Determining the Roles of Sphingolipids in Phagocytosis

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PROJECT SUMMARY Phagocytosis is a conserved cellular process that is essential for innate immune responses, tissue homeostasis and is associated with various pathologies, including microbial susceptibility and autoimmune disorders. Although phagocytosis was first described more than a century ago, most of the molecular mechanisms that govern the different stages of phagocytosis, especially the lipid-protein interaction events, are poorly understood. Studies have shown that phagocytosis involves complex membrane reorganization that requires extensive remodeling of lipids at the cell surface. However, the roles of lipids, such as sphingolipids (SPLs) in phagocytosis, remain mostly unknown. We hypothesize that both structural and bioactive SPLs are critical determinant factors in phagocytosis where they are involved in signaling at the phagocytic synapse and/or help form the biophysical structure of the phagosome through recruiting proteins to the newly formed phagosomes. We recently found that the biosynthesis of sphingomyelin (SM), one of the major SPLs in mammalian cells, is critical for phagocytosis. Whether SM itself or its biosynthetic byproducts are essential for this process is unknown. In this application, we plan to characterize the exact lipid species required for phagocytosis. To this end, we will take chemical and genetic approaches to disrupt SM catabolism at multiple points along its degradative pathway in order to examine the roles of distinct SM-derived lipid species during phagocytosis. To gain insight into the mechanism by which these lipids play roles in this process, we also plan to determine the localization, metabolic conversions, and interacting partners of SPLs during phagocytosis. For this, we will employ the recently reported genetically encoded SM biosensor as well as our novel multi- functional SPL precursor analogs. These tools will allow us to examine the subcellular localization of SPLs in a time- and space-dependent manner. Our functional probes will enable us to identify SPL interacting proteins during the different stages of phagocytosis. An understanding of how these class of lipids enables phagocytosis will reveal insights into this fundamental cellular process and help develop therapeutic strategies to pathologies related to phagocytic disorders. 8