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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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BazGFP overexpression recruits aPKC and PAR-6 early in development but they segregate later. (A) Baz IPs from 2 to 4 h BazGFP overexpressing and WT embryos probed for DE-Cad, aPKC, and Baz. 5% of unbound fractions and full bound fractions analyzed. Irrelevant rabbit serum used as control. (B–D) BazGFP (red) overexpressing embryos at cellularization. Dlg labels furrows. (B) DE-Cad (green), (C) aPKC (green), and (D) PAR-6 (green) at ectopic basal BazGFP puncta (red). (E) Epidermis at gastrulation. PAR-6 (green) and Crb (blue) at apical BazGFP (red). (F) WT epidermis at gastrulation. PAR-6 (green) and Crb (blue) above endogenous Baz (red). (G and H) BazGFP overexpressing embryos. (G) PMGI. PAR-6 (green) and Crb (blue) begin to segregate above BazGFP (red). (H) Stage 10 gut. PAR-6 (green) and Crb (blue) above BazGFP (red). (I) WT PMGI. PAR-6 (green) and Crb (blue) above endogenous Baz (red). Bars, 5 μm.
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fig7: BazGFP overexpression recruits aPKC and PAR-6 early in development but they segregate later. (A) Baz IPs from 2 to 4 h BazGFP overexpressing and WT embryos probed for DE-Cad, aPKC, and Baz. 5% of unbound fractions and full bound fractions analyzed. Irrelevant rabbit serum used as control. (B–D) BazGFP (red) overexpressing embryos at cellularization. Dlg labels furrows. (B) DE-Cad (green), (C) aPKC (green), and (D) PAR-6 (green) at ectopic basal BazGFP puncta (red). (E) Epidermis at gastrulation. PAR-6 (green) and Crb (blue) at apical BazGFP (red). (F) WT epidermis at gastrulation. PAR-6 (green) and Crb (blue) above endogenous Baz (red). (G and H) BazGFP overexpressing embryos. (G) PMGI. PAR-6 (green) and Crb (blue) begin to segregate above BazGFP (red). (H) Stage 10 gut. PAR-6 (green) and Crb (blue) above BazGFP (red). (I) WT PMGI. PAR-6 (green) and Crb (blue) above endogenous Baz (red). Bars, 5 μm.

Mentions: As cellularization proceeds, however, BazGFP displays two different distributions that depend on its expression level. Staining for Baz revealed a wide range of BazGFP accumulation levels, all higher than endogenous Baz (unpublished data). In embryos with the lowest levels, BazGFP remained mainly apical (6/6 embryos; Fig. 4 C, arrow), as does endogenous Baz (Harris and Peifer, 2004). In contrast, in embryos with higher BazGFP levels BazGFP became mislocalized basally along the lateral membrane, often accumulating at the furrow base (10/11 embryos; Fig. 4 D [arrowhead] and Fig. 7, A–C). These differences were also obvious in live imaging. In 6/22 live embryos, BazGFP was retained apically throughout cellularization (Fig. 4 A, 0:23 and 0:27, arrows; Video S1 available at http://www.jcb.org/cgi/content/full/jcb.200505127/DC1). However, in 16/22 embryos, many BazGFP puncta spread basally along the invaginating furrows (Fig. 4 B, 0:14, arrowhead; Video S2 available at http://www.jcb.org/cgi/content/full/jcb.200505127/DC1). This suggests that Baz is normally anchored by an apical scaffold, but at high levels, Baz may saturate the scaffold and excess Baz moves basally with the invaginating furrows.


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

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

BazGFP overexpression recruits aPKC and PAR-6 early in development but they segregate later. (A) Baz IPs from 2 to 4 h BazGFP overexpressing and WT embryos probed for DE-Cad, aPKC, and Baz. 5% of unbound fractions and full bound fractions analyzed. Irrelevant rabbit serum used as control. (B–D) BazGFP (red) overexpressing embryos at cellularization. Dlg labels furrows. (B) DE-Cad (green), (C) aPKC (green), and (D) PAR-6 (green) at ectopic basal BazGFP puncta (red). (E) Epidermis at gastrulation. PAR-6 (green) and Crb (blue) at apical BazGFP (red). (F) WT epidermis at gastrulation. PAR-6 (green) and Crb (blue) above endogenous Baz (red). (G and H) BazGFP overexpressing embryos. (G) PMGI. PAR-6 (green) and Crb (blue) begin to segregate above BazGFP (red). (H) Stage 10 gut. PAR-6 (green) and Crb (blue) above BazGFP (red). (I) WT PMGI. PAR-6 (green) and Crb (blue) above endogenous Baz (red). Bars, 5 μm.
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fig7: BazGFP overexpression recruits aPKC and PAR-6 early in development but they segregate later. (A) Baz IPs from 2 to 4 h BazGFP overexpressing and WT embryos probed for DE-Cad, aPKC, and Baz. 5% of unbound fractions and full bound fractions analyzed. Irrelevant rabbit serum used as control. (B–D) BazGFP (red) overexpressing embryos at cellularization. Dlg labels furrows. (B) DE-Cad (green), (C) aPKC (green), and (D) PAR-6 (green) at ectopic basal BazGFP puncta (red). (E) Epidermis at gastrulation. PAR-6 (green) and Crb (blue) at apical BazGFP (red). (F) WT epidermis at gastrulation. PAR-6 (green) and Crb (blue) above endogenous Baz (red). (G and H) BazGFP overexpressing embryos. (G) PMGI. PAR-6 (green) and Crb (blue) begin to segregate above BazGFP (red). (H) Stage 10 gut. PAR-6 (green) and Crb (blue) above BazGFP (red). (I) WT PMGI. PAR-6 (green) and Crb (blue) above endogenous Baz (red). Bars, 5 μm.
Mentions: As cellularization proceeds, however, BazGFP displays two different distributions that depend on its expression level. Staining for Baz revealed a wide range of BazGFP accumulation levels, all higher than endogenous Baz (unpublished data). In embryos with the lowest levels, BazGFP remained mainly apical (6/6 embryos; Fig. 4 C, arrow), as does endogenous Baz (Harris and Peifer, 2004). In contrast, in embryos with higher BazGFP levels BazGFP became mislocalized basally along the lateral membrane, often accumulating at the furrow base (10/11 embryos; Fig. 4 D [arrowhead] and Fig. 7, A–C). These differences were also obvious in live imaging. In 6/22 live embryos, BazGFP was retained apically throughout cellularization (Fig. 4 A, 0:23 and 0:27, arrows; Video S1 available at http://www.jcb.org/cgi/content/full/jcb.200505127/DC1). However, in 16/22 embryos, many BazGFP puncta spread basally along the invaginating furrows (Fig. 4 B, 0:14, arrowhead; Video S2 available at http://www.jcb.org/cgi/content/full/jcb.200505127/DC1). This suggests that Baz is normally anchored by an apical scaffold, but at high levels, Baz may saturate the scaffold and excess Baz moves basally with the invaginating furrows.

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
Related in: MedlinePlus