Figure 8. Membrane atg8ylation and endolysosomal homeostasis affect retromer-dependent sorting.

(A–C) High content microscopy (HCM) quantification of GLUT1 (immunostaining of endogenous protein) puncta/cell (B) and GLUT1 colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area), (C) in U2OS^WT and U2OS^ATG2A/B-DKO cells treated with or without LLOMe (100 µM) for 45 min. Scale bar, 20 μm. Data, means ± SE (n = 6); one-way ANOVA with Tukey’s multiple comparisons. (D–F) HCM quantification of GLUT1 (immunostaining of endogenous protein) puncta/cell (E) and GLUT1 colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area), (F) in Huh7^WT cand Huh7^VPS37A-KO cells treated with or without LLOMe (100 µM) for 45 min. Scale bar, 20 μm. Data, means ± SE (n = 6); one-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line/condition. (G) Schematic: Membrane atg8ylation maintains membrane homeostasis under basal or stress conditions and, independently of canonical autophagy, affects retromer function. A functional atg8ylation apparatus is required for proper sorting of the retromer cargo GLUT1.

Figure 8—figure supplement 1. High content microscopy (HCM) images of GLUT1.

(A) HCM representative images of GLUT1 puncta; immunostaining of endogenous protein in U2OS^WT and U2OS^ATG2A/B-DKO cells upon treatment with LLOMe (100 µM) for 45 min. (B) HCM representative images of GLUT1 puncta in Huh7^WT and Huh7^VPS37A-KO cells upon treatment with LLOMe (100 µM) for 45 min.

Figure 8—figure supplement 2. Effects of ATG5 knockout on MPR sorting.

(A) Pulse-chase procedure for monitoring CI-MPR trafficking following CI-MPR antibody uptake. (B, C) High content microscopy (HCM) quantification and representative images of Ab-CI-MPR colocalization with TGN46 after 30 min of intracellular sorting in HeLa^WT, HeLa^ATG5-KO, and HeLa^VPS35-KO cells. Yellow masks represent machine-assigned colocalization of Ab-CI-MPR and TGN46. Data, means ± SE (n = 3); one-way ANOVA with Tukey’s multiple comparisons. (D) Confocal images of Ab-CI-MPR antibody localization relative to TGN46 in HeLa^WT, HeLa^ATG5-KO, and HeLa^VPS35-KO cells. (E, F) HCM quantification and representative images of Ab-CI-MPR colocalization with TGN46 after 30 min of intracellular sorting in Huh7^WT and Huh7^ATG5-KO cells. Data, means ± SE (n = 3); unpaired t-test. Each experiment (independent biological repeats; n = 3) consists of machine-identified 500 valid primary objects/cells per well, ≥5 wells/sample. All data collection, processing (object, region of interest [ROI], and target mask assignments) and analyses were computer driven independently of human operators.

Figure 8—figure supplement 3. ATG5 affects retromer assembly.

(A, B) High content microscopy (HCM) quantification of LC3 puncta in Huh7^WT, Huh7^ATG3-KO, Huh7^ATG5-KO, Huh7^ATG7-KO, and Huh7^VPS35-KO cells induced for autophagy in EBSS for 90 min. Plot in C, an example of a whole 96-well plate HCM readout; X-axis, well positions; Y-axis, HCM parameter quantified. Plot in D, nested data from three different plates each one as in C. FM, full medium; EVSS, starvation medium. Data, means ± SE (n = 3); one-way ANOVA with Tukey’s multiple comparisons. Coimmunoprecipitation (co-IP) analysis (C) and quantification (D) of VPS35 and YFP-VPS29 interaction in HeLa^WT, and HeLa^ATG5-KO cells. Data, means ± SE (n = 3), one-way ANOVA with Tukey’s multiple comparisons. Co-IP analysis (E) and quantification (F) of VPS26 and YFP-VPS29 interaction in HeLa^WT, and HeLa^ATG5-KO cells. Data, means ± SE (n = 3); unpaired t-test.