Roles of macroautophagy in Drosophila development

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Macroautophagy, hereafter referred to as autophagy (= “self-eating”), is an evolutionarily conserved and ubiquitous cellular catabolic process responsible for the degradation and recycling of long-lived proteins and organelles. Autophagy enables cell survival during nutrient starvation and other stress conditions, and plays a role in differentiation, tissue remodeling, aging, and immunity. Recent studies have also revealed links between autophagy and lipid metabolism. However, the majority of studies to date have been limited to single cell or cell culture model systems, and the interactions between autophagy and lipid metabolism in complex tissues and organisms during normal development remain ill defined. The long term objective of this research program is to elucidate the functions and regulation of autophagy in normal development in the context of a whole organism. To achieve this goal, we will employ the model system, Drosophila melanogaster, which has evolutionarily conserved autophagy related (Atg) genes. The primary objective of this proposal is to investigate the relationships between autophagy and lipid metabolism in Drosophila. We recently discovered D-TNFAIP8, a novel regulator of both autophagy and lipid metabolism, and here we propose to determine its mechanism of action. We will test our hypothesis that D-TNFAIP8 functions to regulate lipophagy, the degradation of lipids by autophagy. In addition we will utilize our newly developed autophagy tools to identify new Drosophila genes that modulate lipophagy in vitro and in vivo. Directed toward testing our hypothesis and achieving our objectives, we have three specific aims: Aim 1: Determine the roles of D-TNFAIP8 in autophagy and lipid metabolism in multiple cell types and tissues. In this aim we will determine at what step in the autophagy pathway D-TNFAIP8 functions (e.g. autophagosome formation and/or lysosomal turnover) and if D-TNFAIP8 affects lipid biogenesis, storage and/or catabolism. We will determine whether its function in these processes is cell-type-specific, and we will investigate genetic interactions with known components of autophagy and lipid pathways. Aim 2: Elucidate the molecular mechanisms underlying D-TNFAIP8 regulation of autophagy and lipid metabolism. Protein-protein interactions and epistasis analyses will be used to determine if DTNFAIP8 regulates lipid degradation through autophagy or other mechanisms. Aim 3: Identify genes that modulate lipophagy in Drosophila cells and tissues. In this aim, we will use RNAi and genetic approaches coupled with autophagy flux reporters to identify and characterize new lipophagy regulators. This study will provide new knowledge about the relationship between autophagy and other processes important for regulating cellular energy stores and nutrient metabolism. We will gain an understanding for molecular mechanisms involved in the regulation of these processes and characterize potential new regulators of lipophagy in vivo. Since these cellular processes are highly conserved evolutionarily, it is likely that our findings in Drosophila will provide insights into developmental functions of autophagy/lipophagy in other organisms, including humans.