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

  1. Cornelia Biehler
  2. Katheryn E Rothenberg
  3. Alexandra Jette
  4. Helori-Mael Gaude
  5. Rodrigo Fernandez-Gonzalez
  6. Patrick Laprise  Is a corresponding author
  1. Centre de Recherche sur le Cancer, Université Laval, Canada
  2. axe oncologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec-UL, Canada
  3. Institute of Biomedical Engineering, University of Toronto, Canada
  4. Ted Rogers Centre for Heart Research, University of Toronto, Canada
  5. Department of Cell and Systems Biology, University of Toronto, Canada
  6. Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Canada
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Cite this article as: eLife 2021;10:e67999 doi: 10.7554/eLife.67999

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.

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 (Mège 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 apically localized protein Crumbs (Crb) acts as an apical determinant in Drosophila epithelia (Pocha and Knust, 2013; Tepass, 2012; Tepass et al., 1990; Wodarz et al., 1995). The cytoplasmic tail of Crb contains a PSD95/Dlg1/ZO-1 (PDZ)-domain binding motif (PBM), and a Four point one, Ezrin, Radixin, Moesin (FERM)-domain binding motif (FBM) (Klebes and Knust, 2000; Klose et al., 2013; Pocha and Knust, 2013; Tepass, 2012). The PBM recruits PDZ-containing proteins such as Stardust (Sdt), which cooperates with Crb in establishing the apical membrane and promoting its growth (Bachmann et al., 2001; Pocha and Knust, 2013; Tepass and Knust, 1993). The FBM interacts with several FERM domain proteins, including Yurt (Yrt) (Laprise et al., 2006; Tepass, 2009). The latter is localized to the lateral membrane throughout embryogenesis, and supports the stability of this membrane domain during organogenesis (Laprise et al., 2006; Laprise et al., 2009). During terminal differentiation of epithelial cells derived from the ectoderm, Yrt also occupies the apical membrane due to a direct interaction with Crb (Laprise et al., 2006). The apical recruitment of Yrt limits Crb-dependent apical membrane growth to establish a specific apical/basolateral membrane size ratio, which is crucial to defining the morphometric parameters of developing epithelial structures (Laprise et al., 2006; Laprise et al., 2010). Crb displays cooperative functional interactions with atypical protein kinase C (aPKC) and its binding partner Partitioning defective protein 6 (Par-6) (Kempkens et al., 2006; Morais-de-Sá et al., 2010; Tepass, 2012; Whitney et al., 2016). These latter apical proteins act downstream of the small GTPase Cdc42 to promote phospho-dependent inhibition of lateral polarity proteins such as Lethal (2) giant larvae [L(2)gl] and Yrt (Fletcher et al., 2012; Gamblin et al., 2014; Hutterer et al., 2004; Laprise and Tepass, 2011; Peterson et al., 2004; Tepass, 2012). In return, L(2)gl and Yrt repress aPKC function to prevent the spread of apical characteristics to the lateral domain (Bilder et al., 2003; Drummond and Prehoda, 2016; Fletcher et al., 2012; Gamblin et al., 2014; Hutterer et al., 2004; Laprise et al., 2006; Laprise et al., 2009; Tanentzapf and Tepass, 2003; Yamanaka et al., 2006). Mutual antagonism between apical and lateral protein modules thus establishes a sharp boundary between their respective membrane domains (Fletcher et al., 2012; Laprise and Tepass, 2011). Cdc42 also activates p21-activated kinase 1 (Pak1), which has substrates in common with aPKC in fly epithelial cells (Aguilar-Aragon et al., 2018). However, it remains to be determined whether Pak1 targets the full complement of aPKC substrates, and whether phosphorylation by these kinases always has the same functional impact on target proteins and epithelial cell polarity. In particular, the functional relationship linking Pak1 and Yrt remains unexplored.

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) (Handa et al., 2018; 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, DE-cadherin (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., 2000; 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 (Röper, 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.

Results

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 the 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 (Figure 1A, B; Video 1). In contrast, Yrt overexpression increased the retraction velocity after ablation by 43% (Figure 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.

Yrt increases cortical tension.

(A) Epidermal cells in stage 14 Drosophila embryos expressing DE-cad::GFP and shRNA against mCherry or yrt before (cyan in merge) and after (red in merge) laser ablation of a cell-cell interface (white arrowheads). Scale bar, 10 µm. Circular marks visible after ablation indicate where the UV laser passed through the vitelline membrane, creating autofluorescent holes. Bottom panel shows kymographs of interface retraction over time (scale bar, 4 s). Yellow asterisks indicate the position of the tracked vertices. (B) Retraction velocity after laser ablation in embryos expressing shRNA against mCherry (n = 17 junctions in 17 different embryos) or yrt (n = 12). (C) Epidermal cells in stage 14 Drosophila embryos expressing DE-cad::GFP and wild-type levels of Yrt expression (left panel; expression of LacZ as control) or overexpression of FLAG-Yrt (right panel) before (cyan in merge) and after (red in merge) ablation of a cell-cell interface (white arrowheads). Scale bar, 10 µm. (D) Retraction velocity after laser ablation in embryos with wild-type Yrt expression (n = 13 junctions in 13 different embryos) or overexpressing FLAG-Yrt (n = 12). In B and D, error bars indicate the standard deviation (s.d.), and the black bold line denotes the mean. ** p ≤ 0.01, *** p ≤ 0.001 (Mann-Whitney test).

Video 1
yrt depletion decreases retraction velocity.

Video of representative laser ablation experiments in control and yrt knockdown embryos.

Video 2
Yrt overexpression increases retraction velocity.

Video of representative laser ablation experiments in control and Yrt-overexpressing embryos.

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 (Figure 2A–D). In addition, FLAG-Yrt expression resulted in apical enrichment of Sqh in adult ovarian follicle cells (Figure 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 (Figure 3A,B) and the adult ovarian follicular epithelium (mRFP-positive cells; Figure 3C,D). Knockdown of sqh suppressed Yrt-induced apical constriction in (Figure 3E,F). Taken together, these results establish that Yrt promotes Myosin-based 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).

Yrt promotes apical enrichment of Myosin.

(A, B) Expression of Sqh:GFP and shRNA targeting either mCherry or yrt in the embryonic epidermis at stages 13 (A) and 14 (B). Intensity vs. distance is plotted for indicated yellow lines. (C, D) Mean Sqh intensity and heterogeneity of Sqh signal for antero-posterior linescans in stage 13 embryos (C; n = seven sh mCherry, nine sh yrt) and in stage 14 embryos (D; n = 14 sh mCherry, 11 sh yrt). Error bars indicate the standard deviation (s.d.), and black lines denote the mean. *** p < 0.001 (Mann-Whitney test). (E) Mosaic expression of FLAG-Yrt in follicular epithelial cells constitutively expression Sqh::GFP, which was visualized by immunofluorescence. FLAG-Yrt-expressing cells (right panels) are labeled with mRFP, which was co-expressed with LacZ in control clones (left panels). Scale bar represents 5 μm. (F) Quantification of the fluorescence intensity of apical Sqh::GFP in cells expressing the exogenous proteins described in E (stage 5 and 6 follicles were used for quantification). Results were calculated as a ratio between FLAG-Yrt-expressing cells and control cells in the same follicle (n = 18 follicles for the control and n = 19 follicles for FLAG-Yrt). **** p ≤ 0.0001 (one-way ANOVA), error bars indicate s.d.; bold black lines denote the mean in F.

Figure 2—source data 1

Yrt controls Myosin distribution from stage 14 of embryogenesis.

Raw data for Figure 2A–D.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig2-data1-v2.xlsx
Figure 2—source data 2

Yrt increases apical Sqh in the follicular epithelium.

Raw data for Figure 2F.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig2-data2-v2.xlsx
Yrt promotes apical constriction.

(A) Mosaic expression of FLAG-GFP or FLAG-Yrt in the embryonic epidermis at stage 14. Crb immunostaining (magenta) marks the apical domain, whereas immunolabeling of L(2)gl or Discs large 1 (Dlg1) label the lateral membrane. (B) Quantification of apical domain width (n = 13 for FLAG-GFP and n = 15 for FLAG-Yrt; images were acquired from seven individual embryos for each genotype). Results are expressed as the ratio between the width of cells expressing the transgenes and the width of control cells in the same segment (**** p ≤ 0.0001). Scale bar represents 5 μm. (C) Clonal expression of membrane-targeted RFP (mRFP) and LacZ (control; left panels), or mRFP and FLAG-Yrt (F-Yrt; right panels) in the follicular epithelium. Immunostaining of mRFP highlights cells expressing the transgenes, whereas immunolabeling of endogenous Dlg1 (green) marks the lateral membrane of all epithelial cells. (D) Quantification of apical domain width of cells expressing the transgenes listed in C. Results are expressed as the ratio between the width of cells expressing the transgenes and the width of control cells in the same follicle. (E) Mosaic expression of a shRNA targeting sqh (left panels), of FLAG-Yrt (center panels), or expression of both sh-sqh and FLAG-Yrt (right panels). mRFP was co-expressed with these constructs, and used to label positive cells. (F) Quantification of the apical diameter of cells expressing the transgenes indicated in E. Results are expressed as a ratio to the width of control cells. In B, D and F, error bars indicate s.d.; bold black lines denote the mean; n ≥ 20; **** p ≤ 0.0001 (Student’s t-test was used in B, one-way ANOVA for D, F). Follicles shown in C and E were at stage 5. Scale bars represent 5 μm.

