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Cited by 295Developmental Biology and Gene RegulationCellular Mechanics and InteractionsHippo pathway signaling and YAP/TAZ

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

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Harris, Tony J.C., Peifer, Mark·2005·The Journal of Cell Biology
DOI 10.1083/jcb.200505127PMC PMC2171335
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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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Figures

Figure 1. Baz and AJs localize below aPKC and PAR-6 during WT epithelial development. (A) Early epithelial development (Dlg-stained embryos and schematics). At cellularization (left), membrane furrows separate each cell. At gastrulation (right), cells form the ectoderm and body compartments, e.g., the PMGI. (B) Cellularization. Baz (red) and DE-Cad (green) colocalize in apical puncta below the top of furrows (Dlg; blue). (C) Gastrulation. Baz (red) and DE-Cad (green) colocalize in apical puncta below the top of lateral membranes (Dlg; blue). (D and E) Cellularization. (D) aPKC (green) enriched above DE-Cad (red). (E) PAR-6 (green) cytoplasmic and along the entire furrow with slight enrichment above Baz (red). (F–H) Gastrulation. (F) aPKC (green) above DE-Cad (red). (G) PAR-6 (green) above Baz (red). (H) aPKC (green) and PAR-6 (blue) colocalize above Arm (red). (I and J) Stages 7 and 8 PMGI. (I) aPKC (green) and PAR-6 (blue) colocalize above Arm (red). (J) PAR-6 (green) above both Baz (red) and Arm (red, right). (K) Stage 14 gut. Baz (red) below PAR-6 (blue), but above Arm (green). (L) Segmental grooves, stage 14 epidermis. Baz (red) below PAR-6 (blue), but above Arm (green). Bars, 5 μm.
Figure 1. Baz and AJs localize below aPKC and PAR-6 during WT epithelial development. (A) Early epithelial development (Dlg-stained embryos and schematics). At cellularization (left), membrane furrows separate each cell. At gastrulation (right), cells form the ectoderm and body compartments, e.g., the PMGI. (B) Cellularization. Baz (red) and DE-Cad (green) colocalize in apical puncta below the top of furrows (Dlg; blue). (C) Gastrulation. Baz (red) and DE-Cad (green) colocalize in apical puncta below the top of lateral membranes (Dlg; blue). (D and E) Cellularization. (D) aPKC (green) enriched above DE-Cad (red). (E) PAR-6 (green) cytoplasmic and along the entire furrow with slight enrichment above Baz (red). (F–H) Gastrulation. (F) aPKC (green) above DE-Cad (red). (G) PAR-6 (green) above Baz (red). (H) aPKC (green) and PAR-6 (blue) colocalize above Arm (red). (I and J) Stages 7 and 8 PMGI. (I) aPKC (green) and PAR-6 (blue) colocalize above Arm (red). (J) PAR-6 (green) above both Baz (red) and Arm (red, right). (K) Stage 14 gut. Baz (red) below PAR-6 (blue), but above Arm (green). (L) Segmental grooves, stage 14 epidermis. Baz (red) below PAR-6 (blue), but above Arm (green). Bars, 5 μm.CC BY-NC-SA · PubMed Central
Figure 2. Baz acts upstream of aPKC as epithelial polarity is established. (A) WT cellularization. aPKC (red) apical. (B and C) baz m/z mutants. (B) Cellularization. aPKC (red) mislocalized basally. PAR-6 (green) cortical and cytoplasmic. (C) Gastrulation. aPKC (red) still basally mislocalized. PAR-6 (green) has some apical enrichment. (D–F) apkc m/z mutants, cellularization. (D) Cross section. Baz (red) and DE-Cad (green) are apical. (E) Surface view. Baz (red) and DE-Cad (green) colocalize in spot junctions, similar to WT (insets). (F) PAR-6 (green) is cytoplasmic. (G) apkc m/z mutant, stage 8 (late gastrulation). Surface view. Baz (red) and DE-Cad (green) colocalize in discontinuous cortical patches (WT junctions are continuous; insets). Bars, 5 μm.
Figure 2. Baz acts upstream of aPKC as epithelial polarity is established. (A) WT cellularization. aPKC (red) apical. (B and C) baz m/z mutants. (B) Cellularization. aPKC (red) mislocalized basally. PAR-6 (green) cortical and cytoplasmic. (C) Gastrulation. aPKC (red) still basally mislocalized. PAR-6 (green) has some apical enrichment. (D–F) apkc m/z mutants, cellularization. (D) Cross section. Baz (red) and DE-Cad (green) are apical. (E) Surface view. Baz (red) and DE-Cad (green) colocalize in spot junctions, similar to WT (insets). (F) PAR-6 (green) is cytoplasmic. (G) apkc m/z mutant, stage 8 (late gastrulation). Surface view. Baz (red) and DE-Cad (green) colocalize in discontinuous cortical patches (WT junctions are continuous; insets). Bars, 5 μm.CC BY-NC-SA · PubMed Central
Figure 3. Baz is in proximity to the actin and MT cytoskeletons during WT cellularization. (A) Cross section. Baz (red) at the base of the apical actin meshwork (green; actin decorated with the actin binding domain of moesin fused to GFP). (B) Cross section. Apical Baz (red) next to longitudinal MTs (green). (C) Surface view. Apical Baz (red) has minimal overlap with MTs (green). (D) 10 μm deep cross section. Apical Baz (red) next to centrosomes (γ-tubulin, green). (E) Surface view. Apical Baz (red) next to centrosomes (γ-tubulin, green). (F) Schematic of apical Baz in proximity to cytoskeletal cues. Bars, 5 μm.
Figure 3. Baz is in proximity to the actin and MT cytoskeletons during WT cellularization. (A) Cross section. Baz (red) at the base of the apical actin meshwork (green; actin decorated with the actin binding domain of moesin fused to GFP). (B) Cross section. Apical Baz (red) next to longitudinal MTs (green). (C) Surface view. Apical Baz (red) has minimal overlap with MTs (green). (D) 10 μm deep cross section. Apical Baz (red) next to centrosomes (γ-tubulin, green). (E) