Project Summary/Abstract: Lysosomes play essential roles in cell physiology, not only controlling nutrient recycling and cellular growth, but also mediating the proper handling of various cellular stress. Lysosomal dysfunction is associated with aging and many diseases such as lysosomal storage disease, neurodegeneration, and cardiovascular diseases. A hallmark of lysosomal-related diseases is lysosomal membrane permeabilization/damage (LMP) which if not immediately resolved can cause detrimental problems including cell death. We now start to understand that LMP triggers multiple cellular pathways to repair damaged lysosomes. However, none of the previously described pathways appear to be essential for rapid lysosomal repair, suggesting additional repair mechanisms. As an attempt to find such mechanism, we recently designed and executed an unbiased proteomic screen searching for proteins specifically enriched on damaged lysosomes. This screen led to the discovery of the phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway as an essential mechanism for rapid lysosomal repair. We found that LMP stimulates robust production of phosphatidylinositol-4-phosphate (PtdIns4P, PI4P) on damaged lysosomes by type II alpha phosphatidylinositol-4 kinase (PI4K2A). Lysosomal PI4P drives the formation of extensive membrane contacts between the endoplasmic reticulum (ER) and damaged lysosomes by recruiting multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members. The ORPs catalyze subsequent ER-to-lysosomal transport of cholesterol and phosphatidylserine (PS) to mediate rapid membrane repair. While cholesterol by itself increases membrane stability, PS activates ATG2-mediated lipid transport for direct lysosomal repair. The PITT pathway is activated in response to diverse disease-related lysosomal-damaging conditions and is expected to have enormous impact on human pathophysiology. Remarkably, the PITT pathway not only reveals lipid transfer at membrane contacts as a essential mechanism for lysosomal repair, but it also establishes lipid remodeling as a new platform to understand lysosomal quality control. Through three independent projects in the next five years, our lab will continue studying LMP-triggered lysosomal lipid remodeling for better mechanistic understanding of lysosomal quality control and potential therapeutic applications. First, we are purifying lysosomes during and after lysosomal repair to characterize lipid changes by lipidomics, which we believe will identify new lipid messengers important for lysosomal quality control. Second, the PITT-mediated lysosomal cholesterol accumulation provides a great cellular model to study cholesterol transport, and we are particularly interested in the mechanism for cholesterol egress from newly repaired lysosomes. Finally, we are also performing chemical screens using FDA- approved chemical library to search for small molecules that activate or block the PITT pa...