Fig. 3: Mutations in the VPS37A N-terminal hydrophobic membrane-binding motifs impair phagophore targeting of the ESCRT-I complex for autophagosome closure. | Communications Biology

Fig. 3: Mutations in the VPS37A N-terminal hydrophobic membrane-binding motifs impair phagophore targeting of the ESCRT-I complex for autophagosome closure.

From: Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure

Fig. 3

a–h VPS37A KO U-2 OS cells were stably transduced with GFP-tagged VPS37A wildtype (GFP-WT) or a mutant with both 3WLFP and 137FPYL motifs mutated to DDDD (GFP-HMsDM). In b, d the resultant cells were further transduced with pHuji-LC3 and HaloTag (HT)-LC3, respectively. a Immunoblot analysis of whole-cell lysates (input) and immunoprecipitates (GFP-Trap) from the indicated cells. b Confocal images of cells that were transfected with the indicated siRNAs for 45 h and starved for 3 h. Magnified images in the boxed areas are shown in the right panels. GFP-HMsDM signals detected on LC3-positive structures are indicated by dotted circles. Scale bars represent 10 μm, and 1 μm in the magnified images. c Quantification of GFP-VPS37A-positive foci area per cell in b (n ≥ 50) with two-way ANOVA followed by Tukey’s multiple comparisons. d Confocal images of cells that were starved for 3 h in the presence or absence of Bafilomycin A1 (BafA1) and subjected to the HT-LC3 autophagosome completion assay using the indicated membrane-impermeable (MIL) and -permeable (MPL) HaloTag ligands. Scale bars represent 10 μm. e Quantification of the MIL/MPL fluorescence intensity ratio for each cell in the starvation plus BafA1 treatment group (n = 50) with one-way ANOVA followed by Tukey’s multiple comparisons. The data shown are relative to the mean of GFP-WT-expressing cells. f Immunoblot analysis of cells that were starved for 3 h in the presence or absence of BafA1. p62 is a cargo degraded by autophagy, and LC3-II (a covalent conjugation form of LC3 to the amino headgroup of phosphatidylethanolamine lipid) is an autophagic marker. g, h Dot plots of LC3-II and p62 band intensities relative to the mean of untreated GFP-WT-expressing cells (g) and LC3-II and p62 degradation ratios relative to GFP-WT-expressing cells (h) in f (n = 3). Data in g and h are analyzed with two-way and one-way ANOVA followed by Tukey’s multiple comparisons, respectively. All values in the graphs are mean ± SD. ns, not significant.

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