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

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.


Introduction
Simple epithelia form cohesive barriers owing to specialized adherens junctions. For instance, the cadherin-catenin complex, a major component of the zonula adherens (ZA), links cortical actin filaments of neighboring cells indirectly to ensure strong cell-cell adhesion (Harris and Tepass, 2010). Association of the hexameric motor protein non-muscle myosin II (hereafter, myosin) with ZA-associated actin bundles ensures contractility (Mege and Ishiyama, 2017). The ability of epithelial cells to support vectorial transport and secretion adjusts the biochemical environment on both sides of the epithelial layer. These unidirectional functions require the polarization of epithelial cells along the apical-basal axis (Laprise and Tepass, 2011). Reciprocal interactions between the epithelial polarity protein network and ZA components not only define the functional architecture of individual epithelial cells, but also shape epithelial tissues (Tepass, 2012). A classic example is the formation of specialized epithelial structures, such as tubes or acini, resulting from concerted myosin-dependent apical constriction of a group of cells within epithelial sheets (Martin and Goldstein, 2014).
The mammalian Yrt orthologs EPB41L4B and EPB41L5 (also known as EHM2/LULU2 and YMO1/LULU, respectively) support cell migration and invasion, and EPB41L5 is also essential for the epithelial-mesenchymal transition (EMT) Hashimoto et al., 2016;Hirano et al., 2008;Jeong et al., 2019;Lee et al., 2007;Otsuka et al., 2016;Saller et al., 2019;Wang et al., 2006;Yu et al., 2010). Given that Crb and human CRUMBS3 (CRB3) both repress EMT (Campbell et al., 2011;Whiteman et al., 2008), these observations are consistent with the aforementioned concept that fly and mammalian Yrt proteins repress the Crb/CRB3-containing apical machinery (Laprise et al., 2006;Laprise and Tepass, 2011). However, recent findings have shown that depletion of Crb or Yrt causes similar phenotypes in pupal wing cells. Specifically, DEcadherin (DE-cad) staining is fragmented, and F-Actin and Myosin distribution is altered in the absence of these proteins (Salis et al., 2017). This suggests that Yrt controls cell contractility -a premise that awaits formal demonstration. Consistent with this model, it was shown that EPB41L4B and EPB41L5 promote apical constriction (Nakajima and Tanoue, 2010;Nakajima and Tanoue, 2011;Nakajima and Tanoue, 2012). Similar to EPB41L4B and EPB41L5, Drosophila Crb organizes the actomyosin cytoskeleton at cell-cell contacts, thereby supporting ZA integrity and tissue morphogenesis (Letizia et al., 2011;Silver et al., 2019;Tepass, 1996). Crb fulfils this function in ectodermal cells in part through the apical recruitment of the Guanine exchange factor (GEF) Cysts (Cyst), which activates the Rho1-Rho kinase (Rok)-Myosin pathway (Arnold et al., 2017;Rubin et al.;Silver et al., 2019). In contrast, other studies have reported that Crb stabilizes adherens junctions by repressing Myosin activity in amnioserosa cells, a function that requires the FERM-domain protein Moesin (Moe) (Flores-Benitez and Knust, 2015). Moreover, Crb was shown to reduce the membrane residence time of Rok, thereby preventing the formation of a supracellular actomyosin cable in cells expressing high Crb levels at the salivary gland placode boundary where Crb distribution is anisotropic (Roper, 2012;Sidor et al., 2020). These conflicting observations show that the functional relationship linking Crb and Myosin is complex, and likely context-dependent. Whether, and how, Yrt modulates the multifaceted function of Crb in regulating cytoskeletal dynamics remains as an outstanding puzzle. Here, we show that Crb recruits Yrt to the apical membrane to induce Myosin-dependent apical tension in Drosophila epithelial cells. This association and the ensuing increased Myosin activity is repressed by aPKC-dependent phosphorylation of Yrt. In contrast to aPKC, Pak1 maintains the pool of unphosphorylated Yrt via Protein phosphatase 2A (PP2A), thereby supporting Yrt-dependent cortical tension.

