SUMMARY Targeting polarity proteins to mutually exclusive plasma membrane (PM) domains is an essential process for establishing and maintaining cell polarity – a process that is regulated by an ever-expanding interaction network among polarity proteins and their regulators/effectors. In contrast, much less is known about how physical interactions between polarity proteins and the PM contribute to this polarization process. Recent research from the PI’s lab showed that multiple core polarity proteins (“polybasic polarity proteins”) such as Lgl, aPKC, and Dlg are also inherently membrane-binding proteins. They contain so-called polybasic domains that specifically and electrostatically target to the PM by binding to PM phospholipids such as PI4P and PIP2. We have characterized multiple mechanisms that control the polarized electrostatic PM targeting function of polybasic polarity proteins. We have also discovered an unexpected consequence of such widely existing electrostatic PM targeting in cell polarity: PM localization of polybasic polarity proteins is highly susceptible to energetic stresses triggered by hypoxia and ATP inhibition, which induce dynamic turnover of PM PI4P and PIP2. In this proposal, our goal is to further establish electrostatic PM-targeting as a fundamental mechanism regulating cell polarity under both normal and energetic stressed conditions. Specifically, we will investigate: 1) Diverse mechanisms regulating electrostatic PM targeting in cell polarity. We will focus on the regulation of electrostatic PM targeting of basolateral polarity protein Dlg which is controlled by phosphorylation, allosteric regulation of its polybasic domain, and interactions with other basolateral polarity proteins. We will elucidate how allosteric regulation of electrostatic PM targeting acts as a core mechanism by which basolateral polarity proteins control each other’s localization and functions. 2) Control of aPKC phosphorylation by electrostatic PM targeting. aPKC is the key kinase driving cell polarization and its pseudo-substrate region (PSr) is also a polybasic domain that is allosterically controlled by Par-6 for both PM targeting and kinase activity. Using a novel inducible polarization system, we will investigate the hypothesis that Par-6-dependent electrostatic PM targeting of aPKC controls the substrate-specific phosphorylation of aPKC/Par-6, providing a mechanism of spatiotemporal control of aPKC kinase activity in cell polarization. 3) Regulation of electrostatic PM retargeting of polybasic polarity proteins. Despite total loss of the PM targeting function of Lgl and aPKC under hypoxia due to depletion of PM PI4P and PIP2, we found that both proteins always directly retargeted their original basolateral or apical PM domains once reoxygenation started. We will investigate both lipid- and protein-based mechanisms that restore and control such polarized PM retargeting of polybasic polarity proteins. These mechanisms will be critical to...