Journal: Communications Biology

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

doi: 10.1038/s42003-024-06026-7

Figure Lengend Snippet: a – h VPS37A KO U-2 OS cells were stably transduced with GFP-tagged VPS37A wildtype (GFP-WT) or a mutant with both 3 WLFP and 137 FPYL motifs mutated to DDDD (GFP-HMs DM ). 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-HMs DM 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.

Article Snippet: All other reagents were obtained from the following sources: bafilomycin A1 (LC Laboratories, B-1080); 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (BD Bioscience, 564907); digitonin (Sigma-Aldrich, D141); membrane-impermeable HaloTag Ligand (MIL) (Promega, Alexa Fluor 660-conjugated, G8471); membrane-permeable HaloTag Ligand (MPL) (Promega, tetramethylrhodamine-conjugated, G8251); normal goat serum (Sigma-Aldrich, G9023); paraformaldehyde (Electron Microscopy Sciences, 15710); Triton X-100 (Sigma-Aldrich, T8532); XF Plasma Membrane Permeabilizer (XF-PMP) (Seahorse Bioscience, 102504-100).

Techniques: Stable Transfection, Transduction, Mutagenesis, Western Blot, Transfection, Membrane, Fluorescence, Expressing, Conjugation Assay, Marker