Live cell imaging to study the assembly and fate of autophagosomes

Abstract

Autophagy (formerly macroautophagy) is a critical process that occurs in all Eukaryotes. Induction of the pathway results in formation of multilamellar membrane-delimited structures that engulf cytosolic proteins, organelles and intracellular pathogens en masse. Capture and catabolism are integral to the diverse roles of autophagy in recycling components, degrading aggregate-prone proteins, removing damaged organelles, depleting cells of organelles and cytosolic mass, and isolating intracellular pathogens. Despite recent attention, fundamental questions about autophagy remain. How do autophagosomes form? What exact roles do they play in non-starvation conditions? To investigate these topics, I developed live-cell imaging approaches to identify substrates and turnover rates of autophagosomes, and to survey intracellular membranes for putative roles in autophagosome formation. The following studies show that autophagosomes utilize lipid derived from the mitochondria during their formation, and that this is a unique aspect of starvation-induced autophagy. During formation, autophagosomal markers transiently localize to punctae on the surface of mitochondria. A tail-anchored outer mitochondrial membrane protein freely diffuses into the newly forming autophagosome until the two organelles dissociate. Starvation-induced autophagosomes produced in this manner engulf cytosolic contents, are 3-MA-sensitive and persist only transiently in cells before fusing with lysosomes. These findings reveal that the outer mitochondrial membrane serves as a major membrane source for autophagosome biogenesis during starvation. Furthermore, the data define a new intracellular pathway from mitochondria to the autophagosomal/lysosomal system. Following, Chapter 1 provides a general survey of autophagy, Chapter 2 discusses advances in live-cell imaging and its use to identify autophagosome substrates, Chapter 3 discusses monitoring autophagosome turnover by photo pulse-labeling in live cells, Chapter 4 presents data implicating the use of mitochondrial membrane in the biogenesis of autophagosomes, and Chapter 5 reviews the implications of the data herein.