Yrt increases Myosin-dependent cortical tension
It was previously shown that loss of Yrt expression increases cell diameter in pupal tissues (40), thus suggesting that Yrt may promote actomyosin-based contraction to define cell width. To test this hypothesis directly, we measured retraction velocity after laser ablation of cell borders in the Drosophila embryonic epidermis, which reflects Myosin-dependent cortical tension (Hutson et al., 2003). Knockdown of yrt expression decreased the retraction velocity after laser ablation by 49% with respect to controls (Fig. 1A, B; Video 1). In contrast, Yrt overexpression increased the retraction velocity after ablation by 43% (Fig. 1C, D; Video 2). We quantified changes to the material properties of the apical cell surface using a Kelvin-Voigt model to measure the relaxation times of laser ablation experiments, proportional to the viscosity-to-elasticity ratio (Fernandez-Gonzalez et al., 2009). yrt knockdown and Yrt overexpression did not affect tissue viscoelasticity: relaxation times were 12 ± 1 seconds for yrt knockdown vs. 11 ± 2 seconds for controls, and 8 ± 1 seconds for Yrt overexpression vs. 10 ± 1 seconds for controls. Together, our data indicate that Yrt positively regulates cortical tension in the Drosophila embryonic epidermis.
Knockdown of yrt decreased cortical Spaghetti squash (Sqh; the Drosophila Myosin regulatory light chain) localization to cell boundaries without affecting Sqh intensity at the apex of epidermal cells from stage 14 of embryogenesis, a time point coinciding with the apical recruitment of Yrt (Laprise et al., 2006), whereas Yrt depletion had no major impact on Myosin distribution at earlier stages of development ( Fig. 2A-D). In addition, FLAG-Yrt expression resulted in apical enrichment of Sqh in adult ovarian follicle cells (Fig. 2E, F). These data suggest that Yrt controls contractility at the apical domain. Consistent with this premise, mosaic overexpression of FLAG-Yrt induced apical constriction in the embryonic epidermis (Fig. 3A, B) and the adult ovarian follicular epithelium (mRFP-positive cells; Fig. 3C, D). Knockdown of sqh suppressed Yrt-induced apical constriction in (Fig. 3E, F). Taken together, these results establish that Yrt promotes Myosinbased changes in cell mechanics to support cortical tension and/or apical constriction in various epithelial tissues, thereby providing putative molecular insights into how Yrt controls tissue morphogenesis (Franke et al., 2005;Hoover and Bryant, 2002;Laprise et al., 2006). aPKC counteracts Yrt-induced apical constriction by preventing the Crb-dependent apical recruitment of Yrt aPKC phosphorylates Yrt on evolutionarily conserved residues (Gamblin et al., 2014). This prevents Yrt oligomerization, thereby antagonizing its ability to support lateral membrane stability and to restrict apical membrane growth (Gamblin et al., 2018). Expression of membrane-targeted aPKC (aPKC CAAX ) suppressed FLAG-Yrt-induced apical constriction ( Fig. 4A-C, H), showing that aPKC has a broad impact on Yrt function and also alters its ability to promote Myosin activity. However, aPKC CAAX had no effect on apical constriction induced by FLAG-Yrt 5A in which the amino acids targeted by aPKC were replaced by non-phosphorylatable alanine residues [(Gamblin et al., 2014); Fig. 4D, E, H]. In addition, mutation of the aPKC phosphorylation sites into aspartic acids to generate the phosphomimetic Yrt mutant protein FLAG-Yrt 5D (Gamblin et al., 2014) abolished the ability of Yrt to induce apical constriction (Fig. 4F, I). Similarly, another FLAG-Yrt mutant protein unable to oligomerize [FLAG-Yrt FWA ; (Gamblin et al., 2018)] failed to sustain apical constriction (Fig. 4G, I). These results argue strongly that aPKC targets Yrt directly to inhibit its ability to support apical constriction.
To gain further insights on how aPKC impacts on Yrt function in the follicular epithelium, we analyzed Yrt subcellular localization upon modulation of aPKC activity. We observed apical accumulation of Yrt in wild-type cells treated with a chemical inhibitor of aPKC or in aPKC knockdown cells (Fig. 5A, B). These results show that aPKC normally acts to restrict Yrt apical localization, which depends on Crb in the embryonic epidermis and in pupal photoreceptor cells (Laprise et al., 2006). This is also the case in adult follicular epithelial cells, as Yrt failed to accumulate apically in crb mutant cells treated with the aPKC inhibitor (  (Klebes and Knust, 2000)], which prevents the direct association of Yrt and Crb proteins (Laprise et al., 2006). This suggests that Crb plays a pivotal role in Yrt-dependent apical constriction. Accordingly, FLAG-Yrt 5A triggered apical constriction in wild-type cells, whereas it was unable to induce this phenotype in Crb-depleted cells or cells expressing specifically Crb Y10A ( (Gamblin et al., 2014)]. In contrast to aPKC, Pak1 Myr decreased Yrt phosphorylation levels (Fig. 7C). Given that Pak1 is a kinase, this observation is counterintuitive. However, it has been shown that Pak1 can activate the phosphatase PP2A (Staser et al., 2013), thus raising the possibility that the latter targets Yrt downstream of Pak1. Accordingly, Yrt phosphorylation levels were increased strongly in PP2A-A mutant embryos, or in wild-type embryos treated with the PP2A inhibitor Cantharidin Altogether, these data suggest that Pak1 acts upstream of PP2A that antagonizes aPKC function by dephosphorylating Yrt, thereby allowing the latter to induce apical tension ( Fig. 9).

