further tested whether this increase in PI(3)P levels was associated with early endosomes marked by the PI(3)P effector EEA1 2 and observed a significant increase in PI(3)P+ EEA1 vesicles in sgAtg7 and sgAtg16l1 glial cells (Fig 2A and C). Similar results were obtained in sgAtg7 mouse embryonic fibroblasts (MEFs), suggesting that changes in early endosomal PI(3)P as a result of autophagy inhibition are consistent in other cell types (Fig EV2C and D). As observed with Rab5 (Fig 1F and G), EGFR exhibited a higher residency in EEA1+ endosomes upon autophagy inhibition in glial cells (Fig 2D and E). Click here to expand this figure. Figure EV2. Total PI(3)P levels increase in autophagy-inhibited cellsAll cells were serum starved for 4 h before assaying. Western blot analyses of shNf-1/shTp53 glial cells expressing gRNA sequences targeting Atg16l1. 20× magnification of images in Fig 2A. Control, sgAtg7, or sgAtg16l1 cells were treated with 2 ng/ml EGF for 15 min. Cells were then processed for staining using a PI(3)P probe (Alexa 488-labelled 2XFYVE domain). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 100 μm. Control or sgAtg7 MEF cells were treated with 2 ng/ml EGF for 15 min before fixation and staining using EEA1 antibodies and a PI(3)P probe (Alexa 488-labelled 2XFYVE domains). Scale bar: 10 μm. Quantification of PI(3)P+ vesicles per cell and the Pearson's colocalisation coefficient between PI(3)P and EEA1 (in C). Endogenous Beclin-1 was immunoprecipitated from control and sgAtg7 cells that were stimulated with 2 ng/ml EGF for 15 min. Cells were lysed in CHAPS buffer followed by immunoprecipitation of Beclin-1 and analyses by Western blotting. Atg16l1−/− MEFs were reconstituted with wild type (WT) ATG16L1 or a mutant of ATG16L1 containing a deletion in the ATG5 binding domain (residues 1–39, ΔA5). Endogenous VPS34 was immunoprecipitated from these cells following 15 min of EGF (2 ng/ml). Binding partners were assessed by Western blotting. Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two-tailed Student's t-test: *P < 0.05, ***P < 0.001. Download figure Download PowerPoint Figure 2. Autophagy inhibition increases PI(3)P-Positive early endosomesThe following experiments were performed in glial shNf-1/shTp53 glial cells serum starved for 4 h before assaying. Control, sgAtg7 or sgAtg16l1 cells were treated with 2 ng/ml EGF for 15 min. Cells were then processed for staining using anti-EEA1 antibodies and PI(3)P probe (Alexa 488-labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm. Quantification of PI(3)P+ label intensity per cell (in A). Pearson's colocalisation coefficient between PI(3)P and EEA1 (in A). Control and sgAtg7 cells were stimulated 20 ng/ml EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR. Scale bar: 10 μm. Pearson's colocalisation coefficient between EEA1 and EGFR (in D). Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng/ml EGF for 15 min and then lysed in CHAPS-containing detergent buffer and binding partners detected by Western blotting. Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 (in F). Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two-tailed Student's t-test: NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint The production of PI(3)P catalysed by VPS34 is regulated by adaptor proteins that form part of the endocytic or autophagic VPS34 complexes 25-28. To test whether changes in PI(3)P levels upon autophagy inhibition were due to differences in the binding of VPS34 to its adaptor proteins, immunoprecipitations of endogenous VPS34, or its binding partner Beclin-1, were performed from control or sgAtg7 cells. The interactions between VPS34 and its complex components, including Beclin-1, Rubicon, UVRAG and ATG14, were unaltered by autophagy status (Figs 2F and G, and EV2E). Similar results were obtained in MEF cells (Fig EV2F). Together, these findings suggest that the compositions of VPS34 complexes are not altered upon the deletion of autophagy genes. Early endosomes are disrupted in the absence of autophagy machinery and stain positively for Gal8 To further examine the cause of EGFR accumulation at Rab5+ and EEA1+ endosomes, we tested the possibility that damaged early endosomes accumulated in cells that lack autophagy. Recognition of damaged endomembranes can be mediated by a family of glycan-binding proteins known as Galectins that act as "eat-me" signals 45. Of these, Gal3, Gal8 and Gal9 have been involved in the recognition of damaged lysosomes and salmonella-containing vesicles 13-15. To assess whether autophagy-deficient cells accumulate damaged early endosomes that may be recognised by these Galectins, we expressed YFP-tagged Gal3, Gal8 or Gal9 in glial cells and their colocalisation with EEA1 was compared between control and sgAtg7 cells. Figure 3A and B shows that while the colocalisation between EEA1 and Gal3 or Gal9 was