Figure 4.

VAMP7 Regulates Atg16L1 Vesicle Size

(A) HeLa cells were transfected for 20 hr with LC3-CFP (red) and Atg16L1-GFP (green). Confocal colocalization between LC3-CFP and Atg16L1-GFP is shown in white on the image. Yellow arrows show colocalization between LC3 and Atg16L1; white arrows indicate Atg16L1 vesicles with no LC3. Scale bar, 5 μm.

(B) Size distributions of total Atg16L1 vesicles and Atg16L1⁺/LC3⁺ vesicles from images taken in (A) using ImageJ. n = 5 cells from two independent experiments.

(C) HeLa cells transfected with control or VAMP7 siRNA for 4 days were transfected for 20 hr with Atg16L1-GFP. Representative confocal images are shown. Scale bar = 5 μm.

(D) Atg16L1-GFP vesicle sizes from (C). Mean ± SEM of the average size of Atg16L1-GFP vesicles (A.U.: arbitrary unit); three independent experiments; at least 2000 cells were automatically analyzed.

(E) Size distribution of Atg16L1-GFP vesicles from images taken in (C) using ImageJ. n = 4 cells for control and VAMP7 knockdown; two independent experiments.

(F) HeLa cells transfected with control or VAMP7 siRNA for 5 days were starved for 4 hr. Size distribution of endogenous Atg16L1 vesicles is shown. n = 20 cells for control and VAMP7 knockdown; two independent experiments.

(G) Representative images from the experiment described in (F). Arrows indicate endogenous Atg16L1 vesicles. Scale bar, 5 μm.

(H) HeLa cells transfected with control or VAMP7 siRNA for 4 days were transfected for 20 hr with Atg16L1-GFP. The cells were processed for immunogold labeling with anti-GFP antibodies (15 nm gold particles) for electron microscopy. We observed Atg16L1-positive tubular structures in control cells, but not in VAMP7 knockdown cells (right-hand micrographs). Data represent mean ± SEM of the area of Atg16L1-GFP “cluster” (nm²) and the average diameter of individual Atg16L1-GFP vesicles (nm). n = 13 clusters for control. n = 19 clusters for VAMP7^KD. n = 100 single vesicles for control. n = 100 single vesicles for VAMP7^KD.

See also Figure S4.