Image processing and quantification was performed using Fiji software (76); for significance in statistical tests: n.s. not sufficient to antagonize the Par complex. Our data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg. Cell polarity is defined by the coexistence of two distinct spatial identities within MSI-1436 the confines of a single plasma membrane. This process is critical for many cell types, including stem cells, epithelial cells, migratory cells, and immune cells, to carry out their physiological functions (1, 2). Despite the distinct manifestations of polarity in these specialized cells, polarity in each is generated by a common pathway involving a set of conserved protein modules (3C5). Foremost among these are the Par and Scrib modules, consisting of Par-3, Par-6, and atypical protein kinase C (aPKC) for the former and Scribble (Scrib), Discs-large (Dlg), and Lethal giant larvae (Lgl) for the latter (3, 4). These proteins play crucial roles in diverse biological processes and have also been implicated in numerous pathologies, from congenital birth defects to cancer (3, 4, 6). Thus, uncovering their molecular activities is essential to a mechanistic understanding of cell, developmental, and disease biology. A number of studies have provided important insight into the molecular function of the Par module and each of its individual components (7C11). Much of this work derives from epithelial cells and neural stem cells, where the Par module regulates the apical domain and the Scrib module is required to specify the basolateral domain. The core distinction of cortical domains arises MSI-1436 from mutual antagonism between the two modules, centering around interactions between aPKC and Lgl (Fig. 1(((mutant cells (and mutant cells, Dlg localization is normal (mutants, both Scrib and Dlg localizations are unchanged (and or mutants (and mutants (mutants (mutants are not rescued by Scrib or Dlg overexpression (and are stage 5; are stage 7; MSI-1436 and are stage 8; all others are stage 6. n.s. (not significant), 0.05; * 0.05; ** 0.01; **** 0.0001. In contrast to the wealth of mechanistic information about the Par complex, and despite the discovery of the relevant genes decades ago, the molecular mechanisms of basolateral domain specification by the Scrib module are still unknown. All three genes encode large scaffolding proteins CLDN5 containing multiple proteinCprotein interaction domains and lack obvious catalytic activity (13, 20C22). Recent studies have identified novel interacting partners of Scrib module proteins, but few of these interactors have been implicated as regulators MSI-1436 of cell polarity themselves (23, 24). Moreover, few studies have focused on the regulatory relationships within the Scrib module itself, and beyond the well-characterized aPKC-inhibiting function of Lgl, the fundamental molecular activities of Scrib and Dlg remain unknown. In this work, we identify distinct activities of Scrib, Dlg, and Lgl that are required but not sufficient for basolateral polarization, shedding light on the mechanisms that restrict the Par complex to partition the epithelial cell membrane. Results A Linear Hierarchy for Localization but Not Function of Basolateral Polarity Regulators. We used the conserved epithelial features of ovarian follicle cells to study regulation of the basolateral cortical domain (25) (encoding severely truncated or nonfunctional proteins lose polarity, characterized by mixing of apical and basolateral domains and cells form multilayered masses at the poles of the egg chamber (Fig. 1 and mutant follicle cells, both Scrib and Lgl are mislocalized and exhibit hazy, cytoplasmic distributions (Fig. 1 and mutant follicle cells, although Lgl is mislocalized as in mutants, Dlg maintains normal basolateral localization (Fig. 1 and mutant follicle cells, both Scrib and Dlg maintain normally polarized cortical domains (Fig. 1 and or mutant cells and found that this did not modify the phenotype of either mutant (Fig. 1 and mutant phenotype, and Dlg overexpression did not modify the mutant phenotype (Fig. 1 and mutant phenotype (Fig. 1 and and mutant cells remained localized at the cortex and mobile fractions are not changed, it also exhibited increased recovery kinetics (and mutant cells (RNAi cells. (alleles and alleles used in harbors a point mutation.