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A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity.

Gamblin CL, Hardy ÉJ, Chartier FJ, Bisson N, Laprise P - J. Cell Biol. (2014)

Bottom Line: However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined.In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane.The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Biologie Moléculaire, Biochimie Médicale et Pathologie and Centre de Recherche sur le Cancer, Université Laval, and 2 Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, Québec G1R 3S3, Canada.

ABSTRACT
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.

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aPKC-dependent phosphorylation of Yrt is crucial for epithelial cell polarity. (A–D) Cuticle preparation of embryos of the following genotypes: wild type (A), da-GAL4/UAS-Flag-yrtFL (ubiquitous expression of Flag-tagged YrtFL; B), da-GAL4/UAS-Flag-yrt5A (C), and da-GAL4/UAS-Flag-yrt5D (D). Bar, 100 µm (also applies to B–D). (E) Western blot using an anti-Flag antibody showing that all constructs were expressed at similar levels. Actin was used as a loading control. (F) Portion of the ventral ectoderm of control (da-GAL4; driver line used to express Yrt constructs, this line has a wild-type phenotype) stage 12 (St12) embryos costained with Yrt and Crb (left) or with Dlg and aPKC (right). (G–I) Left images show costaining of Flag and Crb, whereas right images depict costaining of Dlg and aPKC in the ectoderm of an embryo expressing Flag-tagged YrtFL (G), an embryo expressing Flag-Yrt5A (H), or an embryo expressing Flag-Yrt5D (I). Arrows in H point to cysts of cells with contracted apexes, whereas arrowheads show cells with reduced Crb and aPKC levels. Bar, 10 µm (also applies to G–I).
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fig3: aPKC-dependent phosphorylation of Yrt is crucial for epithelial cell polarity. (A–D) Cuticle preparation of embryos of the following genotypes: wild type (A), da-GAL4/UAS-Flag-yrtFL (ubiquitous expression of Flag-tagged YrtFL; B), da-GAL4/UAS-Flag-yrt5A (C), and da-GAL4/UAS-Flag-yrt5D (D). Bar, 100 µm (also applies to B–D). (E) Western blot using an anti-Flag antibody showing that all constructs were expressed at similar levels. Actin was used as a loading control. (F) Portion of the ventral ectoderm of control (da-GAL4; driver line used to express Yrt constructs, this line has a wild-type phenotype) stage 12 (St12) embryos costained with Yrt and Crb (left) or with Dlg and aPKC (right). (G–I) Left images show costaining of Flag and Crb, whereas right images depict costaining of Dlg and aPKC in the ectoderm of an embryo expressing Flag-tagged YrtFL (G), an embryo expressing Flag-Yrt5A (H), or an embryo expressing Flag-Yrt5D (I). Arrows in H point to cysts of cells with contracted apexes, whereas arrowheads show cells with reduced Crb and aPKC levels. Bar, 10 µm (also applies to G–I).

