PROJECT SUMMARY Phosphoinositides (PIPs) are minor components of the eukaryotic membrane but major regulators of cellular functions. The seven PIPs are critically involved in nearly every aspect of cell physiology. One of the cellular processes regulated by PIPs is autophagy, a process essential for a broad range of cellular functions and tissue development, and dysregulated in many human diseases. Found on late endosomes and lysosomes, PI(3,5)P2 is necessary for autophagosome maturation, and dysregulation of PI(3,5)P2 biogenesis has been linked to several neurological disorders through defective autophagy. However, the mechanism by which PI(3,5)P2 regulates autophagy is poorly understood. PIP signaling is often mediated by lipid-protein interactions. Our efforts in the last grant cycle have led to the development of a single-molecule assay that detects lipid interaction with proteins in mammalian whole-cell lysates, using which we have discovered widespread PIP interactions within the large family of human pleckstrin homology (PH) domain-containing proteins. XPLN, with dual activities as a RhoA guanine nucleotide exchange factor (GEF) and an endogenous inhibitor of mammalian target of rapamycin complex 2 (mTORC2), has emerged as a novel PI(3,5)P2-interacting protein, and we have also discovered that XPLN regulates autophagy in vivo. Guided by the working hypothesis that XPLN is an effector of PI(3,5)P2 and plays a central role in mediating PIP signaling in the regulation of autophagy, our proposed studies will decipher the biochemical basis of XPLN-PIP interactions and how they control XPLN activity and function. The role of XPLN phosphorylation by protein kinase C will also be investigated. We will ask how those biochemical mechanisms underlie the regulation of autophagy in mammalian cells. Finally, physiological relevance of the new mechanisms will be probed in a mouse model of injury-induced skeletal muscle regeneration, for which autophagy is required. Our expertise in lipid signaling, strong preliminary data, and a unique combination of biochemical, biophysical, cell biology, and animal model approaches will ensure a successful outcome that is likely to have significant impact on the biochemical and functional understanding of PIP signaling and regulation of autophagy.