Cell polarity is a fundamental process in biology whereby a single cell can partition its plasma membrane into two distinct and non-overlapping domains. All cells are polarized, and cell polarity is essential for the development and homeostasis of many tissues and organs. Epithelial cells exhibit a form of polarity called apicobasal polarity that is regulated by the activity of two conserved protein groups: the Par complex, which specifies the apical domain, and the Scrib module, which, in many contexts, is required to specify the basolateral domain. Mutual antagonism between the Par complex and the Scrib module positions the apical-basolateral boundary and primarily involves the apical kinase aPKC and its substrate and inhibitor, the basolateral protein, Lgl. While the mechanisms underlying apical polarity determination by the Par complex are well-studied, the molecular basis for basolateral polarity establishment by the Scrib module proteins, Scrib, Dlg and Lgl are unclear. In this dissertation I investigate the individual functions of Scrib, Dlg and Lgl using genetic manipulations and biochemistry in Drosophila and describe distinct and cooperative activities that give rise to basolateral polarity.
I identified regulatory relationships between Scrib, Dlg and Lgl that govern their localization to the cell cortex and demonstrated that these three proteins possess unique, but cooperative activities that are required but not sufficient to establish the basolateral domain. I found a specific requirement for Dlg to localize Scrib and investigated potential mechanisms, ruling out previously proposed binding interactions and posttranslational modification. I described a requirement for Scrib and Dlg to control Lgl localization by negatively regulating its phosphorylation by aPKC. We proposed a model where Scrib and Dlg ‘protect’ Lgl at the basolateral membrane, activating Lgl’s aPKC-inhibiting potential and limiting apical domain spread.
To define the molecular mechanisms of these observations, I investigated Dlg protein function in detail. I identified a putative PP1-binding motif in the Dlg HOOK domain that was essential for Dlg function, however I did not find evidence for Dlg-PP1 physical interaction and although PP1 regulates Lgl phosphorylation, the ability of Dlg and Scrib to regulate Lgl is largely separate from PP1. However, study of Dlg PP1-binding mutants revealed the requirement of the Dlg HOOK domain in localizing Scrib to the cortex. Using this information and a panel of structure-function constructs, I defined a minimal fragment consisting of only the SH3 and HOOK domains that was necessary and sufficient for function, including Scrib recruitment.
To investigate other mechanisms of Dlg function, I collaborated with Katherine Sharp, a postdoc in the lab, to interrogate Dlg binding partners using APEX2-based proximity proteomics. Our results revealed a large number of nuclear proteins in proximity to Dlg in vivo. We found that a population of endogenous Dlg resides in the nucleus near to the NURF chromatin remodeling complex. Overgrowth of dlg mutant tumors was dependent on NURF complex activity and may result from NURF-dependent activation of Yki target genes. We identified a putative NLS sequence in the Dlg HOOK domain and demonstrated that this motif is essential for Dlg function, but its mutation does not prevent Dlg nuclear entry, suggesting the existence of an alternate NLS.
Together, the work presented in this dissertation constitute an important advance in the understanding of the molecular functions of the Scrib module proteins, Scrib, Dlg and Lgl and provide a plausible model underlying basolateral polarity via our proposed Lgl protection mechanism. These studies set the stage for future investigation of the overarching mechanisms that polarize animal cells and will enable fuller understanding of many fundamental cell polarity-dependent biological processes.