not significantly affected by ATG7 depletion, Gal8 localisation to early endosomes was strongly increased upon ATG7 loss. This suggests that damaged early endosomes may accumulate when autophagosome maturation is inhibited by ATG7 deletion. Moreover, Gal8 labelled EGF+ vesicles in ATG7-deficient cells, indicating that EGFR can localise to this subset of damaged early endosomes (Fig 3C and D). Disruption of early endosomal function can also be induced chemically by monensin treatment in control glial cells resulting in enhanced localisation of Gal8 at early endosomes (Fig 3E and F) 46. Together, these data suggest that damaged early endosomes can accumulate in the absence of autophagy genes or during chemical disruption of endomembranes. Figure 3. Early endosomes stain positively for Gal8 upon autophagy inhibition or monensin treatmentThe following experiments were performed in glial shNf-1/shTp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). Cells transiently expressing YFP-Galectins (YFP-Gal3, YFP-Gal8, or YFP-Gal9) were stimulated with 2 ng/ml EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Galectins (in A). Cells transiently expressing YFP-Gal8 were stimulated with 20 ng/ml Alexa 555-EGF (555-EGF) for 15 min. Scale bar: 10 μm. Quantification of the percentage of YFP-Gal8-labelled vesicles that colocalise with 555-EGF in control or ATG7-deficient cells (in C). Cells transiently expressing YFP-Gal8 were treated 100 μM monensin for 1 h and stimulated with 2 ng/ml EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Gal8 upon monensin treatment (in E). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two-tailed Student's t-test: NS P > 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Autophagy machinery targets early endosomes We hypothesised that damaged early endosomes may be recognised by the autophagy machinery facilitating their targeting to the lysosome system. We tested this during monensin-induced endosomal damage in autophagy-competent cells. Indeed, monensin treatment resulted in the labelling of EEA1+ endosomes with GFP-LC3 stably expressed in control glial or MEF cells but not in those lacking ATG7 (Figs 4A and B, and EV3A and B). The localisation of LC3 to EEA1+ vesicles suggests that the autophagy machinery may be required to recruit lysosomes to early endosomes. Indeed, EEA1+ vesicles stained positive for the lysosomal marker LAMP2 following monensin-induced endomembrane stress in control but not sgAtg7 cells (Fig 4C and D). Interestingly, treatment of cells with bafilomycin A1 to inhibit lysosomal acidification led to the enhanced colocalisation between LC3 and EEA1, suggesting that the lysosomal targeting of early endosomes by the autophagy machinery occurs under basal levels (Fig 4A and B). A potential increase in total early endosome number was not significant between autophagy-competent and autophagy-incompetent cells (Fig EV3C) likely due to high variations in endosome numbers between cells or the existence of compensatory mechanisms that affect endosome biogenesis or maturation. Altogether, these findings suggest that targeting of early endosomes to lysosomes requires components of the autophagy machinery. Figure 4. Autophagy machinery targets early endosomesThe following experiments were performed in glial shNf-1/shTp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). Cells stably expressing GFP-LC3 were treated with 100 μM monensin in the presence or absence of bafilomycin A1 (20 nM) for 1 h and stimulated with 2 ng/ml EGF for 15 min before fixation and immunofluorescence staining against EEA1. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with GFP-LC3 (in A). Cells were treated for 1 h with 100 μM monensin and then stimulated with 2 ng/ml EGF for 15 min. LAMP2 and EEA1 were then detected by immunofluorescence staining. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of the percentage of total EEA1 vesicles that colocalise with LAMP2 (in C). Cells were treated with 2 ng/ml EGF for 15 min before immunofluorescence against endogenous ATG16L1 and EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 puncta that colocalise with ATG16L1 (in E). Cells stably expressing Flag-S-ATG16L1 were treated for 1 h with either 100 μM monensin or 30 μM Dynasore, and then stimulated with 2 ng/ml EGF for 15 min. Cells were then stained by immunofluorescence against EEA1 and Flag tag. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 (in G). Untreated control or sgAtg7 cells, or control cells pre-treated with 100 μM monensin 1 h, stably expressing Flag-S-ATG16L1 were stimulated 20 ng/ml Alexa 555-EGF (555-EGF) for 15 min before fixation and immunofluorescence staining against Flag tag and EEA1. Cells were then imaged by structured illumination microscopy (SIM), and images were reconstructed in Nikon Elements software. Scale bar: 10 μm. Quantification of the percentage of EGF-EEA1 colocalised vesicles that stained triple-positive with ATG16L1 by SIM (in I). Due to the low-throughput nature of this assay, the following cell numbers were counted: control untreated (9 cells), sgAtg7#1 (10 cells), sgAtg7#2 (9 cells) and control + monensin (11 cells). Cells were treated with 2 ng/ml EGF for 15 min before immunofluorescence against endogenous WIPI2 and EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 puncta that colocalise with WIPI2 (in K). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two-tailed Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. The canonical autophagy machinery is required for early endosome targeting MEF cells stably expressing GFP-LC3 were serum starved for 4 h, then treated with 100 μM monensin for 1 h and stimulated with 2 ng/ml EGF for 15 min before immunofluorescence staining against EEA1. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with GFP-LC3 (in A). Box-and-whisker representation of the quantification of the number of EEA1-positive vesicles per cell in control and sgAtg7 cell lines. No significant differences are observed in early endosome numbers between control and autophagy-inhibited cells (control versus sgAtg7#2: P = 0.29, control versus sgAtg7#3: P = 0.43). Glial shNf-1/shTp53 control cells stably expressing Flag-S-ATG16L1 were either serum starved (-FBS) or treated with amino acid-free DMEM (-AA) for 4 h before immunofluorescence staining against Flag tag and EEA1. Scale bar: 10 μm. Glial shNf-1/shTp53 control and sgAtg7 cells stably expressing wild-type ATG16L1 (Flag-S-ATG16L1WT) or LAP-deficient K490A mutant of ATG16L1 (Flag-S-ATG16L1K490A) were serum starved for 4 h followed by stimulation with 2 ng/ml EGF for 15 min and immunofluorescence staining to detect Flag tag and EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles positive for Flag-S-ATG16L1 (in E) with quantification of endogenous ATG16L1 included (see Fig 4E). Atg16l1−/− MEF cells stably expressing wild-type (Flag-S-ATG16L1WT) or LAP-deficient mutant (Flag-S-ATG16L1K490A) of ATG16L1 were serum starved for 4 h followed by stimulation with 2 ng/ml EGF for 15 min and immunofluorescence staining to detect Flag tag and EEA1. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of the percentage of total EEA1 vesicles that stain positive for wild-type or the K490A point mutant of ATG16L1 expressed in Atg16l1−/− MEFs (in G). Atg13−/− MEF cells were treated for 1 h with 100 μM monensin and then stimulated with 2 ng/ml EGF for 15 min. ATG16L1 and EEA1 were then detected by immunofluorescence staining. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles positive for Flag-S-ATG16L1 (in I). Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two-tailed Student's t-test: NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint We further interrogated the recruitment of upstream autophagy players to early endosomes. Staining of EEA1 showed no significant colocalisation with endogenous ATG16L1 in autophagy-competent glial cells (Fig 4E and F) consistent with previously published findings 47. Similarly, induction of autophagy by amino acid starvation did not enhance the colocalisation of EEA1 and ATG16L1 in control cells (Fig EV3D). Intriguingly, a marked increase in the colocalisation between endogenous ATG16L1 and a subset of EEA1+ early endosomes was observed in ATG7-deficient glial cells (Fig 4E and F). This was also observed with Flag-S-tagged ATG16L1 (Fig EV3E and F), demonstrating that both endogenous and stably expressed ATG16L1 show comparable localisation 48. Enhanced colocalisation between ATG16L1 and EEA1 was also observed upon chemically induced endomembrane damage triggered by monensin or Dynasore treatment 46, 49, 50 (Fig 4G and H). This recruitment is less than that observed in ATG7-deficient cells potentially due to the transient localisation of early autophagy machinery during autophagosome maturation, which is arrested by ATG7 deletion 51. Importantly, structured illumination microscopy revealed that ATG16L1 localised to a subpopulation of EEA1+ early endosomes that were also positive for EGF in either sgAtg7- or monensin-treated cells (Fig 4I and J), suggesting that upstream autophagy players can recognise damaged early endosomes where EGFR is arrested. We explored the possibility that the autophagy machinery may be recruited to early endosomes through an LC3-associated phagocytosis (LAP)-like process which occurs on single membranes requiring the core autophagy machinery (such as ATG7 and ATG16L1) but not PI(3)P, WIPI2 or components of the ULK1 complex 52. Assessment of endogenous WIPI2 localisation revealed its positive staining on EEA1+ endosomes in sgAtg7 cells (Fig 4K and L). In addition, a mutant of ATG16L1 that cannot support non-canonical LC3 lipidation on single membranes (ATG16L1K490A) 52 was also recruited t