PROJECT SUMMARY Autophagy is a crucial catabolic pathway by which cellular waste is recycled. Autophagic dysfunction has been implicated in cellular quality control, responses to stress, development, lifespan, and a range of infectious and other diseases in humans, including cancer, neurodegenerative diseases, and diabetes. The crosstalk of organelles including fusion and contact is a critical process involved in autophagosome biogenesis. The exact molecular mechanism for the crosstalk of organelles in autophagy remains far from clear and thus a major topic of investigation. Since organelle crosstalk could act as a switch to spatially and temporally regulate the autophagic flux in human diseases due to its dysregulation, dissecting and controlling the regulatory machinery is essential to understanding the exact roles of autophagy in specific disease contexts. Therefore, studying the molecular mechanism of organelle crosstalk will provide the opportunity to develop new therapeutic strategies in order to control activity of autophagy. We have developed in vitro protein reconstitution systems and live cell super-resolution imaging assays to study the dynamics and interactions of organelles. In this R35 renewal application, based on our preliminary data, we hypothesize that the crosstalk of organelles including fusion and contact regulates autophagosome biogenesis, in which SNAREs, vacuolar protein sorting-associated protein 33A (VPS33A), and Atg9 play a critical role. In the next five years, we will 1) study v- and t- SNAREs and accessory proteins involved in autophagosome maturation; 2) elucidate the role of Atg9 vesicle contact resulting in clustering for early autophagosome biogenesis; 3) analyze and control the crosstalk between Atg9 vesicles and other organelles. Systematical studies on the role of organelle fusion and contact in autophagosome biogenesis through multiple biochemical, biophysical, and cell biological approaches are critical to elucidate detailed molecular mechanisms, which could offer therapeutic advances. Moreover, an attempt to control organelle dynamics and interactions by optogenetic tools would be an important expansion, which is also essential for future drug development.