LC3B-positive vesicles accumulate in large clusters in ATG2 DKO cells. (A) Model for autophagosome growth via direct lipid transport by ATG2. ATG2 moves lipids from a donor membrane to the phagophore membrane, where ATG9 scrambles the lipids to populate both leaflets of the growing bilayer. This model requires that ATG9 be integrated in the expanding phagophore. (B) CLEM combined with FIB-SEM in ATG2 DKO HEK293 cells revealed two accumulations of GFP-LC3B signal (white arrow and inset) that correlated to the site of accumulated vesicles (white arrow on the cell of FIB-SEM image). Scale bars: 5 µm. (C) 3D segmentation of two vesicle clusters. Top: Two vesicle clusters from FIB-SEM (B) at high magnification showing ER structures (red arrows) surrounding the vesicle clusters. Middle: Segmented small vesicle clusters. Bottom: Vesicle clusters with abundant ER contacts (yellow). Scale bar: 250 nm. (D) More FIB-SEM-imaged small vesicle clusters (red arrows) are correlated to the GFP-LC3B positive signals (inset, red arrows). Scale bar: 1 µm. Inset scale bar: 1 µm. (E) 3D segmentation of the vesicle accumulation from the blue box in D. Left: Abundant ER structures are closely surrounding the vesicle cluster (red arrows) on the FIB-SEM image. Middle and right: 3D models are showing the vesicle cluster encaged by the ER structure. Scale bars: 500 nm.