Discussion
Myosin-mediated cell contractility impacts a broad range of cellular processes driving tissue morphogenesis, including apical constriction, convergent extension, and ZA establishment and maintenance (Miao and Blankenship, 2020; Perez-Vale and Peifer, 2020). Our data establish that Yrt increases Myosin-dependent cortical tension in embryonic and adult tissues, thereby providing further insights into how Yrt contributes to organize epithelial tissue architecture. Similarly, a recent study showed that loss of Yrt expression increases cell diameter in pupal wing cells (Salis et al., 2017), thus suggesting that Yrt controls the actomyosin network throughout the fly life cycle.
This function is evolutionarily conserved, as the mammalian Yrt proteins EPB41L4B and EPB41L5 impact actomyosin organization and stimulate apical constriction (Nakajima and Tanoue, 2010;Nakajima and Tanoue, 2011;Nakajima and Tanoue, 2012). Regulation of actomyosin contractility by Yrt likely impacts ZA stability, as suggested by the fragmented distribution of ZA components in Yrt-depleted cells (Salis et al., 2017). This is in line with the fact that Yrt-induced apical constriction depends on Crb, which is also required for organization of junctional Myosin assembly and ZA stability (Pocha and Knust, 2013;Silver et al., 2019;Tepass, 1996).
Our findings showing that Crb and Yrt cooperate in promoting cortical tension may seem at odds with previous studies demonstrating that Yrt inhibits the ability of Crb to support apical membrane growth (Laprise et al., 2006;Laprise et al., 2009). We propose that Yrt is not an inhibitor of Crb function, but rather acts downstream of Crb to specifically promote actomyosin contractility at the expense of other Crb-dependent roles such as apical membrane growth. It is possible that the recruitment of Yrt causes steric hindrance on the short cytoplasmic tail of Crb, thereby preventing the binding of Crb effectors mediating apical membrane growth. In addition, we found that Yrt competes with Moe, which binds to Crb and decreases cortical tension (Flores-Benitez and Knust, 2015;Medina et al., 2002;Salis et al., 2017) Our discoveries indicate that aPKC prevents the Crb-Yrt association through direct phosphorylation of Yrt. Thereby, aPKC excludes Yrt from the apical membrane and represses Yrtinduced apical constriction. aPKC also inhibits the ability of Yrt to restrict Crb-dependent apical membrane growth (Gamblin et al., 2014;Gamblin et al., 2018). This is consistent with the aforementioned model, and with previous reports documenting functional cooperation between aPKC and Crb in establishing the apical domain (Fletcher et al., 2012;Sotillos et al., 2004;Tepass, 2012;Walther and Pichaud, 2010). We have shown that phosphorylation of Yrt on aPKC target sites prevents Yrt self-association, and that oligomerization-defective Yrt mutant proteins are unable to bind Crb (Gamblin et al., 2018). Together, these studies establish that the phosphodependent repression of Yrt oligomerization is a key mechanism by which aPKC controls apicalbasal polarity, cortical tension, and epithelial cell architecture. Our data also show that ectopic localization of aPKC to the lateral domain dislodges Yrt from the membrane, indicating that aPKC controls both Crb-dependent and Crb-independent membrane localization of Yrt. One possibility is that phosphorylation of Yrt by aPKC generates electrostatic repulsion with negatively charged membrane phospholipids, as described for other aPKC substrates (Bailey and Prehoda, 2015). Yrt sequence does not contain a Pak1 consensus motif. Moreover, we found that expression of membrane-targeted Pak1 reduces Yrt phosphorylation levels, whereas membrane-bound aPKC has the opposite effect. This argues that Yrt is not a direct substrate of Pak1, and that this kinase does not phosphorylate the full complement of aPKC substrates within the polarity protein network.
Finally, while Pak1 is required for Yrt-induced apical constriction, aPKC inhibits this function of Yrt. In the light of all of this evidence, we propose that Pak1 promotes the dephosphorylation of aPKC target sites on Yrt to support its functions. We provide evidence that Pak1 acts through the phosphatase PP2A to achieve this function in polarized epithelial cells. PP2A thus has a broad role in cell polarity, as it opposes aPKC and Par-1 signaling to control polarization of neuroblasts and photoreceptor cells (Krahn et al., 2009;Nam et al., 2007;Ogawa et al., 2009). In these cell types, PP2A targets Par6 and/or Baz. This implies that Pak1 may also decrease the phosphorylation level of Baz and/or Par6 in epithelial cells, although the kinase domain of Pak1 can phosphorylate Baz and Par6 in vitro (Aguilar-Aragon et al., 2018). At this point, a cell type-specific or contextdependent regulation of Baz and Par-6 by Pak1 cannot be ruled out. Alternatively, it is possible that Pak1 can both increase or decrease the phosphorylation of specific polarity proteins in regulatory feedback loops, thereby ensuring the delicate equilibrium required for epithelial tissue homeostasis. Combining our data with previous findings (Aguilar-Aragon et al., 2018) suggests that activation of aPKC and Pak1 downstream of Cdc42 has redundant activity on a subset of polarity proteins [e.g. Par6, L(2)gl, Baz], while having opposite effects on Yrt and apical tension.
Identification of the molecular mechanisms selecting the engagement of aPKC or Pak1 downstream of Cdc42 remains an outstanding puzzle, solving of which will provide crucial insights into the understanding of epithelial cell polarity, cortical tension, and tissue morphogenesis.
Overall, our work reveals a novel mechanism regulating cortical tension at the apical domain, which plays a crucial role in developing and adult epithelia. We also refine our understanding of the functional relationship linking Crb and Yrt by establishing that the latter contributes to the plasticity of Crb function rather than acting as an obligatory inhibitor of the apical protein machinery. We also highlight that the roles of aPKC and Pak1 are not fully redundant in epithelial cells, and that these kinases have opposite effects on cortical tension. We also provide evidence that Pak1 can decrease protein phosphorylation in epithelial cells through PP2A, thereby consolidating the broad role played by this phosphatase in cell polarity.

