Systematic Analysis of Autophagy Identifies Atg9 Vesicles as the Origin of the Phagophore

Abstract

Autophagy is a catabolic pathway required for the clearance and recycling of cytoplasmic materials. Upregulation and dysfunction of autophagy contributes to the pathology of cancer and neurogenerative diseases, respectively. To define the molecular mechanisms that control autophagic flux it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of 9 cell lines expressing HaloTagged autophagy factors from their endogenous loci, we systematically analyze the abundance, single-molecule dynamics, and autophagosome association kinetics of a wide variety of autophagy proteins involved in the initiation and maturation of the autophagosome. Our results reveal that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles and tethering of these ATG9 vesicles to donor membranes by ATG2 is a key step in phagophore maturation. In addition, we demonstrate that the overall lifetime of an autophagosome is approximately 160 seconds and the majority of phagophore initiation events fail to produce mature autophagosomes. In total our work establishes a new experimental framework to quantitatively analyze autophagy and demonstrates that ATG9 vesicles are the seeds for autophagosome formation in human cells.

Membrane Contact Sites in Autophagy

“…The PAS is formed in between the vacuole and the ER, while the omegasomes are a subdomain of the ER enriched in phosphatidylinositol-3-phosphate (PtdIns3P) ( Figure 2 B,C) [ 29 , 32 , 33 ]. The concerted action of the Atg1/ULK kinase complex, Atg9/ATG9A-containing vesicles and the PI3K complex at these sites leads to the nucleation of a membranous cistern known as the phagophore or isolation membrane ( Figure 2 A) [ 19 , 20 , 34 , 35 , 36 , 37 ]. The seeding membranes for the phagophore nucleation are not well characterized, and, in addition to the Atg9/ATG9A-containing vesicles, it is likely that several membrane sources contribute, including vesicles derived from the ER, the ER-to-Golgi intermediate (ERGIC) and/or the recycling endosomes [ 35 , 36 , 37 , 38 , 39 , 40 ].…”

“…The concerted action of the Atg1/ULK kinase complex, Atg9/ATG9A-containing vesicles and the PI3K complex at these sites leads to the nucleation of a membranous cistern known as the phagophore or isolation membrane ( Figure 2 A) [ 19 , 20 , 34 , 35 , 36 , 37 ]. The seeding membranes for the phagophore nucleation are not well characterized, and, in addition to the Atg9/ATG9A-containing vesicles, it is likely that several membrane sources contribute, including vesicles derived from the ER, the ER-to-Golgi intermediate (ERGIC) and/or the recycling endosomes [ 35 , 36 , 37 , 38 , 39 , 40 ]. The PtdIns3P synthesized at the phagophore by the PI3K complex is key for the subsequent recruitment of the rest of the ATG machinery.…”