Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling

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.

A Western blot analyses of shNf-1/shTp53 glial cells expressing gRNA sequences targeting Atg16l1. B 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 lm. C 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 lm. D Quantification of PI(3)P + vesicles per cell and the Pearson's colocalisation coefficient between PI(3)P and EEA1 (in C). E 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. F Atg16l1 À/À MEFs were reconstituted with wild type (WT) ATG16L1 or a mutant of ATG16L1 containing a deletion in the ATG5 binding domain (residues 1-39, DA5).
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. Figure EV3. The canonical autophagy machinery is required for early endosome targeting.
A MEF cells stably expressing GFP-LC3 were serum starved for 4 h, then treated with 100 lM 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 lm. B Quantification of percentage of total EEA1 vesicles that colocalise with GFP-LC3 (in A). C 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). D 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 lm. E Glial shNf-1/shTp53 control and sgAtg7 cells stably expressing wild-type ATG16L1 (Flag-S-ATG16L1 WT ) or LAP-deficient K490A mutant of ATG16L1 (Flag-S-ATG16L1 K490A ) 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 lm. F Quantification of percentage of total EEA1 vesicles positive for Flag-S-ATG16L1 (in E) with quantification of endogenous ATG16L1 included (see Fig 4E). G Atg16l1 À/À MEF cells stably expressing wild-type (Flag-S-ATG16L1 WT ) or LAP-deficient mutant (Flag-S-ATG16L1 K490A ) 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 lm. H Quantification of the percentage of total EEA1 vesicles that stain positive for wild-type or the K490A point mutant of ATG16L1 expressed in Atg16l1 À/À MEFs (in G). I Atg13 À/À MEF cells were treated for 1 h with 100 lM 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 lm. J 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.  Figure EV4. Targeting of EGFR to late endosomes and Rab4 + recycling endosomes remain intact in the absence of autophagy.
A Cells were treated with 20 ng/ml Alexa 555-EGF (555-EGF) for the indicated times before fixation. 555-EGF was detected using the ImageXpress high-content imaging platform, and representative images of 555-EGF are shown. Scale bar: 100 lm. B Following high-content imaging of 555-EGF and DAPI, 555-EGF uptake relative to cell number was quantified and made relative in each cell line to background readings from unstimulated cells (t = 0 min) (in A). C Control or sgAtg7 glial shNf-1/shTp53 cells were either untreated, serum starved (-FBS) for 4 h, or amino acid starved (-AA) for 2 h and LysoSensor probe added for 1 h. Shown are quantifications of LysoSensor fluorescence intensity per cell normalised to untreated control cells. D Cells were stimulated with 20 ng/ml EGF for the indicated times before EGFR levels were analysed by Western blotting. E Densitometry analyses of Western blotting showing EGFR degradation with measurements made relative to EGFR levels in unstimulated cells (t = 0 min) (in D). F Cells were stimulated with 20 ng/ml 555-EGF for 15 min before immunofluorescence staining against endogenous Rab7. White arrows indicate colocalisation. Scale bar: 10 lm. G Quantification of Pearson's colocalisation coefficient between 555-EGF and Rab7 (in F). H Schematic representation of cell surface protein biotinylation assay to assess endocytosis and recycling rates (see Fig 6C and D). I Cells stably expressing mCherry-Rab4 were stimulated with 20 ng/ml EGF for 5 or 15 min and then fixed and stained by immunofluorescence against EGFR. White arrows indicate colocalisation. Scale bar: 10 lm. J Quantification of Pearson's colocalisation coefficient between mCherry-Rab4 and EGFR (in I). K Cells were stimulated with Alexa 555-transferrin (555-Tfn, 20 ng/ml) for 15 min and chased with media without transferrin for 15 min. Cells were then stained by immunofluorescence against Rab11. Scale bar: 10 lm. L Quantification of Pearson's colocalisation coefficient between 555-Tfn and Rab11 (in K).
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. ◀ Figure EV5. Analyses of EGFR signalling and levels in various cell lines upon autophagy inhibition.
A Glial shNf-1/shTp53 sgAtg3 cells were either left untreated (+serum) or serum starved for 4 h and then stimulated with 2 ng/ml EGF for 15 min before analysis by Western blotting. B Glial shNf-1/shTp53 shAtg13 cells were serum starved for 4 h and then stimulated with 2 ng/ml EGF for 15 or 30 min before analysis by Western blotting. C Glial EGFRvIII-expressing control and sgAtg7 cells were serum starved for 4 h and then stimulated with 2 ng/ml EGF before analysis by Western blotting. D Atg16l1 À/À MEFs were reconstituted with wild-type (WT) ATG16L1 or with a mutant of ATG16L1 with deletion of the ATG5-binding domain (residues 1-39, DA5).
Cells were either untreated or serum starved for 4 h and then stimulated with 2 ng/ml EGF for 10 or 30 min before signalling pathway analyses by Western blotting. E Total EGFR levels in untreated sgAtg7 and sgAtg3 MEFs were analysed by Western blotting. F-I The following cells were serum starved for 4 h followed by stimulation with 2 ng/ml EGF before Western blot analyses: HT29 control and crATG7 (F); MCF-7 control and sgATG5 (G); A549 control and sgATG5 (H); and U2OS control and sgATG5 (I). J Representative images of propidium iodide staining in control or sgAtg7 glial shNf-1/shTp53 or EGFRvIII-expressing cells that were serum starved for 24 h in the presence or absence of 20 ng/ml EGF. Scale bar: 100 lm. K Quantification of the experiment in J with propidium iodide relative fluorescence units (RFU) made relative to cell number as assessed by DAPI RFU measured by a fluorescence plate reader. These values were then made relative to the values in the negative control setting (untreated cells, UT).
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.