PROJECT SUMMARY Autophagy is used by all cells to deliver cytoplasmic material to the lysosome for degradation. Significantly, autophagy has been implicated in several human diseases, including inflammatory disorders, cancer and neurodegeneration. Most of what we know about the regulation of autophagy is based on pioneering studies in yeast that defined the core autophagy machinery, but recent studies in animals have revealed that autophagy can possess different regulatory mechanisms in distinct cell types. Our research program aims to understand how autophagy is regulated under physiological conditions, and here we investigate developmentally programmed autophagy in the Drosophila intestine. This system possesses several advantages for these studies, including robust genetic, genomic and cell biological tools that enable sophisticated cellular analyses at single cell resolution. We have focused on studying autophagy in midgut enterocyte cells of the intestine as a model. Developing midgut cells require autophagy for proper cell death and degradation. Importantly, these cells are the only existing model to study how distinct cargoes, including endoplasmic reticulum (ER) and mitochondria, are selectively cleared by autophagy within a single cell in a physiological context. Our future research program contains 4 projects that will address key questions in the autophagy field, with a specific focus on how specific organelle cargoes are distinguished and selected for autophagy. What genes regulate mitochondrial clearance by autophagy? What genes are required for clearance of endoplasmic reticulum by autophagy? Are mitochondrial and ER cargoes differentially selected for autophagic clearance within a single cell during development? What genes and mechanisms control endosome-dependent and Atg gene-independent pathway for the clearance of ER? These proposed studies will address a critical gap in our knowledge about the cell context- specific mechanisms that regulate autophagy and cargo recruitment within a developing animal. Given the strong conservation of autophagy mechanisms between Drosophila and mammals, we expect that what we discover will provide insight into the diversity of mechanisms that control autophagy in humans, and how alterations in autophagy in different cell contexts may lead to disease. Furthermore, an understanding of the diversity of mechanisms that control autophagy in animals is essential knowledge for the design of rationale strategies to target autophagy for disease therapies.