Mentions: To address the functional impact of the aPKC-dependent phosphorylation of Yrt in vivo, we generated transgenic flies expressing either Flag-tagged full-length Yrt (YrtFL) or the nonphosphorylatable mutant Yrt5A (full-length Yrt carrying S348A, S358A, T379A, S387A, and S392A mutations). Analysis of cuticle integrity revealed that overexpression of YrtFL had a dominant effect and resulted in the formation of large holes in the ventral epidermis and impairment of head epidermis morphogenesis (Fig. 3, A and B). Although expressed at a level similar to YrtFL (Fig. 3 E), Yrt5A was associated with a much more dramatic phenotype characterized by epithelial tissue collapse resulting in dispersed grains of cuticle (Fig. 3 C). Expression of Yrt5A phenocopies a complete loss of critical positive regulators of the apical domain, including Crb, Stardust, and aPKC (Tepass et al., 1990; Tepass and Knust, 1993; Harris and Peifer, 2007). This suggests that the Yrt mutant resistant to phosphorylation by aPKC has an enhanced ability to antagonize the apical machinery and, thus, disrupts the integrity of the apical domain. Accordingly, Crb and aPKC expression was severely reduced in most ectodermal cells of embryos expressing Yrt5A, and the lateral proteins Discs large (Dlg) and Yrt (Yrt5A) lined the entire circumference of cells lacking aPKC and Crb (compare Fig. 3, H [arrowheads] with F). Residual Crb and aPKC staining marked the contact point of contracted apexes of cells forming rosettes (Fig. 3 H, arrows). Yrt5A and Dlg invaded these constricted apical domains in stage 12 embryos. In contrast, overexpressed YrtFL was mostly restricted to the lateral domain along with Dlg at the same stage of embryogenesis (Fig. 3 G). This shows that segregation of lateral and apical proteins and exclusion of Yrt from the apical membrane require phosphorylation of Yrt by aPKC, which neutralizes Yrt activity at the apical membrane. In agreement with this model, reduction of aPKC levels or activity exacerbated the phenotype associated with Yrt overexpression, whereas aPKC overexpression had the opposite effect (Fig. S3, A–L). In addition, expression of the phosphomimetic Yrt5D mutant (S348D, S358D, T379D, S387D, and S392D) had a weaker impact compared with overexpression of YrtFL and Yrt5A. Yrt5D expression produced mild epithelial morphogenesis defects characterized by the presence of small ventral holes, but development of anterior structures and apical–basal polarity were normal (Fig. 3, D, E, and I). In comparison to YrtFL and Yrt5A, which were mainly associated with the plasma membrane, Yrt5D showed a more diffuse distribution and accumulated in the cytoplasm (Fig. 3, G–I). This suggests that phosphorylation by aPKC prevents cortical localization of Yrt. Collectively, these results demonstrate that aPKC-dependent phosphorylation of Yrt is a critical event for proper epithelial cell polarization. Specifically, aPKC antagonizes Yrt functions to preserve the integrity of the apical domain and epithelial tissue organization.


A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity.

Gamblin CL, Hardy ÉJ, Chartier FJ, Bisson N, Laprise P - J. Cell Biol. (2014)

aPKC-dependent phosphorylation of Yrt is crucial for epithelial cell polarity. (A–D) Cuticle preparation of embryos of the following genotypes: wild type (A), da-GAL4/UAS-Flag-yrtFL (ubiquitous expression of Flag-tagged YrtFL; B), da-GAL4/UAS-Flag-yrt5A (C), and da-GAL4/UAS-Flag-yrt5D (D). Bar, 100 µm (also applies to B–D). (E) Western blot using an anti-Flag antibody showing that all constructs were expressed at similar levels. Actin was used as a loading control. (F) Portion of the ventral ectoderm of control (da-GAL4; driver line used to express Yrt constructs, this line has a wild-type phenotype) stage 12 (St12) embryos costained with Yrt and Crb (left) or with Dlg and aPKC (right). (G–I) Left images show costaining of Flag and Crb, whereas right images depict costaining of Dlg and aPKC in the ectoderm of an embryo expressing Flag-tagged YrtFL (G), an embryo expressing Flag-Yrt5A (H), or an embryo expressing Flag-Yrt5D (I). Arrows in H point to cysts of cells with contracted apexes, whereas arrowheads show cells with reduced Crb and aPKC levels. Bar, 10 µm (also applies to G–I).
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Related In: Results  -  Collection

