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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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Baz positioning involves a saturable, apical scaffold, and actin. (A) In 6/22 embryos, BazGFP forms apical puncta at early cellularization (0:00 min, arrow) and stays apical as furrows pass the base of the nuclei (0:11, arrow), reach full length (0:23, arrow) and as gastrulation begins (0:27, arrow; Video S1). (B) In 16/22 embryos, BazGFP accumulates apically (0:00, arrow), but many BazGFP puncta move basally with the furrows (0:14, arrowhead; furrows at base of nuclei; Video S2). Some BazGFP puncta remain basal at full furrow length (0:23, arrowhead), but in 14/16 embryos BazGFP is repositioned apically by gastrulation (0:31, arrow; Video S3). (C and D) Fixed mid-cellularization, BazGFP-expressing embryos imaged with the same settings. 6/6 embryos with the lowest BazGFP levels had mainly apical BazGFP (C, arrow). 10/11 embryos with higher BazGFP levels had ectopic basal BazGFP (D, arrowhead). (E–G) CD treatment. (E) Endogenous Baz (red) shifts basally but largely remains furrow associated (DE-Cad; green). Note relatively normal furrows and colocalization of Baz and DE-Cad (arrowhead). (F) With severe furrow defects (note wide furrow spacing), basal Baz is largely cortical. (G) Some actin is detected (green; actin decorated with the actin binding domain of moesin fused to GFP) but without specific enrichment at Baz accumulations (red, arrowhead). (H) Colchicine also leads to basal endogenous Baz. Bars, 5 μm.
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fig4: Baz positioning involves a saturable, apical scaffold, and actin. (A) In 6/22 embryos, BazGFP forms apical puncta at early cellularization (0:00 min, arrow) and stays apical as furrows pass the base of the nuclei (0:11, arrow), reach full length (0:23, arrow) and as gastrulation begins (0:27, arrow; Video S1). (B) In 16/22 embryos, BazGFP accumulates apically (0:00, arrow), but many BazGFP puncta move basally with the furrows (0:14, arrowhead; furrows at base of nuclei; Video S2). Some BazGFP puncta remain basal at full furrow length (0:23, arrowhead), but in 14/16 embryos BazGFP is repositioned apically by gastrulation (0:31, arrow; Video S3). (C and D) Fixed mid-cellularization, BazGFP-expressing embryos imaged with the same settings. 6/6 embryos with the lowest BazGFP levels had mainly apical BazGFP (C, arrow). 10/11 embryos with higher BazGFP levels had ectopic basal BazGFP (D, arrowhead). (E–G) CD treatment. (E) Endogenous Baz (red) shifts basally but largely remains furrow associated (DE-Cad; green). Note relatively normal furrows and colocalization of Baz and DE-Cad (arrowhead). (F) With severe furrow defects (note wide furrow spacing), basal Baz is largely cortical. (G) Some actin is detected (green; actin decorated with the actin binding domain of moesin fused to GFP) but without specific enrichment at Baz accumulations (red, arrowhead). (H) Colchicine also leads to basal endogenous Baz. Bars, 5 μm.

Mentions: To assess how Baz is positioned during cellularization, we first performed time lapse imaging of embryos expressing UAS-driven BazGFP under the control of a maternal GAL4 driver. BazGFP is functional as it rescues baz mutant follicle cells and embryos (Benton and St Johnston, 2003a). We imaged the apical–basal axis of the epithelium in embryo cross sections. At early cellularization, BazGFP shows low level, even accumulation on nascent furrows (Fig. 4 A, B, 0:00, arrows). BazGFP then coalesces into punctate accumulations (Fig. 4 A, 0:11, arrow), closely resembling endogenous Baz (Harris and Peifer, 2004). This confirms that Baz is recruited apically during the earliest stages of polarity establishment.


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

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

Baz positioning involves a saturable, apical scaffold, and actin. (A) In 6/22 embryos, BazGFP forms apical puncta at early cellularization (0:00 min, arrow) and stays apical as furrows pass the base of the nuclei (0:11, arrow), reach full length (0:23, arrow) and as gastrulation begins (0:27, arrow; Video S1). (B) In 16/22 embryos, BazGFP accumulates apically (0:00, arrow), but many BazGFP puncta move basally with the furrows (0:14, arrowhead; furrows at base of nuclei; Video S2). Some BazGFP puncta remain basal at full furrow length (0:23, arrowhead), but in 14/16 embryos BazGFP is repositioned apically by gastrulation (0:31, arrow; Video S3). (C and D) Fixed mid-cellularization, BazGFP-expressing embryos imaged with the same settings. 6/6 embryos with the lowest BazGFP levels had mainly apical BazGFP (C, arrow). 10/11 embryos with higher BazGFP levels had ectopic basal BazGFP (D, arrowhead). (E–G) CD treatment. (E) Endogenous Baz (red) shifts basally but largely remains furrow associated (DE-Cad; green). Note relatively normal furrows and colocalization of Baz and DE-Cad (arrowhead). (F) With severe furrow defects (note wide furrow spacing), basal Baz is largely cortical. (G) Some actin is detected (green; actin decorated with the actin binding domain of moesin fused to GFP) but without specific enrichment at Baz accumulations (red, arrowhead). (H) Colchicine also leads to basal endogenous Baz. Bars, 5 μm.
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Related In: Results  -  Collection

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fig4: Baz positioning involves a saturable, apical scaffold, and actin. (A) In 6/22 embryos, BazGFP forms apical puncta at early cellularization (0:00 min, arrow) and stays apical as furrows pass the base of the nuclei (0:11, arrow), reach full length (0:23, arrow) and as gastrulation begins (0:27, arrow; Video S1). (B) In 16/22 embryos, BazGFP accumulates apically (0:00, arrow), but many BazGFP puncta move basally with the furrows (0:14, arrowhead; furrows at base of nuclei; Video S2). Some BazGFP puncta remain basal at full furrow length (0:23, arrowhead), but in 14/16 embryos BazGFP is repositioned apically by gastrulation (0:31, arrow; Video S3). (C and D) Fixed mid-cellularization, BazGFP-expressing embryos imaged with the same settings. 6/6 embryos with the lowest BazGFP levels had mainly apical BazGFP (C, arrow). 10/11 embryos with higher BazGFP levels had ectopic basal BazGFP (D, arrowhead). (E–G) CD treatment. (E) Endogenous Baz (red) shifts basally but largely remains furrow associated (DE-Cad; green). Note relatively normal furrows and colocalization of Baz and DE-Cad (arrowhead). (F) With severe furrow defects (note wide furrow spacing), basal Baz is largely cortical. (G) Some actin is detected (green; actin decorated with the actin binding domain of moesin fused to GFP) but without specific enrichment at Baz accumulations (red, arrowhead). (H) Colchicine also leads to basal endogenous Baz. Bars, 5 μm.
Mentions: To assess how Baz is positioned during cellularization, we first performed time lapse imaging of embryos expressing UAS-driven BazGFP under the control of a maternal GAL4 driver. BazGFP is functional as it rescues baz mutant follicle cells and embryos (Benton and St Johnston, 2003a). We imaged the apical–basal axis of the epithelium in embryo cross sections. At early cellularization, BazGFP shows low level, even accumulation on nascent furrows (Fig. 4 A, B, 0:00, arrows). BazGFP then coalesces into punctate accumulations (Fig. 4 A, 0:11, arrow), closely resembling endogenous Baz (Harris and Peifer, 2004). This confirms that Baz is recruited apically during the earliest stages of polarity establishment.

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