Figure 7.
Autophagy Stimulation Induces Atg16L1 Vesicle Homotypic Fusion
(A) HeLa cells transiently transfected for 20 hr with Atg16L1-GFP were treated as indicated with rapamycin (1 μg/ml) for 1 hr or starved for 1 hr. Representative confocal pictures are shown. Scale bars, 5 μm.
(B) Quantification of the number of Atg16L1-GFP vesicles per cell using ImageJ from the pictures obtained in (A). Mean ± SD of the number of Atg16L1-GFP vesicle per cell; three independent experiments; n = 20 cells per experiment.
(C) HeLa cells transiently transfected for 20 hr with Atg16L1-GFP were treated as indicated with rapamycin (1 μg/ml) for 1 hr or starved for 1 hr and subjected to live-cell imaging. Data are mean ± SD of the number of fusion events per cell in 10 min from two independent experiments. n = 5 cells for untreated condition, n = 7 cells for starvation condition, and n = 5 cells for rapamycin condition.
(D) The velocity of the Atg16L1-GFP vesicles from (C) using ImageJ manual tracking plugin. Means ± SEM of the velocity of ten Atg16L1 vesicles.
(E) Model of Atg16L1 precursor maturation. Atg16L1 precursors (Atg16L1+/LC3−) formed, in this example, from the plasma membrane undergo homotypic fusion via a VAMP7-containing SNAREs complex. Atg16L1 precursor homotypic fusion leads to LC3 acquisition and therefore maturation toward phagophores (Atg16L1+/LC3+) and fully formed autophagosomes (Atg16L1−/LC3+).