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Distinct Activities of Scrib Module Proteins Organize Epithelial Polarity

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Distinct Activities of Scrib Module Proteins Organize Epithelial Polarity Distinct activities of Scrib module proteins organize epithelial polarity Mark J. Khourya and David Bildera,1 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Edited by Margaret T. Fuller, Stanford University School of Medicine, Stanford, CA, and approved April 15, 2020 (received for review October 25, 2019) A polarized architecture is central to both epithelial structure and In contrast to the wealth of mechanistic information about the function. In many cells, polarity involves mutual antagonism Par complex, and despite the discovery of the relevant genes between the Par complex and the Scribble (Scrib) module. While decades ago, the molecular mechanisms of basolateral domain molecular mechanisms underlying Par-mediated apical determina- specification by the Scrib module are still unknown. All three tion are well-understood, how Scrib module proteins specify the genes encode large scaffolding proteins containing multiple basolateral domain remains unknown. Here, we demonstrate de- protein–protein interaction domains and lack obvious catalytic pendent and independent activities of Scrib, Discs-large (Dlg), and activity (13, 20–22). Recent studies have identified novel inter- Lethal giant larvae (Lgl) using the Drosophila follicle epithelium. acting partners of Scrib module proteins, but few of these Our data support a linear hierarchy for localization, but rule out interactors have been implicated as regulators of cell polarity previously proposed protein–protein interactions as essential for themselves (23, 24). Moreover, few studies have focused on the polarization. Cortical recruitment of Scrib does not require palmi- regulatory relationships within the Scrib module itself, and be- toylation or polar phospholipid binding but instead an indepen- yond the well-characterized aPKC-inhibiting function of Lgl, the dent cortically stabilizing activity of Dlg. Scrib and Dlg do not fundamental molecular activities of Scrib and Dlg remain un- directly antagonize atypical protein kinase C (aPKC), but may in- known. In this work, we identify distinct activities of Scrib, Dlg, stead restrict aPKC localization by enabling the aPKC-inhibiting and Lgl that are required but not sufficient for basolateral po- activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each re- larization, shedding light on the mechanisms that restrict the Par quired, all three together are not sufficient to antagonize the Par complex to partition the epithelial cell membrane. complex. Our data demonstrate previously unappreciated diver- DEVELOPMENTAL BIOLOGY sity of function within the Scrib module and begin to define the Results elusive molecular functions of Scrib and Dlg. A Linear Hierarchy for Localization but Not Function of Basolateral Polarity Regulators. We used the conserved epithelial features of epithelia | polarity | Drosophila | Par complex | Scrib module Drosophila ovarian follicle cells to study regulation of the baso- lateral cortical domain (25) (SI Appendix, Fig. S1 A–C). Cells ell polarity is defined by the coexistence of two distinct mutant for null alleles of scrib, dlg,orlgl encoding severely Cspatial identities within the confines of a single plasma truncated or nonfunctional proteins lose polarity, characterized membrane. This process is critical for many cell types, including by mixing of apical and basolateral domains and cells form multilayered masses at the poles of the egg chamber (Fig. 1 B–E stem cells, epithelial cells, migratory cells, and immune cells, to – carry out their physiological functions (1, 2). Despite the distinct and SI Appendix, Fig. S1 I K) (20, 21, 26). Importantly, we fo- manifestations of polarity in these specialized cells, polarity in cused our analysis on the central follicle epithelium, where polarity-deficient cells retain relatively normal morphology that each is generated by a common pathway involving a set of con- served protein modules (3–5). Foremost among these are the Par and Scrib modules, consisting of Par-3, Par-6, and atypical pro- Significance tein kinase C (aPKC) for the former and Scribble (Scrib), Discs- large (Dlg), and Lethal giant larvae (Lgl) for the latter (3, 4). To enable their physiological functions, cells must polarize their These proteins play crucial roles in diverse biological processes plasma membrane. In many epithelia, polarity is regulated by and have also been implicated in numerous pathologies, from balanced activity of the apical Par complex and basolateral congenital birth defects to cancer (3, 4, 6). Thus, uncovering Scribble module. While the former is understood in molecular their molecular activities is