Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex

Abstract

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1-PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1 and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.

aPKC regulates apical constriction to prevent tissue rupture in the Drosophila follicular epithelium

“… 45 Alternatively, aPKC may target other actomyosin regulators or function through other apical polarity proteins implicated in the regulation of apical actomyosin contractility, such as Crumbs and Lulu2/Yurt. 24 , 41 , 45 , 46 , 47 , 48 In line with recent work showing aPKC prevents apical constriction by repressing apical Yurt accumulation, 36 we observed Yurt apical enrichment after optogenetic inactivation of aPKC .…”

“…Recent work showed that aPKC prevents Yurt mislocalization to the apical membrane of follicle cells, where Yurt binding to Crumbs induces apical constriction. 36 We therefore tested Yurt and Crumbs response in tissues clonally expressing UAS-LARIAT and exposed to light for 40 min. Crumbs largely maintained its apical localization, despite partial co-localization with the GFP::aPKC clusters ( Figures S2 C and S2D).…”

“…aPKC is essential for apical-basal polarity and thereby provides spatial cues that position the actomyosin network apically. Other apical polarity proteins can also act as positive regulators of apical constriction, such as Crumbs associated with Yurt 36 or Cysts, 41 and Cdc42, which activates MyoII through MRCK. 24 , 42 Thus, it is anticipated that whenever a genetic perturbation of aPKC disrupts apical-basal polarity, it would also prevent apical constriction.…”

“… 71 N/A Mouse monoclonal anti-phospho-Histone H3 (pH3) Ser 10 (1:1000) Cell Signalling Cat# 9706; RRID: AB_331748 Rabbit anti-phospho-Histone H3 (pH3) Ser10 (1:2000) Merck Millipore Cat# 06-570; RRID: AB_310177 Rabbit anti-aPKC (c20, 1:2000) Santa Cruz Biotechnology Cat# sc-208; RRID: AB_2168668 Mouse anti-Crumbs (1:50) Developmental Studies Hybridoma Bank Cat# cq4; RRID: AB_528181 Rabbit anti-Yurt (1:2000) Biehler et al. 36 RRID: AB_2568494 Mouse anti-α-Tubulin (DM1A, 1:10 000) Santa Cruz Biotechnology Cat# sc-32293; RRID: AB_628412 Alexa Fluor 568-conjugated goat anti-rabbit Thermo Fisher Scientific Cat# A-11036; RRID: AB_10563566 Alexa Fluor 647-conjugated goat anti-rabbit Thermo Fisher Scientific Cat# A-31573; RRID: AB_2536183 Alexa Fluor 647-conjugated goat anti-mouse Thermo Fisher Scientific Cat# A-31571; RRID: AB_162542 Chemicals, peptides, and recombinant proteins Insulin solution from bovine pancreas Sigma-Aldrich Cat# I0516 Schneider’s Insect Medium Sigma-Aldrich Cat# S0146 Phalloidin-FITC Sigma-Aldrich Cat# P5282 Phalloidin-TRITC Sigma-Aldrich Cat# P1951 Phalloidin-CruzFluor647 Santa Cruz Biotechnology Cat# sc-363797 Cell Mask Orange Plasma Membrane Stain Thermo Fisher Cat# C10045 Latrunculin A Sigma-Aldrich Cat# 428021 …”

“…Alternatively, for F-actin staining of antibody-stained samples, egg chambers were incubated for 30 minutes with Phalloidin diluted in PBT and washed three times for 10 minutes with PBT before mounting. We used the following primary and secondary antibodies: rabbit anti-phospho-Histone H3 (pH3) Ser10 (1:2000; Upstate Biotechnology), mouse anti-Crumbs (1:50; DSHB), rabbit anti-Yurt (1:2000; Biehler et al., 36 gift from Patrick Laprise, Centre de Recherche sur le Cancer, Québec ), Alexa Fluor 568-conjugated goat anti-rabbit (Invitrogen; 1:300), Alexa Fluor 647-conjugated anti-rabbit (Invitrogen) and Alexa Fluor 647-conjugated anti-mouse (Invitrogen). For Crumbs staining, fixed egg chambers were denatured with guanidine hydrochloride after the post-fixation washes with PBT: we rinsed samples twice in 50 mM Tris-HCl, pH 7.5, before incubating for 20 min in 6M guanidine hydrochloride prepared in 50 mM, pH 7.5, washing three times for 10 min with PBT and proceeding to block and stain with the antibodies as described.…”