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 (aPKCCAAX) suppressed FLAG-Yrt-induced apical constriction (Figure 4A–C,H), showing that aPKC has a broad impact on Yrt function and also alters its ability to promote Myosin activity. However, aPKCCAAX had no effect on apical constriction induced by FLAG-Yrt5A in which the amino acids targeted by aPKC were replaced by non-phosphorylatable alanine residues [(Gamblin et al., 2014; Figure 4D,E,H)]. In addition, mutation of the aPKC phosphorylation sites into aspartic acids to generate the phosphomimetic Yrt mutant protein FLAG-Yrt5D (Gamblin et al., 2014) abolished the ability of Yrt to induce apical constriction (Figure 4F,I). Similarly, another FLAG-Yrt mutant protein unable to oligomerize [FLAG-YrtFWA; (Gamblin et al., 2018)] failed to sustain apical constriction (Figure 4G,I). These results argue strongly that aPKC targets Yrt directly to inhibit its ability to support apical constriction.

aPKC represses Yrt-induced apical constriction.

(A–G) Clonal expression of the indicated proteins in the follicular epithelium. Clones were positively labeled with mRFP or Yrt staining. (H, I) Quantification of the apical diameter of cells expressing the transgenes indicated in A–E (H) or F, G (I). Results are expressed as a ratio to the width of control cells in the same follicle (stage 5 and 6 follicles were used for quantification). Scale bars represent 5 μm. **** p ≤ 0.0001 (one-way ANOVA), error bars indicate s.d.; bold black lines denote the mean, n ≥ 19.

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 (Figure 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 (Figure 5C,D; crb mutant cells are positively labeled with GFP). Similarly, Yrt was absent from the apical membrane upon inhibition of aPKC in cells exclusively expressing a mutant Crb protein containing an amino acid substitution inactivating its FBM [CrbY10A; (Figure 5E,F; 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-Yrt5A triggered apical constriction in wild-type cells, whereas it was unable to induce this phenotype in Crb-depleted cells or cells expressing specifically CrbY10A (Figure 5G–J). Although our data establish that Crb and Yrt cooperate in promoting apical constriction, apical localization of Yrt in cells with suboptimal aPKC activity is not sufficient to induce prominently this phenotype (Figure 5A,B). Yrt and Moe, another FERM-domain protein binding to Crb, have opposite effects on Myosin-induced cortical tension [this study; (Flores-Benitez and Knust, 2015; Laprise et al., 2006; Médina et al., 2002; Salis et al., 2017)]. It is thus possible that the amount of Yrt relocalized to the apical domain upon inhibition of aPKC is not sufficient to outcompete Moe, whereas Yrt overexpression reaches the threshold required to displace most Moe proteins from Crb. Accordingly, inhibition of aPKC in Moe knockdown cells caused apical constriction, in contrast to what was observed in control cells (Figure 6A,B). In addition, expression of active Moe (Karagiosis and Ready, 2004) suppressed Yrt-induced apical constriction (Figure 6C,D), thereby confirming that these proteins are involved in a competitive functional interaction. Together, these results establish that aPKC precludes cortical tension by repressing the Yrt–Crb association, and that Moe antagonizes Yrt function.

Crb is essential for Yrt-dependent apical constriction.

(A) Panels depict immunofluorescence of Yrt (green in merge) and Fasciclin 3 (Fas3; lateral marker, magenta) in the follicular epithelium treated for 2 hr with the aPKC inhibitor CRT-006-68-54. (B) aPKC knockdown cells (mRFP positive) were immunostained for Yrt. (C) crb11A22 (null allele) homozygous mutant clones were produced in adult crb/+ female flies (mutant clones are GFP positive). Dissected ovaries were incubated with the aPKC inhibitor CRT-006-68-54 prior to fixation and Yrt immunostaining. (D) Quantification of apical Yrt intensity in control or crb mutant cells within the same follicle in presence of the aPKC inhibitor CRT-006-68-54. (E) Analysis of Yrt localization in control or crb11A22 mutant cells expressing exogenous CrbY10A (GFP-positive cells) exposed to the aPKC inhibitor CRT-006-68-54. (F) Quantification of apical Yrt staining in control and crb null cells expressing exogenous CrbY10A treated with the aPKC inhibitor. (G) FLAG-Yrt5A was specifically expressed in crb mutant cell clones (right panels). Mosaic expression of FLAG-Yrt5A in control cells or crb mutant clones were used as controls (middle and left panels, respectively). Clones were labeled with GFP (left panels) or by Yrt immunostaining (middle and right panels). Dlg1 staining was used to label the lateral membrane. (H) Quantification of the apical domain width of cells expressing the transgenes listed in G. (I) Immunostaining of Yrt and Dlg1 in follicular epithelial cells expressing FLAG-Yrt5A and exogenous wild-type Crb (left panels), or FLAG-Yrt5A together with CrbY10A (right panels). (J) Quantification of the apical domain width of cells expressing the transgenes listed in I. Results are expressed as the ratio between the width of cells expressing the transgenes and the width of control cells in the same follicle. Stage three follicles were depicted in A and B, whereas panels C, E, G, and I display stage five follicles. In D, F, H, and J error bars indicate s.d.; bold black lines denote the mean; n ≥ 20; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 (one-way ANOVA). All scale bars represent 5 μm.

Moe suppresses Yrt-induced apical constriction.

(A) Mosaic knockdown of Moe in absence or presence of the aPKC inhibitor CRT-006-68-54. Clones are positively labeled with membrane-targeted RFP. (B) Quantification of apical domain width of knockdown cells in absence or presence of the aPKC inhibitor CRT-006-68-54. Results were calculated as a ratio between clonal cells and control cells in the same follicle. (C) Clonal expression of FLAG-Yrt, Myc-MoeT559D (Karagiosis and Ready, 2004), or both proteins (clones are labeled with mRFP). (D) Quantification of the apical domain width of cells expressing the transgenes listed in C. Results are expressed as the ratio between the width of cells expressing transgenes and the width of control cells in the same follicle (quantification was performed using stage five follicles). In B and D, error bars indicate s.d.; bold black lines denote the mean; n ≥ 18; * p ≤ 0.05, **** p ≤ 0.0001 (one-way ANOVA). All scale bars represent 5 μm.

Pak1 and PP2A decrease Yrt phosphorylation and are required for Yrt-induced apical constriction

It was recently shown that aPKC and Pak1 share common phosphorylation targets, namely the polarity proteins L(2)gl, Bazooka (Baz), Par6, and Crb (Aguilar-Aragon et al., 2018). It was also proposed that aPKC and Pak1 act redundantly on these substrates (Aguilar-Aragon et al., 2018). However, it remains to be determined whether Pak1 impacts the function of other aPKC substrates such as Yrt. In addition, it is unclear if aPKC and Pak1 function is fully redundant in epithelial cells, or whether these kinases also have specific roles. Overexpression of Par6 together with aPKCCAAX, which accumulated ectopically at the lateral membrane (Figure 7A), strongly reduced membrane localization of Yrt (Figure 7A,B). Expression of membrane-targeted Pak1 [Pak1Myr; (Noren et al., 2000)] suppressed Yrt cortical release induced by aPKC and Par6 (Figure 7A,B). This result raises the intriguing possibility that Pak1 opposes aPKC function, a premise that we explored by investigating Yrt phosphorylation upon overexpression of these kinases. Overexpression of Par6 and aPKCCAAX is associated with an upward shift in the gel migration profile of Yrt (Figure 7C). This resulted from increased Yrt phosphorylation, as treatment of samples with the λ Phosphatase prior to electrophoresis abolished the impact of aPKCCAAX on Yrt gel mobility (Figure 7C; Gamblin et al., 2014). In contrast to aPKC, Pak1Myr decreased Yrt phosphorylation levels (Figure 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 (Figure 7D,E). Strikingly, inhibition of PP2A suppressed the impact of Pak1Myr expression on Yrt phosphorylation (Figure 7F), arguing that PP2A acts downstream of Pak1.

Pak1 and PP2A control the phosphorylation level of Yrt.

(A) Yrt immunostaining in control follicular epithelial cells or clones of cells expressing aPKCCAAX together with Par6, Pak1Myr, or aPKCCAAX with Par6 and Pak1Myr. Clones were positively labeled with RFP or aPKC immunostaining in stage four follicles. Scale bar represents 5 μm. (B) Quantification of lateral Yrt staining intensity in genotypes described in A. Error bars indicate s.d.; bold black lines denote the mean; n ≥ 16; **** p ≤ 0.0001 (one-way ANOVA). (C) Control embryos, embryos overexpressing Par-6 and aPKCCAAX, or embryos expressing Pak1Myr were homogenized. Samples were incubated or not with the λ phosphatase and processed for SDS-PAGE. Western blotting using anti-Yrt antibodies showed the migration profile of phosphorylated (pYrt) or unphosphorylated Yrt, whereas β-Tubulin (βTub) was used as loading control. (D) Western blots showing the migration profile of Yrt (pYrt stands for phosphorylated Yrt) extracted from wild-type (WT) or PP2A-A mutant embryos. The expression level of PP2A-A is also shown, and Actin was used as loading control. (E) Wild-type embryos were treated or not with the PP2A inhibitor Cantharidin, homogenized, and processed for SDS-PAGE. Western blotting using control samples shows the phosphorylation levels of Yrt (pYrt), whereas samples incubated with λ Phosphatase prior to gel electrophoresis show unphosphorylated Yrt. β-Tubulin (βTub) was used as loading control. (F) Embryos overexpressing Pak1Myr were treated or not with PP2A inhibitor and homogenized. Western blotting using anti-Yrt antibodies showed the migration profile of phosphorylated (pYrt) or unphosphorylated Yrt. λ Phosphatase was used as a positive control of Yrt dephosphorylation, and β-Tubulin (βTub) was used as loading control.

Figure 7—source data 1

aPKC and Pak1 control Yrt localization.

Raw data for Figure 7B.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig7-data1-v2.xlsx
Figure 7—source data 2

aPKC and Pak1 control Yrt phosphorylation.

Original scan for Figure 7C.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig7-data2-v2.pdf
Figure 7—source data 3

PP2A dephosphorylates Yrt.

Original scan for Figure 7D.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig7-data3-v2.pdf
Figure 7—source data 4

Pak1 acts upstream of PP2A.

Original scan for Figure 7E,F.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig7-data4-v2.pdf
Figure 7—source data 5

Loading controls.

Original scan of loading controls.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig7-data5-v2.pdf
Figure 7—source data 6

Annotated blots.

Region of each blot shown in Figure 7C–F are highlighted with a yellow box.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig7-data6-v2.pdf

We then tested the functional impact of Pak1- and PP2A-dependent modulation of Yrt phosphorylation. Chemical inhibition of Pak1 or PP2A, or knockdown of their expression suppressed apical constriction induced by FLAG-Yrt (Figure 8A–H). Of note, FLAG-Yrt5A maintained its ability to induce apical constriction in the presence of Cantharidin, or when Pak1 or PP2A were depleted (Figures 8C–D,E–H). Pak1 and PP2A are thus dispensable for Yrt-induced apical constriction when aPKC target sites on Yrt are mutated to non-phosphorylatable residues. 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 (Figure 9).

Pak1 and PP2A are essential for Yrt-induced apical constriction.

(A) Stage four follicles clonally overexpressing FLAG-Yrt were incubated with the vehicle DMSO or the Pak1 inhibitor (IPA-3; Pak1 inh.) prior to fixation and immunostaining with antibodies directed against Yrt and Dlg1. (B) Quantification of the apical diameter of cells expressing transgenes indicated in A incubated or not with IPA-3. Results are expressed as the ratio between the width of cells expressing the transgenes and the width of control cells in the same follicle. (C) Immunostaining of Dlg1 in follicles displaying mosaic expression of a shRNA targeting Pak1 and the indicated FLAG-Yrt constructs. Clones were labeled with mRFP or Yrt staining. (D) Quantification of the diameter of cells expressing the exogenous proteins described in C. Results were calculated as a ratio between transgene expressing cells and control cells within the same follicle. (E) Follicles containing cell clones expressing FLAG-Yrt or FLAG-Yrt5A (mRFP-positive cells) were incubated or not with the PP2A inhibitor Cantharidin (PP2A inh.) prior to immunostaining with indicated antibodies. (F) Quantification of the apical diameter of cells expressing transgenes indicated in E. Results are expressed as the ratio between the width of cells expressing the transgenes and the width of control cells in the same follicle. (G) Panels depict follicular epithelium displaying mosaic knockdown of PP2A-A (left panels), mosaic expression of FLAG-Yrt (middle panels), or the combination of the indicated FLAG-Yrt constructs with a shRNA targeting PP2A-A (middle and right panels). Immunostaining of mRFP or Yrt highlights cells expressing the transgenes, whereas immunolabeling of endogenous Dlg1 shows the lateral domain of all epithelial cells. (H) Quantification of the apical diameter of cells expressing transgenes indicated in G. Results are expressed as the ratio between the width of cells expressing the transgenes and the width of control cells in the same follicle. B, D, F, and H, error bars indicate s.d.; bold black lines denote the mean; n ≥ 16; * p ≤ 0.05, **** p ≤ 0.0001 (one-way ANOVA). (C, F and G) imaged follicles were at stages 5 or 6. All scale bars represent 5 μm.

Figure 8—source data 1

Pak1 and PP2A promote apical constriction.

Raw data for Figure 8B,D,F,H.

https://cdn.elifesciences.org/articles/67999/elife-67999-fig8-data1-v2.xlsx
Model: Yrt is a multifunctional protein promoting cortical tension downstream of Crb.

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 Yrt-induced contractility. The equilibrium between aPKC and Pak1–PP2A activities thus balances Yrt and Crb functions.

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; Médina et al., 2002; Salis et al., 2017). Our model is that the numerous functions of Crb are specified by the presence or absence of different FERM domain proteins, which competitively binds to Crb. This paradigm is further supported by previous findings showing that Crb controls cell growth through recruitment of the FERM domain protein Expanded (Ex) (Ling et al., 2010; Robinson et al., 2010). Thus, fine regulation of the incorporation of different FERM-domain proteins within distinct Crb complexes most likely dictates the biological output downstream of Crb. This ordered equilibrium may explain the functional plasticity of Crb, which can both increase or decrease Myosin-dependent contractility to accommodate dynamic regulation of epithelial cell polarity, cell–cell adhesion, and tissue morphogenesis (Bajur et al., 2019; Flores-Benitez and Knust, 2015; Letizia et al., 2011; Röper, 2012; Sidor et al., 2020; Silver et al., 2019).

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 Yrt-induced 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 phospho-dependent repression of Yrt oligomerization is a key mechanism by which aPKC controls apical–basal 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).

Cdc42 is a key regulator of epithelial cell polarity acting upstream of aPKC and Pak1 (Aguilar-Aragon et al., 2018; Pichaud et al., 2019). These kinases share common substrates and have redundant functions in apical–basal polarity regulation (Aguilar-Aragon et al., 2018). However, 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 context-dependent 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.

Materials and methods

Drosophila genetics

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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 hr at 37°C on two consecutive days. Ovaries were dissected and processed 48 hr after the second heat shock. Tables 1 and 2 depict the stock list and the complete genotype of flies used in this study, respectively.

Table 1
List of fly stocks used in this study.
GenotypeSourceIdentifier
D. melanogaster: UAS-FLAG-YurtReference (Gamblin et al., 2014)N/A
D. melanogaster: UAS-FLAG-Yurt[5A]Reference (Gamblin et al., 2014)N/A
D. melanogaster: UAS-FLAG-Yurt[FWA]Reference (Gamblin et al., 2018)N/A
D. melanogaster: UAS-FLAG-Yurt[5D]Reference (Gamblin et al., 2014)N/A
D. melanogaster: UAS-sh yurtDrosophila Bloomington Stock Centre# 36118
D. melanogaster: UAS-sh squashDrosophila Bloomington Stock Centre# 32439
D. melanogaster: UAS-aPKC[CAAX]Reference (Sotillos et al., 2004)N/A
D. melanogaster: UAS-aPKC[CAAX], Par6Gift from T. Harris (Harris and Tepass, 2010)N/A
D. melanogaster: UAS-sh mCherryDrosophila Bloomington Stock Centre# 35785
D. melanogaster: FRT82b, crb[11a22]Drosophila Bloomington Stock Centre# 3448
D. melanogaster: UAS-sh aPKCDrosophila Bloomington Stock Centre# 34332
D. melanogaster: UAS-sh Pak1Drosophila Bloomington Stock Centre# 41714
D. melanogaster: UAS-Pak[myr]Drosophila Bloomington Stock Centre# 8804
D. melanogaster: foscrb[Y10A]; FRT82b, crb[11a22]Reference (Klose et al., 2013)N/A
D. melanogaster: UAS-LacZDrosophila Bloomington Stock Centre# 8529
D. melanogaster: sh Pp2a-ADrosophila Bloomington Stock Centre# 55050
D. melanogaster: endo-E-Cadherin::GFPReference (Huang et al., 2009)N/A
D. melanogaster: sqh-sqh::GFPReference (Royou et al., 2004)N/A
D. melanogaster: matαtub67;15 GAL4Reference (Staller et al., 2013)N/A
D. melanogaster: daughterless-GAL4Drosophila Bloomington Stock Centre# 27608
D. melanogaster: hs-FLP;; tub-FRT-Gal80-FRT-Gal4, UAS–mRFPGift from Y. Bellaiche, Institut Curie, PSL Research University, Paris, France.N/A
D. melanogaster: hs-FLP; tub-GAL4, UAS-GFP; FRT82b, tub-GAL80Gift from N. Perrimon, Harvard Medical School, Boston, USAN/A
D. melanogaster: hs-FLP; sqh-Sqh::GFP; tub>Gal80>Gal4, UAS–mRFPGift from Y. Bellaiche, Institut Curie, PSL Research University, Paris, France.N/A
D. melanogaster: wild typeDrosophila Bloomington Stock Centre# 25210
D. melanogaster: PP2A-A[EP2332] / CyoDrosophila Bloomington Stock Centre# 1704
D. melanogaster: Patched-GAL4Drosophila Bloomington Stock Centre# 65661
D. melanogaster: UAS-myc-Moesin[T559D]Drosophila Bloomington Stock Centre# 8630
D. melanogaster: UAS-sh MoesinDrosophila Bloomington Stock Centre# 8629
D. melanogaster: UAS-FLAG-GFPReference (Gamblin et al., 2018)N/A
Table 2
Detailed genotypes.
Figure 1
Aendo-E-Cadherin::GFP / +; UAS-sh mCherry / matαtub67;15 GAL4
endo-E-Cadherin::GFP / +; UAS-sh yrt / matαtub67;15 GAL4
Bendo-E-Cadherin::GFP / +; UAS-sh mCherry / matαtub67;15 GAL4
endo-E-Cadherin::GFP / +; UAS-sh yrt / matαtub67;15 GAL4
Cendo-E-Cadherin::GFP / UAS-LacZ; daughterless-GAL4 / +
endo-E-Cadherin::GFP / +; UAS-FLAG-Yrt / daughterless-GAL4
Dendo-E-Cadherin::GFP / UAS-LacZ; daughterless-GAL4 / +
endo-E-Cadherin::GFP / +; UAS-FLAG-Yrt / daughterless-GAL4
Figure 2
Asqh-Sqh::GFP; UAS-sh mCherry / matαtub67;15 GAL4
sqh-Sqh::GFP ; UAS-sh yrt / matαtub67;15 GAL4
Bsqh-Sqh::GFP; UAS-sh mCherry / matαtub67;15 GAL4
sqh-Sqh::GFP ; UAS-sh yrt / matαtub67;15 GAL4
Csqh-Sqh::GFP; UAS-sh mCherry / matαtub67;15 GAL4
sqh-Sqh::GFP ; UAS-sh yrt / matαtub67;15 GAL4
Dsqh-Sqh::GFP; UAS-sh mCherry / matαtub67;15 GAL4
sqh-Sqh::GFP ; UAS-sh yrt / matαtub67;15 GAL4
Ehs-FLP; sqh-Sqh::GFP; UAS-LacZ / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP; sqh-Sqh::GFP; UAS-FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Fhs-FLP; sqh-Sqh::GFP; UAS-LacZ / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP; sqh-Sqh::GFP; UAS-FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Figure 3
APatched-GAL4 / +; UAS-FLAG-GFP / +
Patched-GAL4 / +; IAS-FLAG-Yrt / +
BPatched-GAL4 / +; UAS-FLAG-GFP / +
Patched-GAL4 / +; IAS-FLAG-Yrt / +
Chs-FLP / +; ; UAS- LacZ / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Dhs-FLP / +; ; UAS- LacZ / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Ehs-FLP / +; sh sqh / +; UAS- LacZ / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh LexA / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh sqh / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Fhs-FLP / +; sh sqh / +; UAS- LacZ / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh LexA / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh sqh / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Figure 4
Ahs-FLP / +; ; UAS- aPKC[CAAX] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Bhs-FLP / +; UAS- LacZ / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Chs-FLP / +; ; UAS-aPKC[CAAX], UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Dhs-FLP / +; UAS- LacZ / +; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Ehs-FLP / +; ; UAS-aPKC[CAAX], UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Fhs-FLP / +; ; UAS- FLAG-Yrt[5D] / tub-FRT-GAL80-FRT-Gal4, UAS–mRFP
Ghs-FLP / +; ; UAS- FLAG-Yrt[FWA] / tub-FRT-GAL80-FRT-Gal4, UAS–mRFP
Hhs-FLP / +; ; UAS- aPKC[CAAX] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS- LacZ / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS-aPKC[CAAX], UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS- LacZ / +; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS-aPKC[CAAX], UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Ihs-FLP / +; UAS- LacZ / +; + / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-Gal4, UAS–mRFP
hs-FLP / +; ; UAS- FLAG-Yrt[5D] / tub-FRT-GAL80-FRT-Gal4, UAS–mRFP
hs-FLP / +; ; UAS- FLAG-Yrt[FWA] / tub-FRT-GAL80-FRT-Gal4, UAS–mRFP
Figure 5
Awild type ovaries treated with DMSO or aPKC inhibitor
Bhs-FLP / +; ; sh aPKC / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Chs-FLP; tub-GAL4, UAS-GFP / +; FRT82b, crb[11a22] / FRT82b, tub-GAL80 treated with aPKC inhibitor
Dhs-FLP; tub-GAL4, UAS-GFP / +; FRT82b, crb[11a22] / FRT82b, tub-GAL80 treated with aPKC inhibitor
Ehs-FLP; tub-GAL4, UAS-GFP / fosCrb[Y10A]; FRT82b, crb[11a22] / FRT82b,tub-GAL80 treated with aPKC inhibitor
Fhs-FLP; tub-GAL4, UAS-GFP / fosCrb[Y10A]; FRT82b, crb[11a22] / FRT82b,tub-GAL80 treated with aPKC inhibitor
Ghs-FLP; tub-GAL4, UAS-GFP / UAS-LacZ; FRT82b, crb[11a22] / FRT82b,tub-GAL80
hs-FLP; tub-GAL4, UAS-GFP / UAS-FLAG-Yrt[5A]; FRT82b / FRT82b,tub-GAL81
hs-FLP; tub-GAL4, UAS-GFP / UAS-FLAG-Yrt[5A]; FRT82b, crb[11a22] / FRT82b,tub-GAL80
Hhs-FLP; tub-GAL4, UAS-GFP / UAS-LacZ; FRT82b, crb[11a22] / FRT82b,tub-GAL80
hs-FLP; tub-GAL4, UAS-GFP / UAS-FLAG-Yrt[5A]; FRT82b / FRT82b,tub-GAL81
hs-FLP; tub-GAL4, UAS-GFP / UAS-FLAG-Yrt[5A]; FRT82b, crb[11a22] / FRT82b,tub-GAL80
Ihs-FLP; tub-GAL4, UAS-GFP / UAS-FLAG-Yrt[5A], fosCrb; FRT82b, crb[11a22] / FRT82b,tub-GAL80
hs-FLP; tub-GAL4, UAS-GFP / UAS-FLAG-Yrt[5A], fosCrb[Y10A]; FRT82b, crb[11a22] / FRT82b,tub-GAL80
Jhs-FLP; tub-GAL4, UAS-GFP / UAS-FLAG-Yrt[5A], fosCrb; FRT82b, crb[11a22] / FRT82b,tub-GAL80
hs-FLP; tub-GAL4, UAS-GFP / UAS-FLAG-Yrt[5A], fosCrb[Y10A]; FRT82b, crb[11a22] / FRT82b,tub-GAL80
Figure 6
Ahs-FLP / +; ; UAS-sh moe / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP treated with H2O or aPKC inhibitor
Bhs-FLP / +; ; UAS-sh moe / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP treated with H2O or aPKC inhibitor
Chs-FLP / +; UAS- myc-Moesin[T559D] / +; + / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS- LacZ / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS- myc-Moesin[T559D] / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Dhs-FLP / +; UAS- myc-Moesin[T559D] / +; + / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS- LacZ / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS- myc-Moesin[T559D] / +; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Figure 7
Ahs-FLP / +; ; UAS- LacZ / tub-FRT-Gal80-FRT-Gal4, UAS–mRFP
hs-FLP / +; ; UAS-aPKC[CAAX], Par6 / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS-Pak[Myr] / +; + / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS-Pak[Myr] / +; UAS-aPKC[CAAX], Par6 / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Bhs-FLP / +; ; UAS- LacZ / tub-FRT-Gal80-FRT-Gal4, UAS–mRFP
hs-FLP / +; ; UAS-aPKC[CAAX], Par6 / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS-Pak[Myr] / +; + / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; ; UAS-Pak[Myr] / +; UAS-aPKC[CAAX], Par6 / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
CDaughterless-GAL4 / +; UAS-FLAG-GFP / Daughterless-GAL4
Daughterless-GAL4 / +; UAS-aPKC[CAAX], Par6 / Daughterless-GAL4
Daughterless-GAL4 / UAS-Pak[myr]; + / Daughterless-GAL4
D wild type
Pp2A-29b[EP2332]
E wild type embryos treated with DMSO or PP2A inhibitor
FDaughterless-GAL4 / UAS-Pak[myr]; + / Daughterless-GAL4 treated with DMSO or PP2A inhibitor
Figure 8
Ahs-FLP / +; ; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP treated with DMSO or Pak1 inhibitor
Bhs-FLP / +; ; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP treated with DMSO or Pak1 inhibitor
Chs-FLP / +; sh Pak1; + / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh Pak1; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh Pak1; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Dhs-FLP / +; sh Pak1; + / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh lexA; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh Pak1; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh lexA; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh Pak1; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Ehs-FLP / +; ; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP treated with DMSO or PP2A inhibitor
hs-FLP / +; ; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP treated with DMSO or PP2A inhibitor
Fhs-FLP / +; ; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP treated with DMSO or PP2A inhibitor
hs-FLP / +; ; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP treated with DMSO or PP2A inhibitor
Ghs-FLP / +; ; sh Pp2A-29b / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS-FLAG-Yrt; sh Pp2A-29b / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS-FLAG-Yrt[5A]; sh Pp2A-29b / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
Hhs-FLP / +; ; sh Pp2A-29b / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh lexA; UAS- FLAG-Yrt / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS-FLAG-Yrt; sh Pp2A-29b / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; sh lexA; UAS- FLAG-Yrt[5A] / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP
hs-FLP / +; UAS-FLAG-Yrt[5A]; sh Pp2A-29b / tub-FRT-GAL80-FRT-GAL4, UAS–mRFP

Laser ablation for measurement of mechanical properties

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Stage 14 embryos were dechorionated for 2 min 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 s 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 modeled 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:

Lt=D1-e-tτ

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 τ:

τ=μE

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.

Immunofluorescence

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Dissected ovaries or dechorionated embryos were fixed for 15 min in 4% paraformaldehyde or heat fixed (aPKC, Yrt, Crb, and Arm stainings) as previously described (Gamblin et al., 2014). Fixed ovaries and embryos were blocked for 1 hr in NGT (2% normal goat serum, 0.3% Triton X-100 in PBS). Ovaries and embryos were incubated with the following primary antibodies overnight at 4°C: mouse anti-Dlg1 [4F3, Developmental Studies Hybridoma Bank at the University of Iowa (DSHB)], 1:25 dilution; rabbit anti-Yrt, 1:1000 (Biehler et al., 2020); guinea-pig anti-Yrt (Laprise et al., 2006), 1:250; mouse anti-GFP (Roche, Sigma Aldrich), 1:200; rabbit anti-GFP (A-11122, Invitrogen) 1:200; mouse anti-Fas3 (7G10, DSHB), 1:10; rabbit anti-RFP (600-401-379, Rockland), 1:400; rabbit anti-Lgl (d-300, Santa Cruz), 1:100; rabbit anti-aPKC (C-20, Santa Cruz Biotechnology), 1:200; rat anti-Crb (Sollier et al., 2015), 1:250. Ovaries and embryos were then washed three times in PBT (0.3% Triton X-100 in PBS) before and after incubation with secondary antibodies (1:400 in NGT, 1 hr at room temperature), which were conjugated to Cy3 (Jackson ImmunoResearch Laboratories) or Alexa Fluor 488 (Molecular Probes). Ovarioles were mechanically separated and mounted in Vectashield mounting medium (Vector Labs), and imaged with a LSM700 confocal (63× Apochromat lens with a numerical aperture of 1.40). Embryos were mounted in Vectashield mounting medium and imaged with the Zeiss LSM700 confocal using LD C-Apochromat 40x 1.1 NA water Korr objective. Images were uniformly processed with ImageJ (National Institutes of Health).

Phosphatase assays

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Dechorionated embryos were homogenized in ice-cold lysis buffer (1% Triton X-100, 50 mM TRIS-HCl pH 7.5, 5% glycerol, 150 mM NaCl, 1 mM PMSF, 0.5 μg/mL aprotinin, 0.7 μg/mL pepstatin, and 0.5 μg/mL leupeptin). Lysates were cleared by centrifugation at 4°C, and 400 units of λ Phosphatase (New England Biolabs) was added to 30 μg of proteins extracted from embryos. 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.

Western blotting

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Dechorionated embryos were homogenized in ice-cold lysis buffer (1% Triton X-100, 50 mM TRIS HCl pH 7.5, 5% glycerol, 100 mM NaCl, 50 mM NaF, 5 mM EDTA pH 8, 40 mM β-glycerophosphate, 1 mM PMSF, 0.5 μg/mL aprotinin, 0.7 μg/mL pepstatin, 0.5 μg/mL leupeptin and 0.1 mM orthovanadate) and processed for SDS-PAGE and western blotting as previously described (Laprise et al., 2002). Primary antibodies used: Rabbit anti-Yrt (Biehler et al., 2020), 1:10,000; mouse anti-β−Tubulin (E7, DSHB), 1:2000; rabbit anti-aPKC (C-20, Santa Cruz Biotechnology), 1:2000; rabbit anti-PP2A-A (Krahn et al., 2009), 1:10,000; mouse anti-Actin (NB-100–74340, Novus Biologicals), 1:2000. HRP-conjugated secondary antibodies were from GE Healthcare and used at a 1:2000 dilution.

Chemical treatment of embryos

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Dechorionated embryos were incubated under agitation for 1 hr 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.

Chemical treatment of ovaries

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Dissected ovaries were treated for 2 hr with inhibitors of aPKC (CRT-006-68-54, Bio-Techn Sales Corporation, 10 μM) or Pak1 (IPA-3, Millipore Sigma, 50 μM) as described (Chartier et al., 2012).

Measurements of apical domain size and quantification of fluorescence intensity

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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 Institutes of Health). Control cells facing labeled cells on the opposite side of the follicle were used as control. Results are expressed as the ratio between measurements in labeled cells and control cells. A minimum of 15 follicles were analyzed in each condition. A similar approach was used to analyse apical domain width of epidermal cells expressing FLAG-GFP (control) or FLAG-Yrt in stage 13 embryos (n ≥ 13 from 7 different embryos). FLAG-GFP- or FLAG-Yrt-negative cells in the same embryonic segment were used as control. Statistical differences between groups were determined using one-way ANOVA (apical domain size) or Student’s t-test (fluorescence intensity).

Analysis of myosin distribution in the embryonic epidermis

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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 (5 slices of 0.5 μm each) of the epidermis. A 20 μm line was drawn along the anterior-posterior axis of the tissue in each embryo to calculate mean intensity and heterogeneity (Zulueta-Coarasa and Fernandez-Gonzalez, 2018) of the GFP signal. Here, heterogeneity is defined as:

standarddeviation(intensity)mean(intensity)

This measurement captures the variability within each linescan, which increases when there are clear peaks and troughs.

Data availability

Source data have been provided for all figures and are available as supporting files.

References

Decision letter

  1. Michel Bagnat
    Reviewing Editor; Duke University, United States
  2. Anna Akhmanova
    Senior Editor; Utrecht University, Netherlands

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

[Editors' note: this paper was reviewed by Review Commons.]

Acceptance summary:

In this manuscript, the authors investigated how the balance of apical and lateral membrane domains is regulated in epithelial cells of the Drosophila embryonic epidermis. They found that the protein Yurt, which controls the function of the apical determinant Crumbs, is regulated by a balance of phosphorylation/de-phosphorylation by PP2A and aPKC. Their results revealed that these interactions reciprocally regulate apical contractility and polarization, thereby providing a homeostatic mechanism that may link cell polarity and tissue mechanics.

https://doi.org/10.7554/eLife.67999.sa1

Author response

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Atypical protein kinase C (aPKC) and p21-activated kinase 1 (Pak1) are kinases acting downstream of Cdc42 that have many common substrates and redundant activity on target proteins and epithelial cell polarity. In this work, authors find that they have opposing effects on Yurt. Unphosphorylated Yurt is recruited to the apical membrane by Crumbs and increases Myosin dependent apical tension. While aPKC-dependent phosphorylation of Yurt represses this apical recruitment, Pak1 mediated activation of Protein phosphatase 2A dephosphorylates Yurt thereby increasing apical localization and tension.

This is an interesting manuscript that presents novel insights into how cortical tension is regulated by Yurt. The experimental data broadly support the conclusions and the discovery that aPKC and Pak1 have opposite effects on Yurt is a welcome observation in light of the recent claim that these kinases are redundant with each other. The manuscript could be improved in several ways, but the first two are not essential for publication:

1. All of the experiments involve artificial conditions in which Yurt is over-expressed. However, aPKC is active in most if not all epithelia and Yurt is therefore restricted to the lateral domain. Is there any evidence that changes in the apical recruitment of Yurt modify cortical tension in wild-type cells during development? Demonstrating that the apical localisation of Yurt is developmentally regulated and plays a role in morphogenesis would substantially increase the significance of their results.

We fully agree with reviewer 1 that demonstrating the importance of the pathway that we described in a physiological process during development would greatly increase the significance of our study. We have previously showed that the apical recruitment of Yrt is developmentally regulated (Laprise et al., 2006). Yrt is confined to the lateral membrane in embryonic epithelial tissues from the end of cellularization till stage 13. At stage 14, Yrt is recruited apically by Crb and localizes to both apical and lateral membranes in the epidermis. Specifically, Yrt is enriched at lateral septate junctions, and also co-localizes with Crb within the marginal zone (Laprise et al., 2006; Laprise et al., 2009). We found that knockdown of yrt decreased cortical Sqh levels at the apex of epidermal cells from stage 14 of embryogenesis, whereas Yrt depletion had no major impact of Myosin distribution at earlier stages of development (Figure 2 of the revised manuscript). This strongly argues that the apical recruitment of Yrt confers to the latter the ability to control Myosin-dependent function. In addition, Yrt-depleted cells showed delayed elongation along the dorso-ventral axis at stage 14. This suggests that the Yrt-dependent recruitment of Myosin contributes to cell elongation along the dorsal-ventral axis in preparation for dorsal closure, thereby providing a putative molecular and cellular basis explaining defective dorsal closure in yrt mutant embryos (Hoover and Bryant, 2002; Laprise et al., 2006).

2. Along similar lines, does unphosphorylated Yurt increase cortical tension laterally when localised there or does the recruitment of Myosin also require Crumbs?

Yrt only impacts Myosin distribution following its apical recruitment in the embryonic epidermis. Morever, Yrt overexpression recruits Myosin specifically at the apical membrane (Figure 2E, F) in follicle cells. This recruitment requires the apical protein Crb (Figure Author response image 1). Consistent with these results, overexpression of wild type Yrt or nonphosphorylatable Yrt5A decreases cell diameter exclusively at the cell apex, a function that requires Crb (Figure 3A-D; 5G, H). In addition, mutation of the FERM-binding domain of Crb, which is required for apical recruitment of Yrt (Laprise et al., 2006), abolished Yrtinduced apical constriction (Figure 5I, J). We thus believe that Yrt primarily acts downstream of Crb to increase contractility at the apical domain, and that Yrt has a limited role in promoting cortical tension at the lateral domain. Our data thus propose that Crb-bounded Yrt promotes cortical tension (this study), whereas lateral Yrt maintains lateral membrane identity and controls septate junction permeability [Figure 9 of the revised manuscript; (Laprise et al., 2006; Laprise et al., 2009)].

Author response image 1
Yrt is a multifunctional protein promoting cortical tension downstream of Crb.

Mosaic expression of FLAG-Yrt (F-Yrt) with a control shRNA or a shRNA targeting crb in follicular epithelial cells constitutively expressing Sqh::GFP, which was visualized by immunofluorescence. FLAG-Yrt-expressing cells (middle and right panels) are labeled with mRFP. The lower part of the figure shows quantification of apical Sqh intensity in cells expressing the transgenes listed above. Results are presented as a ratio between clonal cells and control cells in the same follicle (see Author response image 2; *P £ 0.05, **P £ 0.01).

3. Most of the images are tiny and show only small regions of the embryo or follicular epithelium, which makes it very difficult to tell which part of the tissue these come from. Cortical tension varies with position and developmental stage in the follicle cell layer (see for example Balaji et al. (2019) Development: 146, dev171256), and it is therefore important to specify both, either by showing a larger region of each egg chamber or by putting this information in the Figure legends. To illustrate this point, figure 2J is meant to show that cells treated with the aPKC inhibitor have a reduced apical area, but the righthand image looks like wild-type cells at either the anterior or posterior end of the egg chamber, where the epithelium is more curved.

We thank reviewer 1 for mentioning the work of Balaji et al. Of note, we only used previtellogenic follicles for quantification (stage 3-6 follicles; this information is available in the paper, see p.20). At these stages, follicles display limited elongation, and all follicular epithelial cells are cuboidal. This suggests a relatively homogenous tension across the tissue. Accordingly, Balaji et al. showed that differential Myosin distribution and the ensuing modulation of tension in follicular epithelial cells mostly occur between stage 6 to 9 when egg chambers lengthen, and epithelial cells acquire different morphologies along the antero-posterior axis. In addition, we haven’t observed variation in phenotypes related to cell position upon Yrt overexpression. However, we recognize that cortical tension may vary with position within the follicle, even at pre-vitellogenic stages. This is why we always selected cells facing mutant/overexpressing cell clones in the follicle as control (mirror image). In this way, control cells are at the same developmental stage and occupy a position within the follicle with similar physical constrains. This is explained in Methods and illustrated in Figure Author response image 2. In parallel to acquiring high magnification images, which facilitate visualization of apical constriction in the paper, we systematically imaged the entire follicle (or a large portion of it) for quantification of apical domain width of control and mutant/transgene-expressing cells. This enabled us to provide the stage of all follicles in figure legends of the revised manuscript, as requested by reviewer 1.

Author response image 2
We used mosaic analysis in the Drosophila follicular epithelium.

The impact of clonal gene depletion and/or protein overexpression on apical constriction was assessed by measuring apical domain width of cells within clones (pink cells) and control cells occupying an equivalent position on the other side of the follicle (grey cells). Results were expressed as the ratio between the width of clonal cells and the width of control cells in the same follicle.

4. Although the panels present examples that prove the relevant point for the specific figure, there are some inconsistencies across figures. For example, Yurt localises apically in Figure 2K, but there is no obvious apical constriction.

It is true that inhibition or knockdown of aPKC (shown as Figure 5A, B in the revised manuscript) is not sufficient to significantly induce apical constriction although Yrt is partially relocalized apically under these conditions. As explained in the discussion (see pages 12 and 13 of the manuscript), our model is that Yrt competes with other FERM domain proteins, such as Moesin (Moe), for binding to Crb. Of note, Yrt and Moe have opposite effects on Myosin activity downstream of Crb [this study; (Flores-Benitez and Knust, 2015; Laprise et al., 2006; Medina et al., 2002; Salis et al., 2017)]. It is thus possible that the amount of Yrt relocalized to the apical domain upon inhibition of aPKC is not sufficient to fully outcompete Moe, whereas Yrt overexpression reaches the threshold required to displace most Moesin molecules. Accordingly, we found that inhibition of aPKC in Moe knockdown cells caused apical constriction, in contrast to what was observed in control cells (Figure 6A, B of the revised manuscript). In addition, we found that expression of active Moe suppresses Yrt-induced apical constriction (Figure 6C, D), thereby supporting the concept that these proteins are involved in a competitive functional interaction. These data provide further support to our model that Crb functions are specified by FERM domain proteins that competitively associate with Crb to form distinct complexes.

Specific comments:

1. Figure 2L/2O : It is not clear if Yurt is relocalizing in 2L/O in the control cells on aPKC inhibitor treatment. Quantification along with a better image would help readers appreciate this change.

We provided better images along with quantification confirming that the apical relocalization of Yrt upon inhibition of aPKC requires Crb with an intact FERM-domain binding motif (Figure 5C-F of the revised manuscript).

2. Figure 3B – Top right: The Yrt/ aPKC signal is not clear. It would help to provide a better image.

We replaced Figure 3B (now 7A in the revised manuscript), which confirms that expression of aPKCCAAX together with Par6 reduces membrane association of Yrt. This has also been quantified (see Figure 7B of the revised manuscript).

3) The evidence that PAK1 regulates Yrt phosphorylation rests almost entirely on the treatment with a Pak1 inhibitor, as the expression of myristolated Pak1 has little effect on Yurt phosphorylation (Figure 3C). Since inhibitors often have off target effects, it would help to confirm this results by making pak1 mutant clones.

We showed that shRNA-mediated knockdown of Pak1 suppresses apical constriction induced by Yrt overexpression (Figure 8C, D of the revised manuscript). This complements the use of the Pak1 inhibitor (Figure 8A, B) and demonstrates that Pak1 is required for Yrtdependent apical constriction. To supplement Figure 3C (now Figure 7C) and further support our conclusion that Pak1 sustains Yrt function by reducing its phosphorylation on aPKC target sites, we expressed Yrt5A with a control shRNA or a shRNA targeting Pak1 (the S/T residues targeted by aPKC are mutated to non-phosphorylatable A residues in Yrt5A). We found that Yrt5A induces apical constriction in Pak1-depleted cells whereas wild type Yrt is unable to do so in absence of Pak1. This solidifies our model indicating that Pak1 support Yrt function by preventing phosphorylation of aPKC target sites. This result was added as Figure 8C-D of the revised manuscript.

Reviewer #1 (Significance (Required)):

This is an interesting manuscript that presents novel insights into how cortical tension is regulated by Yurt. The experimental data broadly support the conclusions and the discovery that aPKC and Pak1 have opposite effects on Yurt is a welcome observation in light of the recent claim that these kinases are redundant with each other.

All of the experiments involve artificial conditions in which Yurt is over-expressed. However, aPKC is active in most if not all epithelia and Yurt is therefore restricted to the lateral domain. Is there any evidence that changes in the apical recruitment of Yurt modify cortical tension in wild-type cells during development? Demonstrating that the apical localisation of Yurt is developmentally regulated and plays a role in morphogenesis would substantially increase the significance of their results.

Along similar lines, does unphosphorylated Yurt increase cortical tension laterally when localised there or does the recruitment of Myosin also require Crumbs?

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary

The manuscript by Biehler et al. addresses the role and regulation of Yrt in promoting cortical tension and apical constriction. The authors show using laser ablation that Yrt is necessary and sufficient to promote cortical tension. Moreover, Yrt overexpression results in accumulation of Myosin and apical cell constriction. Furthermore, Yrt's ability to promote apical constriction depends on Crb and is counteracted by the kinase aPKC. Finally, overexpression of a membrane-targeted form of the kinase Pak1 decreases Yrt phosphorylation in a PP2A (a phosphatase) dependent manner. PP2A antagonizes aPKC's function on Yrt. The authors conclude that Pak1-induced PP2A activity antagonizes aPKC to promote Yrt-induced apical constriction.

Major comments

1. Figure 1 A-D It is unclear why the measured retraction velocities of the two controls in B and D are so different. The difference in mean retraction velocity between the two controls exceeds the differences in mean retraction velocity between controls and experiments. The authors should comment (or repeat the laser ablation experiments). The authors also should show the retraction as a function of time after ablation.

The differences in retraction velocities in our original data set could be explained by the large gap in time (~6 months) between the experiments, which could have affected laser power and other experimental conditions. We repeated all the laser ablation experiments within 2 weeks and replaced the data in Figure 1. There is now no significant difference between the controls in Figures 1B and 1D. As before, yrt knockdown resulted in slower retraction velocities (reduced cortical tension), and Yrt overexpression increased retraction velocities (increased tension). We included kymographs of the junction ablation experiments in Figures 1A and 1C to show retraction over time.

2. Figure 1E,F It is unclear how yrt-dependent changes in viscosity relate to alterations in cortical tension. The authors should clarify.

We updated our calculations of viscoelasticity of the tissue and found that there was no significant effect of Yrt manipulation on tissue viscosity. Previously, we fitted the individual retraction vs. time curves with the Kelvin-Voigt model out to 68 seconds following ablation. However, we realized that the goodness of fit decreased significantly when we included data beyond 40 seconds after laser ablation (Author response image 3, left). The reason for this is that the Kelvin-Voigt model does not fit well the stress relaxation that occurs at longer time scales. Thus, we fit the initial 36 seconds post-ablation of the newly acquired data, finding that the goodness of fit provides R values (correlation between data and model) consistently greater than 0.96 (Author response image 3, right). With this improved calculation, we found no effect of Yrt manipulation on relaxation time, and thus no effect on tissue viscosity: 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. Thus, changes in recoil velocity can be attributed to changes in cortical tension.

Author response image 3
Kelvin-Voigt model fits retraction over time better at shorter time scales.

(Left) A sample set of retraction vs. time data (points) fit with the Kelvin-Voigt model (line). The accuracy of the fit for the initial recoil is sacrificed in an attempt to fit the longer stress relaxation response. (Right) The same data fit with the Kelvin-Voigt model only up to 36 seconds post-ablation (dashed line). The fit for the initial recoil response is significantly more accurate.

3. Figure 3A The use of a PAK1 inhibitor is not convincing. The authors should use pak1 mutant clones (or clones expressing sh-RNA targeting Pak1 ,see Figure 4) that allow a direct comparison of Yrb subcellular distribution in control and pak1 mutant cells.

We found that shRNA-mediated knockdown of Pak1 and inhibition of Pak1 activity with the chemical inhibitor IPA-3 both suppressed Yrt-induced apical constriction (Figure 8A-D of the revised manuscript). Combination of these complementary approaches confirms that Pak1 is required to sustain Yrt function, thereby supporting our model. In the original manuscript, we also showed that IPA-3 releases Yrt from the lateral membrane. We were not able to convincingly reproduce this phenotype by knocking down Pak1. Our hypothesis is that residual Pak1 in Pak1-knockdown cells is sufficient to maintain some Yrt at the lateral membrane. However, we cannot exclude that IPA-3 may have additional targets impacting Yrt localization as suggested by reviewer 2. We thus decided to remove this result from the manuscript to avoid any possible confusion. Importantly, we showed that expression of active Pak1Myr suppresses aPKC-induced displacement of Yrt from the membrane (see Figure 7B of the revised manuscript for quantification), further supporting our model that Pak1 and aPKC have antagonistic roles in regulating Yrt localization and function. Moreover, our data indicate that Yrt5A induces apical constriction in Pak1-depleted cells whereas wild type Yrt is unable to do so in absence of Pak1 (Figure 8C-D of the revised manuscript; the S/T residues targeted by aPKC are mutated to non-phosphorylatable A residues in Yrt5A). This indicates that Pak1 is dispensable for Yrt-induced apical constriction when aPKC target sites on Yrt are mutated to non phosphorylatable A residues. Altogether, our data indicates that Pak1 supports Yrt function by preventing phosphorylation of aPKC target sites.

Minor comments

4. Title: The title appears to be too general. A more informative title reflecting the interplay between Yrb/aPKC/Pak1 should be chosen.

The new title is: Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex.

5. Abstract: "…we show that Yurt is not a general inhibitor of crumbs…" This should be rephrased as the authors only address one specific function of Crb in their manuscript.

The sentence now reads as follow: ‘Here, we show that Yurt also increases Myosin dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells.’

6. Figure 1 The authors should clarify why they measured tension in one epithelium (embryonic epidermis) and did the remaining analyses in another epithelium (follicle epithelium).

We measured tension in the embryonic epidermis because this is a well-established model for such experiments, and our experimental setup is adapted to this tissue. The apical surface of the embryonic epidermis faces the exterior of the embryo. In contrast, the apical surface of the follicular epithelium faces the interior of the egg chamber. Using the embryonic epidermis for laser ablation assays facilitates laser focusing on apical and subapical junctional regions, minimizing the loss of laser power caused by light travelling through cells. The laser power lost would be significantly greater if we targeted (sub)apical regions in the follicular epithelium.

We then moved to the follicular epithelium to monitor apical constriction, as cell organization in this tissue is well suited for visualization of the apical-basal axis. In addition, defects in tissue architecture are much less pronounced in the follicular epithelium upon alteration of epithelial polarity or cell contractility, thereby facilitating the analysis of cell shape and integrity. Additionally, for genetic manipulations, the follicular epithelium provides a much more tractable experimental system in which clonal analysis is more effective than in the embryo. However, we have data showing that Yrt also induces apical constriction in the embryonic epidermis similar to what we observed in the follicular epithelium, showing that Yrt promotes apical constriction in both embryonic and adult tissues. This result was added to the manuscript as Figure 3A, B.

7. Page 8, top "…demonstrate that the Crb-YRT complex promotes cortical tension and apical constriction." The authors did not measure cortical tension in this context. The authors should rephrase their conclusion more carefully.

We rephrased this part of the manuscript, which is now: ‘Together, these results demonstrate that the Crb–Yrt complex promotes apical constriction’.

8. Figure 3C should indicate aPKC-CAAX + Par6 and not only aPKC-CAAX.

This has been fixed.

9. It would be helpful for the reader if the authors could provide a summary/model of their work as an additional panel to Figure 4.

We added a schematic model summarizing our findings (Figure 9 in the manuscript).

10. Discussion: The data referred to as 'unpublished results' should be shown in supplementary figures.

We think that showing these results would break the flow of the paper. We simply removed the part of the discussion referring to these unpublished data, which are not necessary to support our conclusions. In addition, a published paper referenced in the discussion shows similar results (Salis et al., 2017). This also shortens the discussion, as requested by reviewer 2 (see 11, below).

11. Discussion: The discussion is long and should be written more concisely. The mere repetition of the data already shown in the results part should be minimized.

We trimmed the discussion and removed unnecessary/repetitive parts.

Reviewer #2 (Significance (Required)):

Significance

The manuscript provides a conceptual advance in the understanding of how interactions between regulatory proteins control mechanical tension and cell shape in Drosophila epithelia. Previous work showed that (i) Yrt is essential for epithelial polarity (Laprise, 2009), (ii) Yrt subapical localization depends on Crb (Salis, 2017), (iii) Yrt function is antagonized by aPKC (Gamblin, 2018; Gamblin, 2014) and (iv) Yrt promotes apical cell constriction in the pupal wing (Salis, 2017). The manuscript now adds two players (Pak1, PP2A) to the regulation of Yrt activity and clarifies Yrt's role in epithelial morphogenesis.

Readership

The manuscript might be of interest to researchers studying cell polarity and epithelial morphogenesis in animals.

Field of expertise

Drosophila

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this manuscript Biehler and co-workers analyzed the role of Yurt (Yrt) a protein know to anatgonize the role of Crumbs (Crb) in apical membrane growth. Using Drosophila (embryos and egg chambers) as a model system they found that Yrt promotes myosinIImediated apical constriction and that aPKC prevents Crb-Yrt interaction through direct phosphorylation of Yrt, which results in Yrt dissociation from the apical surface and repression of apical constriction. In contrast, the kinase Pak1 promotes Yrt dephosphorylation, through activation of the phosphatase PP2A, facilitating apical constriction. In summary this manuscript provides evidence for a role for Yrt in apical constriction but it remains unclear how this is related to the role of Crumbs in limiting apical membrane growth.

Comments:

1) The snapshot of the laser ablation experiments presented in Figure 1 demonstrate the presence of large fluorescence aggregate after photoablation (panel A and C). This suggests that rather that punctual cutting of junctional structures the laser light might have caused the production of cavitation bubbles. No videos for these experiments have been provided making it difficult to evaluate the validity of these perturbations. Also, it is unclear how many times these experiments have been repeated, n=17 junctions, does this refer to 17 junctions in the same embryo? If so, it must be repeated at least in three different embryos.

The circular marks that are visible following laser ablation are not cavitation bubbles, but auto fluorescent holes made by the laser in the vitelline membrane, the protective sac that encloses the Drosophila embryo. To show this, we have attached both a maximum intensity projection of the tissue (the approach used to generate the images shown in Figure 1A and 1C) and the individual Z-slices that make up the projected image in an embryo expressing DE-cadherin::GFP (Author response image 4). The first slice labelled Z = 0 µm is the plane containing the vitelline membrane. The vitelline membrane plane displays weak autofluorescence and the bright-rimmed hole caused by laser ablation. The outline of the hole is bright due to ultraviolet-induced photoactivation of the vitelline membrane. Subsequent slices move basally into the epidermis. The slice at Z = 0.5 µm is the plane in which the ablation laser was focused, corresponding to the plane of adherens junctions. Because the surface of the embryo is curved, travelling further in Z brings adherens junctions further from the center of the image into focus. When the maximum intensity projection is generated, the image shows the circular ring apparently in the plane of the severed junction, but this is just a result of the projection. Note that over time (Video 1 and 2), the intensity of the mark on the vitelline membrane decays, consistent with it being the result of UV-induced photoactivation. In addition, the fluorescent mark on the vitelline membrane does not move despite movements of the cells (Video 1, 2), further confirming that the fluorescent mark is in a static structure (the vitelline membrane is glued to the coverslip). We offer a brief explanation of the circular fluorescent marks in the Figure 1 legend.

Author response image 4
Autofluorescent holes in the vitelline membrane generated by laser ablation.

The leftmost image shows the maximum intensity projection of a section of epidermal tissue immediately following ablation. The rest of the images are individual confocal slices starting at the plane of the vitelline membrane (Z = 0 um) where the hole caused by ablation is in focus, and moving basally into the tissue. Note that the ablated adherens junction is in focus at the plane Z = 0.5 um.

We have also included videos of the laser ablation experiments as supplemental material.

The number of experiments reported corresponds to the number of junctions severed. Only one junction was ablated in any given embryo. Therefore, n = 17 junctions indicates that 17 embryos were imaged with one junction ablated in each embryo. We have adjusted the figure legend to clarify this point.

2. Experiments have been performed in two different model epithelia, however there is no explanation of why some experiments were done in one system and some in the other.

Please refer to comment no. 6 of reviewer 2 for a detailed answer to this point.

3. The authors should specify for each of the panel presented in how many embryos/egg chambers were the experiments repeated. It seems all the quantifications were made using multiple cells form one sample.

We used multiple independent samples. This was clarified in both methods and figure legends.

Reviewer #3 (Significance (Required)):

The results presented in this stud expand our knowledge on how polarity proteins regulate epithelial cell polarization and cell contractility but are overall rather incremental compared to what it is known already, which limits my overall enthusiasm.

Referees cross-commenting

In light of reviewer 2 and my comments, I think it will be particularly important that the authors clarify the laser ablation experiments as detailed in the report.

References

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Karagiosis, S.A., and D.F. Ready. 2004. Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis. Development. 131:725-732.

Laprise, P., S. Beronja, N.F. Silva-Gagliardi, M. Pellikka, A.M. Jensen, C.J. McGlade, and U. Tepass. 2006. The FERM protein Yurt is a negative regulatory component of the Crumbs complex that controls epithelial polarity and apical membrane size. Developmental cell. 11:363-374.

Laprise, P., K.M. Lau, K.P. Harris, N.F. Silva-Gagliardi, S.M. Paul, S. Beronja, G.J. Beitel, C.J. McGlade, and U. Tepass. 2009. Yurt, Coracle, Neurexin IV and the Na(+),K(+)-ATPase form a novel group of epithelial polarity proteins. Nature. 459:1141-1145.

Laprise, P., and U. Tepass. 2011. Novel insights into epithelial polarity proteins in Drosophila. Trends Cell Biol.

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https://doi.org/10.7554/eLife.67999.sa2

Article and author information

Author details

  1. Cornelia Biehler

    1. Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
    2. axe oncologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec-UL, Québec, Canada
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Katheryn E Rothenberg

    1. Institute of Biomedical Engineering, University of Toronto, Toronto, Canada
    2. Ted Rogers Centre for Heart Research, University of Toronto, Toronto, Canada
    Contribution
    Data curation, Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8191-7528
  3. Alexandra Jette

    1. Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
    2. axe oncologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec-UL, Québec, Canada
    Contribution
    Formal analysis, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5423-2374
  4. Helori-Mael Gaude

    1. Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
    2. axe oncologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec-UL, Québec, Canada
    Contribution
    Data curation, Investigation
    Competing interests
    No competing interests declared
  5. Rodrigo Fernandez-Gonzalez

    1. Institute of Biomedical Engineering, University of Toronto, Toronto, Canada
    2. Ted Rogers Centre for Heart Research, University of Toronto, Toronto, Canada
    3. Department of Cell and Systems Biology, University of Toronto, Toronto, Canada
    4. Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, Canada
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0770-744X
  6. Patrick Laprise

    1. Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
    2. axe oncologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec-UL, Québec, Canada
    Contribution
    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    Patrick.Laprise@crchudequebec.ulaval.ca
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9785-4376

Funding

Canadian Institutes of Health Research (MOP-142236)

  • Patrick Laprise

Canadian Institutes of Health Research (MOP-156279)

  • Rodrigo Fernandez-Gonzalez

Canadian Institutes of Health Research

  • Katheryn E Rothenberg

Fonds de Recherche du Québec - Santé

  • Cornelia Biehler

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors would like to acknowledge A Wodarz, S Campuzano, T Harris, E Knust, D St-Johston, Y Bellaiche, N Perrimon, the Bloomington Drosophila Stock Center, the Drosophila Genomics Resource Center, and the Developmental Studies Hybridoma Bank for reagents. Flybase was used as an important database for this work. We also thank C Gamblin for critical reading of the manuscript. This work was supported by operating grants from the Canadian Institute of Health Research (CIHR) to PL (MOP-142236) and RFG (MOP-156279). CB is supported by a scholarship from FRQ-S, and K R is a postdoctoral CIHR grantee.

Senior Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewing Editor

  1. Michel Bagnat, Duke University, United States

Publication history

  1. Received: March 2, 2021
  2. Accepted: June 30, 2021
  3. Accepted Manuscript published: July 2, 2021 (version 1)
  4. Version of Record published: July 15, 2021 (version 2)

Copyright

© 2021, Biehler et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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