PROJECT SUMMARY/ABSTRACT The plasma membrane (PM) is a bustling hub of transport, signaling and structural functions that sustain life at the cellular level. Failure to appropriately choreograph these parallel functions contributes to the pathogenesis of many diseases. Selective regulation of PM function relies on the cytosolic leaflet phosphoinositide (PPIn) lipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 is a master regulator, recruiting and/or activating scores of proteins controlling PM permeability, vesicular traffic, cytoskeletal assembly and receptor-driven signaling. Human diseases from cancer to rare monogenic disorders are known to be associated with aberrant PI(4,5)P2 levels, which result from disrupted PI(4,5)P2 synthesis or breakdown. That said, the sheer number of PI(4,5)P2-regulated functions has made it challenging to identify which functions contribute to disease phenotype, let alone attempt therapeutic intervention. Therefore, the long-term goal of the lab's research is to develop a detailed, mechanistic understanding of how PPIn metabolism couples to individual physiological processes; ultimately, we aim to use this knowledge to develop strategies that modulate or restore PI(4,5)P2 regulation of individual functions that are aberrant in disease. Our goal in this application is to define three distinct mechanisms by which metabolism of PM PI(4,5)P2 or its signaling products couple to multiple physiological processes. In the first project, we leverage our recent discovery of a PM PI(4,5)P2 homeostatic mechanism to experimentally tune the lipid level in cells: this will allow us for the first time to determine the PI(4,5)P2 concentration-dependence of key PM functions. This will reveal which functions are most affected by pathogenic PI(4,5)P2 levels and thus likely drive disease phenotypes – a crucial first step in devising therapeutic interventions. In the second project, we will identify lipid transfer proteins (LTPs) that couple PPIn removal from membranes to their degradation by PPIn phosphatases located in other organelles. We will employ a synthetic biology approach in which LTPs deplete ectopic mitochondrial PPIn in live cells. Identifying LTPs coupled to different PPIn pools will reveal new targets to prevent PPIn accumulation in monogenic disease where phosphatase activity is lost. In the third project, we will define the distinct PI3K signaling landscapes driven by the two downstream lipid products of PI(4,5)P2: PIP3 and PI(3,4)P2. We will define the unique spatiotemporal and effector protein activation profiles of these lipids in live cells, using our unique molecular tools to precisely detect and chemogenetically manipulate the lipids in cell culture. Crucially, this will identify spatiotemporal and effector protein activation profiles that are unique to each lipid. This knowledge will be vital to eliminate on-target adverse effects of proven PI3K therapeutics. Collectively, at the conclusion of thi...