Tracking the transition from an ATG9A vesicle to an autophagosome

ABSTRACT

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.

Abbreviations

ATG: autophagy-related proteins; ULK1/2: Unc-51-Like activating Kinases; PI3Ks: Phosphoinositide 3-Kinases; ATG2A: autophagy-related protein 2A; ATG5: autophagy-related protein 5; ATG16: autophagy-related protein 16; ATG8: autophagy-related protein 8; U2OS: human bone osteosarcoma epithelial cell; LC3B: microtubule-associated protein 1A/1B Light Chain 3B; GABARAPL1: GABA type A Receptor-Associated Protein Like 1; ATG9A: autophagy-related protein 9A; ATG13: autophagy-related protein 13; SQSTM1: Sequestosome-1/p62; WIPI2: WD repeat domain, Phosphoinositide Interacting 2; PI3P: Phosphoinositide-3-phosphate.

KEYWORDS:

• Autophagy
• ATG9A vesicles
• single-particle tracking
• genome editing
• quantitative imaging

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by a grant from the National Institutes of Health (DP2 GM142307) to J.C. Schmidt. J.C. Schmidtwas a Damon Runyon Dale F. Frey Scientist supported (in part) by the Damon Runyon Cancer Research Foundation (DFS-24-17).