During macroautophagy (hereafter referred to as autophagy), a crescent-shaped membrane known as the phagophore elongates to engulf damaged cargo and seals to generate a double-membrane vesicle called the autophagosome. While much of the core machinery regulating autophagy induction and phagophore nucleation and elongation has been identified, the machinery for phagophore closure is largely unknown. The goal of this application is to define the molecular mechanism by which the phagophore seals to generate the completed autophagosome. Phagophore closure is a critical step in autophagy that is required for lysosomal fusion and efficient degradation and recycling of autophagic cargo. The sealing of phagophores has misguidedly been described as a membrane fusion event when the process of generating the inner and outer autophagosomal membrane actually requires membrane scission. Notably, the topology of phagophore closure is similar to ESCRT (endosomal sorting complexes required for transport)-mediated scission, in which the ESCRT machinery mediates membrane fission from within the membrane neck. While ESCRT defects accumulate immature autophagosomal structures in C. elegans, Drosophila and mammals, a molecular understanding for the phenotype remains unclear. Exploiting the topology of LC3 during autophagosome biogenesis, we established a novel HaloTag-LC3 based assay to differentiate phagophores, autophagosomes and autolysosomes. Using this assay, we have identified ESCRT-III as a candidate regulator of phagophore closure. We hypothesize that phagophore-associated factors assemble a novel adaptor for the recruitment and assembly of ESCRT-III filaments for phagophore closure. We will test our hypotheses in the following Specific Aims: (1) to define the core ESCRT machinery required for phagophore closure; (2) to identify the upstream factor(s) that serve as an autophagy-specific adaptor for ESCRT-III assembly during phagophore closure.
Autophagy is an intracellular recycling process during which a cup-shaped membrane expands around damaged material and seals to form a double-membrane vesicle known as the autophagosome. While much of the core machinery regulating the early steps of autophagosome biogenesis has been characterized, the mechanism by which the membrane seals to generate the completed autophagosome is unknown and, here, we seek to elucidate this process. As autophagy maintains cellular homeostasis in response to stress and dysregulated autophagy is implicated in the pathogenesis of many human diseases, including neurodegeneration, cancer, heart disease, liver disease, immunity, and aging, understanding the molecular mechanism of autophagy is of great importance.
