Tracking the transition from an ATG9A vesicle to an autophagosome

Live-cell imaging is a well-established tool to gain insights into the dynamics of autophagosome formation. We have recently generated a panel of human cancer cell lines that express HaloTagged autophagy-related (ATG) proteins from their endogenous loci, providing a highly sensitive tool to study autophagosome biogenesis. Using these cell lines in combination with automated autophagic foci tracking we dissected the molecular mechanisms of phagophore initiation and expansion. Our work supports a model in which phosphorylations by the Unc-51-Like activating Kinases (ULK1/2) and phosphoinositide 3-kinases (PI3Ks) complexes trigger the recruitment of autophagy factors to mobile ATG9A-positive vesicles transforming them into phagophores. Only a small fraction, approximately 10–20%, of these phagophores expand into autophagosomes, suggesting that autophagosome biogenesis is inefficient. Finally, we demonstrate that ATG2A recruitment commits mobile phagophores to autophagosome formation via tethering to donor membranes and promoting the conjugation of human ATG8 homologues.