Fig. 5: Elevation of WIPI1 protein abundance enhances phagophore formation.

a Correlative light (left panel) electron microscopy (middle, right panel) of U2OS cells stably expressing GFP-WIPI1. Elongated, perinuclear autophagic membranes decorated with WIPI1 (left panel) were found to represent the presence of multiple phagophore-like membranes (indicated with white arrows, right panel). Scale bars: 20 μm (left panel), 5 μm (middle panel), 2 μm (right panel). b Time-course analyses of WIPI1 puncta formation after transient inhibition of PI3P synthesis. Stable U2OS GFP-WIPI1 cells were pretreated with LY294002 (100 µM) for 3 h, followed by starvation for 5, 15, 30, 60, 90 min as indicated. Representative images were acquired by confocal LSM (scale bar: 20 µm). The numbers of WIPI1 puncta cells (middle panel) and the numbers of cells harbouring elongated, perinuclear autophagic membranes decorated with GFP-WIPI1 (right panel) were quantified by fluorescence microscopy (one-way ANOVA with Dunnett’s post-hoc test, mean ± SD, 300 cells from n = 3). c Stable U2OS GFP-WIPI1 cells were starved and analysed by live-cell microscopy. Still images from Supplementary Video 1 displaying omegasome-like structures decorated with GFP-WIPI1 are displayed. d GFP-WIPI1-positive phagophores are sensitive to the inhibition of PI3P synthesis. U2OS cells stably expressing GFP-WIPI1 were starved with or without bafilomycin A1 (BafA1) and subjected to live-cell microscopy. Approximately 3 h after autophagy induction by starvation, LY294002 (100 µM) was added. The disappearance of WIPI1-positive perinuclear phagophores after LY294002 treatment was determined by live-cell microscopy and representative still images are shown (left panels, scale bar: 20 µm). Quantifications are provided in the right panels (Welch’s t test, mean ± SD, n = 8 videos for starved conditions, n = 5 videos for starved/BafA1 conditions). e Rhodamine-PE GUVs containing PI(3)P (representative images, left panels, scale bar: 5 µm) or PI(3,5)P₂ (images shown with controls in Supplementary Fig. 7) were incubated with native protein extracts from U2OS cells stably expressing GFP-WIPI1 or GFP, along with parental U2OS cells, followed by confocal LSM imaging. CellProfiler-based image analysis was used to measure the GFP intensity on GUV edges (right panels, Kruskal–Wallis with Dunn’s post-hoc testing, up to 404 GUVs analysed from n = 3 for each condition). Supplementary material is available (Supplementary Fig. 7; Supplementary Video 1; Supplementary Data 1). P values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant.