Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology

First Advisor

Melia, Thomas

Abstract

Autophagosomes are transient organelles which grow from a seed vesicle to encapsulate a target cargo and subsequently fuse with the lysosome to degrade its contents. This process occurs rapidly and requires the concerted effort of a host of unique membranes and proteins in a complicated cascade, which I detail in chapter I. Deletion of the lipid transport proteins ATG2A and ATG2B results in the accumulation of most of the preceding autophagic membranes and proteins in a large compartment. The ATG2 DKO compartment is advantageous as a model for the phagophore assembly site (PAS) for the following reasons: it is not transient, it is much larger than the WT PAS, and it captures the phagophore (or nascent autophagosome) in an early, immature state that is otherwise hard to image. Using this tool, I have identified and characterized the membranes which directly contribute to autophagosome biogenesis. In chapter II of this dissertation, I discovered using unbiased proximity labeling and genetic silencing techniques that RAB1 and ARFGAP1 positive membranes localize to the PAS and are essential for autophagosome biogenesis. These ERGIC proteins colocalize in ATG2 DKO cells with autophagy enzymes which function on the phagophore, suggesting that ERGIC membranes are recruited at an early stage of autophagosome biogenesis. In chapter III, I increased the functional resolution at the ATG2 DKO compartment by developing two tools which lead to the segregation of autophagic membranes. Using this increased resolution, I unambiguously identified and partially characterized the proteome of four membrane types (all identified for the first time using transmembrane proteins) involved in autophagosome biogenesis through a combination of light and electron microscopy. The first three membranes – ATG9A vesicles, STX17/STX16 containing protophagophores, and VAMP4 vesicles – were characterized using the first tool, RavZ C258A, which shields ATG8 proteins from interacting with their native partners. Overexpression of this protein sequesters ATG8 containing membranes into one group, leaving only the small VAMP4 positive vesicles in a remaining matrix of cargo adaptors. Under these conditions, the ATG9A signal colocalized with the SNARE proteins STX17 and STX16 and corresponded exclusively to cup shaped protophagophores, suggestive of a fusion event. The fourth membrane – a subdomain of the ER positive for VAPA and lipid synthesis enzymes – was characterized by the overexpression of FIP200, which results in the invasion of this membrane into the ATG2 DKO compartment. These findings suggest that phagophores expand through a mixture of vesicle fusion and lipid transport. In chapter IV, I demonstrate that LRRK2, a protein which is commonly mutated in Parkinson’s Disease, binds the autophagic cargo adaptor P62 when in the active conformation. When overexpressed in ATG2 DKO cells, LRRK2 localizes to the ATG2 DKO compartment whereas it is cytosolic in WT cells. Perturbations which led to an increased concentration of the active conformation led to an enrichment of LRRK2 in the ATG2 DKO compartment whereas the inactive conformation remained cytosolic. Furthermore, locking overexpressed LRRK2 in the active conformation leads to the polymerization of LRRK2 on microtubules, to which structures P62 is also recruited. In summary, this dissertation leverages the ATG2 DKO compartment to establish a framework for how diverse membranes and proteins organize at the PAS. This work reshapes our understanding of the earliest stages of autophagosome formation by revealing RAB1 membranes as a source for autophagosome biogenesis, resolving the identities and functions of four distinct membrane populations, and defining a mechanistic relationship between LRRK2 and P62. These findings clarify longstanding questions about membrane origin and phagophore expansion and open new conceptual directions for connecting autophagy to broader cellular processes and disease.

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