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

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.

AMPK Regulates Phagophore-to-Autophagosome Maturation

“…In fluorescence microscopy images of eukaryotic cells, autophagosomes appear as bright cytoplasmic puncta, also referred to as foci 35 . Using time-lapse imaging, it is possible to monitor the rate of foci formation, their lifetime and mobility 9 . These metrics provide valuable insights into the dynamics of autophagic flux and the molecular mechanisms underlying autophagosome biogenesis.…”

“…These metrics provide valuable insights into the dynamics of autophagic flux and the molecular mechanisms underlying autophagosome biogenesis. To study autophagy in human cancer cells, we have developed a cell line panel in which the HaloTag is introduced at the endogenous loci of the autophagy factors ATG13, ULK1, WIPI2 and ATG2A 9 . This panel of cell lines provides a sensitive tool to detect autophagosome formation.…”

“…The autophagosome is a vesicle enclosed by two lipid membranes, and its initiation and assembly are tightly controlled by autophagy genes (ATGs) 1 . One model for autophagosome biogenesis proposes that ATG9 containing vesicles serve as the seed for recruitment of other autophagy factors 9–11 . ATG9 is the only known transmembrane protein among the ATGs and has lipid scramblase activity, transferring phospho-lipids between the two lipid bilayer leaflets 11,12 .…”

“…ATG9 is the only known transmembrane protein among the ATGs and has lipid scramblase activity, transferring phospho-lipids between the two lipid bilayer leaflets 11,12 . Mobile ATG9 vesicles are specified to form autophagosomes by a phospho-lipid signaling cascade, enabling the recruitment of autophagy machinery required for autophagosome maturation 9–11 . Key maturations factors include the scaffold protein WIPI2 and the ATG16L1-ATG12-ATG5 complex, which acts like a ubiquitin ligase conjugating phosphatidylethanolamine to ATG8-like proteins (LC3, GABARAP) 1,13,14 .…”

“…To systematically analyze how amino acids, glucose, and growth factors regulate phagophore initiation and maturation into autophagosomes, we used our recently developed collection of cell lines expressing HaloTagged autophagy factors from their endogenous loci 9 . This approach allows us to analyze autophagic flux by determining the initiation and maturation kinetics of autophagosomes in living cells, avoiding artifacts due to protein overexpression 32,33 .…”