Drosophila genetics
The UAS-GAL4 system was employed to drive the expression of transgenes (Brand et al., 1994).
Mosaic analysis in the follicular epithelium was performed using the Flippase recognition target (FRT)/Flippase (FLP) system in which the FLP recombinase was under the control of a heat shock promoter (hsFLP) (Chou and Perrimon, 1996;Golic and Lindquist, 1989). Newly hatched females were heat-shocked for 3 h at 37°C on two consecutive days. Ovaries were dissected and processed 48 h after the second heat shock. Table 1 and 2 depict the stock list and the complete genotype of flies used in this study, respectively.

Laser ablation for measurement of mechanical properties
Stage 14 embryos were dechorionated for 2 minutes in 50% bleach, washed with water, and glued on a coverslip using heptane glue (Scepanovic et al., 2021). Ablations were induced using a pulsed Micropoint N2 laser (Andor) tuned to 365 nm on a Revolution XD spinning disk confocal microscope. The laser produces 120 μJ pulses at durations of 2-6 ns each. For ablation of cell junctions, 10 consecutive laser pulses were delivered to a single spot along a cell interface. Images were acquired every 4 s before and after ablation of a single interface using a 60x oil immersion lens (Olympus, NA 1.35). The positions of the tricellular vertices connected by the ablated interface were manually tracked prior to and for 36 seconds following ablation using the image analysis platform SIESTA (Fernandez-Gonzalez and Zallen, 2011;Leung and Fernandez-Gonzalez, 2015).
To measure retraction velocity, the change in distance between the tricellular vertices was measured comparing images acquired immediately before and after ablation and divided by the time elapsed between the two images. To quantify the viscoelastic properties of the tissue, we modelled the laser ablation results as the damped recoil of an elastic fibre, using a Kelvin-Voigt mechanical-equivalent circuit (Fernandez-Gonzalez et al., 2009). Briefly, the Kelvin-Voigt circuit models cell interfaces as a spring (elasticity) and a dashpot (viscosity) configured in parallel. As a result, the distance between the ends of the interface when tension is released can be quantified as: where L(t) is the distance between the tricellular vertices at the end of the severed interface at time t after ablation, D is the asymptotic value of the distance between the tricellular junctions, and the relaxation time : = is the ratio of the viscosity (μ) to the elasticity (Ε) of the tissue. Statistical differences between groups were measured using the nonparametric Mann-Whitney U test.
The volume of the reaction mix was completed to 30 µl with the MetalloPhosphatase buffer (New England Biolabs) containing 1 mM of MnCl2 prior to a 30 min incubation at 30°C. The reaction was stopped by addition of Laemmli's buffer.

Chemical treatment of embryos
Dechorionated embryos were incubated under agitation for 1 h at room temperature in a 0.9% NaCl solution supplemented with 100 µM of Cantharidin under an octane phase (1:1). Embryos were then washed three times in PBT.

Measurements of apical domain size and quantification of fluorescence intensity
Stage 3−6 follicles were imaged, and the apical diameter or fluorescence intensity of genetically modified cells (positively labeled with mRFP or GFP) was measured using ImageJ (National

Analysis of Myosin distribution in the embryonic epidermis
To analyze myosin distribution in epidermal cells in stage 13 and stage 14 embryos, image stacks were acquired of embryos expressing sqh:GFP and either shRNA targeting mCherry or yrt.
Maximum intensity projections were generated from slices representing the apical domain (        C, F and G, imaged follicles were at stages 5 or 6. All scale bars represent 5 µm. Yrt is localized to the lateral membrane where it prevents the spread of apical characteristics in differentiating epithelial cells and controls the occluding function of septate junctions in fully differentiated cells (Laprise et al., 2006;Laprise et al., 2009;Laprise and Tepass, 2011;Sollier et al., 2015). Yrt also occupies the apical domain owing to its direct interaction with Crb (Laprise et al., 2006). Yrt promotes Crb-dependent cortical tension (this study) in contrast to Moe that represses Myosin function downstream of Crb (Flores-Benitez and Knust, 2015;Salis et al., 2017).
Hence, these proteins contribute to the functional plasticity of Crb, and a fine regulation of their association with Crb is thus required to define and stabilize the functional architecture of epithelial cells. Our data indicate that aPKC plays a key role in this process through phospho-dependent exclusion of Yrt from the apical domain. In contrast, Pak1 promotes Yrt dephosphorylation through activation of PP2A. The Pak1-PP2A module, which opposes aPKC function, is thus essential for