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The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila.

Harris TJ, Peifer M - J. Cell Biol. (2005)

Bottom Line: Adherens junctions (AJs) often direct this polarity, but we previously found that Bazooka (Baz) acts upstream of AJs as epithelial polarity is first established in Drosophila.Surprisingly, we found that Baz localizes to an apical domain below its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms.These results reveal key steps in the assembly of the apical domain in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. tonyh@email.unc.edu

ABSTRACT
Cell polarity is critical for epithelial structure and function. Adherens junctions (AJs) often direct this polarity, but we previously found that Bazooka (Baz) acts upstream of AJs as epithelial polarity is first established in Drosophila. This prompted us to ask how Baz is positioned and how downstream polarity is elaborated. Surprisingly, we found that Baz localizes to an apical domain below its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms. In fact, Baz positioning is independent of aPKC and PAR-6 relying instead on cytoskeletal cues, including an apical scaffold and dynein-mediated basal-to-apical transport. AJ assembly is closely coupled to Baz positioning, whereas aPKC and PAR-6 are positioned separately. This forms a stratified apical domain with Baz and AJs localizing basal to aPKC and PAR-6, and we identify specific mechanisms that keep these proteins apart. These results reveal key steps in the assembly of the apical domain in Drosophila.

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Crb is required to segregate aPKC and PAR-6 from AJs after cellularization. Stage 10 zygotic crb mutants. In embryo surface views (A) and cross section (B), PAR-6 (green), and aPKC (blue) colocalize with fragmented AJs (Arm, red). (C) Gut. PAR-6 (green) and aPKC (blue) intermix with AJs (Arm, red). In WT, they are above AJs (C, insets). Bars, 5 μm.
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fig8: Crb is required to segregate aPKC and PAR-6 from AJs after cellularization. Stage 10 zygotic crb mutants. In embryo surface views (A) and cross section (B), PAR-6 (green), and aPKC (blue) colocalize with fragmented AJs (Arm, red). (C) Gut. PAR-6 (green) and aPKC (blue) intermix with AJs (Arm, red). In WT, they are above AJs (C, insets). Bars, 5 μm.

Mentions: Crb plays a key role in maintaining the integrity of AJs and the apical domain after cellularization (Grawe et al., 1996; Tepass, 1996). We thus hypothesized that as Crb accumulates during gastrulation it may act to segregate Baz and AJs from aPKC and PAR-6. To test this, we analyzed the distribution of apical cues in crb2 mutants. In stages 9 and 10 of crb2 zygotic mutants, AJs fragment and become randomly positioned around the cell cortex and along the basolateral membrane as epidermal cells dissociate. We previously found that Baz colocalizes with AJ fragments (Harris and Peifer, 2004). Now we asked whether aPKC and PAR-6 localize at these fragments in the absence of Crb. Indeed, both aPKC and PAR-6 are recruited to fragmented AJs in the epidermis of crb2 mutants (aPKC shows greater enrichment at these sites; Fig. 8 A, B, arrows). We next analyzed the gut epithelium of crb2 mutants, which, in contrast to the epidermis, maintains basic epithelial structure at stage 10. Here, Arm, aPKC, and PAR-6 are extensively intermixed in the apical domain and colocalize in lateral puncta (Fig. 8 C, arrows), in contrast to their segregation in the WT gut (Fig. 8 C, inset). These data suggest that Baz, AJs, aPKC, and PAR-6 have direct or indirect affinities for one another, and that Crb helps segregate Baz and AJs from aPKC and PAR-6 after cellularization.


The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila.

Harris TJ, Peifer M - J. Cell Biol. (2005)

Crb is required to segregate aPKC and PAR-6 from AJs after cellularization. Stage 10 zygotic crb mutants. In embryo surface views (A) and cross section (B), PAR-6 (green), and aPKC (blue) colocalize with fragmented AJs (Arm, red). (C) Gut. PAR-6 (green) and aPKC (blue) intermix with AJs (Arm, red). In WT, they are above AJs (C, insets). Bars, 5 μm.
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fig8: Crb is required to segregate aPKC and PAR-6 from AJs after cellularization. Stage 10 zygotic crb mutants. In embryo surface views (A) and cross section (B), PAR-6 (green), and aPKC (blue) colocalize with fragmented AJs (Arm, red). (C) Gut. PAR-6 (green) and aPKC (blue) intermix with AJs (Arm, red). In WT, they are above AJs (C, insets). Bars, 5 μm.
Mentions: Crb plays a key role in maintaining the integrity of AJs and the apical domain after cellularization (Grawe et al., 1996; Tepass, 1996). We thus hypothesized that as Crb accumulates during gastrulation it may act to segregate Baz and AJs from aPKC and PAR-6. To test this, we analyzed the distribution of apical cues in crb2 mutants. In stages 9 and 10 of crb2 zygotic mutants, AJs fragment and become randomly positioned around the cell cortex and along the basolateral membrane as epidermal cells dissociate. We previously found that Baz colocalizes with AJ fragments (Harris and Peifer, 2004). Now we asked whether aPKC and PAR-6 localize at these fragments in the absence of Crb. Indeed, both aPKC and PAR-6 are recruited to fragmented AJs in the epidermis of crb2 mutants (aPKC shows greater enrichment at these sites; Fig. 8 A, B, arrows). We next analyzed the gut epithelium of crb2 mutants, which, in contrast to the epidermis, maintains basic epithelial structure at stage 10. Here, Arm, aPKC, and PAR-6 are extensively intermixed in the apical domain and colocalize in lateral puncta (Fig. 8 C, arrows), in contrast to their segregation in the WT gut (Fig. 8 C, inset). These data suggest that Baz, AJs, aPKC, and PAR-6 have direct or indirect affinities for one another, and that Crb helps segregate Baz and AJs from aPKC and PAR-6 after cellularization.

Bottom Line: Adherens junctions (AJs) often direct this polarity, but we previously found that Bazooka (Baz) acts upstream of AJs as epithelial polarity is first established in Drosophila.Surprisingly, we found that Baz localizes to an apical domain below its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms.These results reveal key steps in the assembly of the apical domain in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. tonyh@email.unc.edu

ABSTRACT
Cell polarity is critical for epithelial structure and function. Adherens junctions (AJs) often direct this polarity, but we previously found that Bazooka (Baz) acts upstream of AJs as epithelial polarity is first established in Drosophila. This prompted us to ask how Baz is positioned and how downstream polarity is elaborated. Surprisingly, we found that Baz localizes to an apical domain below its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms. In fact, Baz positioning is independent of aPKC and PAR-6 relying instead on cytoskeletal cues, including an apical scaffold and dynein-mediated basal-to-apical transport. AJ assembly is closely coupled to Baz positioning, whereas aPKC and PAR-6 are positioned separately. This forms a stratified apical domain with Baz and AJs localizing basal to aPKC and PAR-6, and we identify specific mechanisms that keep these proteins apart. These results reveal key steps in the assembly of the apical domain in Drosophila.

Show MeSH