Figure 2.
Clustering of autophagy initiation factors, ATG9A, and LC3B-II to vesicles and cup-like pre-phagophores in the absence of ATG2. (A) TEM reveals morphologies of clustered membranes in ATG2 DKO cells and nearby surrounding ER (black arrows). Zoomed fields at the bottom highlight critical features. Left: Heterogenous small vesicles embedded in a dense matrix in the center of the cluster. Center: Cup-shaped membranes at the periphery of the cluster (red arrows in the main field). Right: Focal accumulation of ferritin particles around peripheral vesicles (yellow asterisks in the main field). Scale bar: 500 nm. Inset scale bars: 100 nm. (B) Immunofluorescence of ATG2 DKO cells comparing the distribution of autophagy biogenesis factors ATG9A, p62, WIPI2, and FIP200 with LC3B. ATG9A, p62, and LC3B span the breadth of each fluorescence cluster as demonstrated with individual line scans, while WIPI2 and FIP200 are enriched at the periphery of each fluorescence cluster. Single slices from confocal Z-stacks. All line scans correspond to the white dashed line in the insets. Scale bars: 10 µm. Inset scale bars: 3 µm. (C) Immunofluorescence of GFP-LC3B expressing WT HEK293 cells showing endogenous localization of ATG9A in relation to LC3B (white arrows) in control and bafilomycin A1 treated cells (0.1 µM for 2 h). Maximum intensity projections of AiryScan confocal images. Scale bars: 10 µm. Inset scale bars: 3 µm. (D) Immunofluorescence of endogenous LC3B in WT and ATG9A KO HEK293 cells. The ATG9A KO does not show bafilomycin A1-dependent accumulation of autophagosomes, nor support LC3B-II formation (Fig. S2 A). Maximum intensity projections of confocal images. Scale bars: 10 µm.

Clustering of autophagy initiation factors, ATG9A, and LC3B-II to vesicles and cup-like pre-phagophores in the absence of ATG2. (A) TEM reveals morphologies of clustered membranes in ATG2 DKO cells and nearby surrounding ER (black arrows). Zoomed fields at the bottom highlight critical features. Left: Heterogenous small vesicles embedded in a dense matrix in the center of the cluster. Center: Cup-shaped membranes at the periphery of the cluster (red arrows in the main field). Right: Focal accumulation of ferritin particles around peripheral vesicles (yellow asterisks in the main field). Scale bar: 500 nm. Inset scale bars: 100 nm. (B) Immunofluorescence of ATG2 DKO cells comparing the distribution of autophagy biogenesis factors ATG9A, p62, WIPI2, and FIP200 with LC3B. ATG9A, p62, and LC3B span the breadth of each fluorescence cluster as demonstrated with individual line scans, while WIPI2 and FIP200 are enriched at the periphery of each fluorescence cluster. Single slices from confocal Z-stacks. All line scans correspond to the white dashed line in the insets. Scale bars: 10 µm. Inset scale bars: 3 µm. (C) Immunofluorescence of GFP-LC3B expressing WT HEK293 cells showing endogenous localization of ATG9A in relation to LC3B (white arrows) in control and bafilomycin A1 treated cells (0.1 µM for 2 h). Maximum intensity projections of AiryScan confocal images. Scale bars: 10 µm. Inset scale bars: 3 µm. (D) Immunofluorescence of endogenous LC3B in WT and ATG9A KO HEK293 cells. The ATG9A KO does not show bafilomycin A1-dependent accumulation of autophagosomes, nor support LC3B-II formation (Fig. S2 A). Maximum intensity projections of confocal images. Scale bars: 10 µm.

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