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fig3: aPKC-dependent phosphorylation of Yrt is crucial for epithelial cell polarity. (A–D) Cuticle preparation of embryos of the following genotypes: wild type (A), da-GAL4/UAS-Flag-yrtFL (ubiquitous expression of Flag-tagged YrtFL; B), da-GAL4/UAS-Flag-yrt5A (C), and da-GAL4/UAS-Flag-yrt5D (D). Bar, 100 µm (also applies to B–D). (E) Western blot using an anti-Flag antibody showing that all constructs were expressed at similar levels. Actin was used as a loading control. (F) Portion of the ventral ectoderm of control (da-GAL4; driver line used to express Yrt constructs, this line has a wild-type phenotype) stage 12 (St12) embryos costained with Yrt and Crb (left) or with Dlg and aPKC (right). (G–I) Left images show costaining of Flag and Crb, whereas right images depict costaining of Dlg and aPKC in the ectoderm of an embryo expressing Flag-tagged YrtFL (G), an embryo expressing Flag-Yrt5A (H), or an embryo expressing Flag-Yrt5D (I). Arrows in H point to cysts of cells with contracted apexes, whereas arrowheads show cells with reduced Crb and aPKC levels. Bar, 10 µm (also applies to G–I).
Mentions: To address the functional impact of the aPKC-dependent phosphorylation of Yrt in vivo, we generated transgenic flies expressing either Flag-tagged full-length Yrt (YrtFL) or the nonphosphorylatable mutant Yrt5A (full-length Yrt carrying S348A, S358A, T379A, S387A, and S392A mutations). Analysis of cuticle integrity revealed that overexpression of YrtFL had a dominant effect and resulted in the formation of large holes in the ventral epidermis and impairment of head epidermis morphogenesis (Fig. 3, A and B). Although expressed at a level similar to YrtFL (Fig. 3 E), Yrt5A was associated with a much more dramatic phenotype characterized by epithelial tissue collapse resulting in dispersed grains of cuticle (Fig. 3 C). Expression of Yrt5A phenocopies a complete loss of critical positive regulators of the apical domain, including Crb, Stardust, and aPKC (Tepass et al., 1990; Tepass and Knust, 1993; Harris and Peifer, 2007). This suggests that the Yrt mutant resistant to phosphorylation by aPKC has an enhanced ability to antagonize the apical machinery and, thus, disrupts the integrity of the apical domain. Accordingly, Crb and aPKC expression was severely reduced in most ectodermal cells of embryos expressing Yrt5A, and the lateral proteins Discs large (Dlg) and Yrt (Yrt5A) lined the entire circumference of cells lacking aPKC and Crb (compare Fig. 3, H [arrowheads] with F). Residual Crb and aPKC staining marked the contact point of contracted apexes of cells forming rosettes (Fig. 3 H, arrows). Yrt5A and Dlg invaded these constricted apical domains in stage 12 embryos. In contrast, overexpressed YrtFL was mostly restricted to the lateral domain along with Dlg at the same stage of embryogenesis (Fig. 3 G). This shows that segregation of lateral and apical proteins and exclusion of Yrt from the apical membrane require phosphorylation of Yrt by aPKC, which neutralizes Yrt activity at the apical membrane. In agreement with this model, reduction of aPKC levels or activity exacerbated the phenotype associated with Yrt overexpression, whereas aPKC overexpression had the opposite effect (Fig. S3, A–L). In addition, expression of the phosphomimetic Yrt5D mutant (S348D, S358D, T379D, S387D, and S392D) had a weaker impact compared with overexpression of YrtFL and Yrt5A. Yrt5D expression produced mild epithelial morphogenesis defects characterized by the presence of small ventral holes, but development of anterior structures and apical–basal polarity were normal (Fig. 3, D, E, and I). In comparison to YrtFL and Yrt5A, which were mainly associated with the plasma membrane, Yrt5D showed a more diffuse distribution and accumulated in the cytoplasm (Fig. 3, G–I). This suggests that phosphorylation by aPKC prevents cortical localization of Yrt. Collectively, these results demonstrate that aPKC-dependent phosphorylation of Yrt is a critical event for proper epithelial cell polarization. Specifically, aPKC antagonizes Yrt functions to preserve the integrity of the apical domain and epithelial tissue organization.

Bottom Line: However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined.In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane.The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Biologie Moléculaire, Biochimie Médicale et Pathologie and Centre de Recherche sur le Cancer, Université Laval, and 2 Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, Québec G1R 3S3, Canada.

ABSTRACT
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.

Show MeSH
Related in: MedlinePlus