essential to a mechanistic un- detail, little is known about how the latter works. We identify distinct functions of the three Scribble module proteins, sepa- derstanding of cell, developmental, and disease biology. rating independent roles in a localization hierarchy from co- A number of studies have provided important insight into the operative roles in apical polarity antagonism, and showing that molecular function of the Par module and each of its individual they are not together sufficient to specify basolateral identity. – Drosophila components (7 11). Much of this work derives from This work establishes an essential basis for a mechanistic un- epithelial cells and neural stem cells, where the Par module derstanding of this core polarity machinery that controls processes regulates the apical domain and the Scrib module is required to ranging from stem cell divisions to organ morphogenesis across specify the basolateral domain. The core distinction of cortical animal species. domains arises from mutual antagonism between the two mod- ules, centering around interactions between aPKC and Lgl Author contributions: M.J.K. and D.B. designed research; M.J.K. performed research; (Fig. 1A). In the apical domain, aPKC phosphorylates Lgl on M.J.K. analyzed data; and M.J.K. and D.B. wrote the paper. three residues within a polybasic domain, causing it to dissociate The authors declare no competing interest. from the plasma membrane (12–14). Conversely, Lgl inhibits This article is a PNAS Direct Submission. aPKC kinase activity and localization along the basolateral cor- Published under the PNAS license. tex (15–17). Many details of Par protein activities and their See online for related content such as Commentaries. outcomes are now understood, including specific protein–protein 1To whom correspondence may be addressed. Email: [email protected]. interactions in dynamic complexes, their structural basis, post- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ translational modifications, and the kinetic order of events dur- doi:10.1073/pnas.1918462117/-/DCSupplemental. ing apical polarization (18, 19). First published May 15, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1918462117 PNAS | May 26, 2020 | vol. 117 | no. 21 | 11531–11540 Downloaded by guest on September 30, 2021 aPKC aPKC A B Dlg C Dlg DNA DNA Apical Par6 P P P P aPKC Baz Lgl WT scrib1 Basolateral aPKC aPKC DEScrib Dlg Dlg DNA DNA Scrib Lgl dlgm52 lgl27S3 n.s. F I L **** ******** n.s. scrib2 aPKC scrib2 lglRNAi aPKC * n.s. ** G J lgl27S3 aPKC lgl27S3 dlgRNAi aPKC H K dlgm52 aPKC dlgRNAi scrib2 aPKC MOQScrib Dlg Dlg dlgm52 scrib1 lgl27S3 N Lgl PRLgl Scrib dlgm52 scrib1 lgl27S3 STU dlgRNAi,>Lgl aPKC scrib2, >Lgl aPKC dlgm52, >Scrib aPKC V W X scrib2, >Dlg aPKC lgl27S3, >Scrib aPKC lglRNAi, >Dlg aPKC Fig. 1. Functional relationships within the Scrib module. (A) Simplified schematic representation of epithelial polarity interactions. Polarity phenotypes of WT (B), scrib (C), dlg (D), and lgl (E) follicle cells: Mutants exhibit polarity loss, characterized by mixing of apical and basolateral domains and multilayering of the epithelium. Compared to single mutants (F–H), double-depleted combinations (I–K) do not show an enhanced apical expansion phenotype. (L) Quan- tification of aPKC mislocalization phenotype in single and double mutants. aPKC spread represented as ratio of lateral:apical intensity in single cells. Lo- calization of Scrib module proteins: Both Scrib and Lgl show hazy, cytoplasmic mislocalization in dlg mutant cells (M and N). In scrib mutant cells, Dlg localization is normal (O), while Lgl is mislocalized (P). In lgl mutants, both Scrib and Dlg localizations are unchanged (Q and R). Overexpression of Lgl does not rescue apical polarity defects in dlg or scrib mutants (S and T). Scrib overexpression cannot rescue dlg mutants (U) nor can Dlg overexpression rescue scrib mutants (V). lgl mutants are not rescued by Scrib or Dlg overexpression (W and X). (Scale bars, 10 μm.) One-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SD, data points are measurements from single cells. White line indicates mutant cells and/or overexpression clones in this and all subsequent figures. Follicles in D, F, and R are stage 5; H, J, P, and S are stage 7; K and O are stage 8; all others are stage 6. n.s. (not significant), P *>P0.05; < 0.05; **P < 0.01; ****P < 0.0001. allows accurate monitoring of protein localization. We first validated RNAi transgene targeting a second gene. However, we asked whether Scrib, Dlg, and Lgl have independent as well as saw no differences between single mutant cells and cells double- shared functions in epithelial polarity. We generated follicle cells depleted for scrib dlg, scrib lgl, and lgl dlg, and lateral aPKC simultaneously mutant for one of the genes and expressing a spread was
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