Quantitative analysis of autophagy reveals the role of ATG9 and ATG2 in autophagosome formation

Author:

Broadbent David G.123ORCID,Barnaba Carlo1ORCID,Perez Gloria I.1ORCID,Schmidt Jens C.14ORCID

Affiliation:

1. Institute for Quantitative Health Science and Engineering, Michigan State University 1 , East Lansing, MI, USA

2. College of Osteopathic Medicine, Michigan State University 2 , East Lansing, MI, USA

3. Michigan State University 3 Department of Physiology, , East Lansing, MI, USA

4. Michigan State University 4 Department of Obstetrics and Gynecology, , East Lansing, MI, USA

Abstract

Autophagy is a catabolic pathway required for the recycling of cytoplasmic materials. To define the mechanisms underlying autophagy it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of cell lines expressing HaloTagged autophagy factors from their endogenous loci, we analyzed the abundance, single-molecule dynamics, and autophagosome association kinetics of autophagy proteins involved in autophagosome biogenesis. We demonstrate that autophagosome formation is inefficient and ATG2-mediated tethering to donor membranes is a key commitment step in autophagosome formation. Furthermore, our observations support the model that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles, and that the ULK1 complex and PI3-kinase form a positive feedback loop required for autophagosome formation. Finally, we demonstrate that the duration of autophagosome biogenesis is ∼110 s. In total, our work provides quantitative insight into autophagosome biogenesis and establishes an experimental framework to analyze autophagy in human cells.

Funder

National Institutes of Health

Damon Runyon Cancer Research Foundation

Publisher

Rockefeller University Press

Subject

Cell Biology

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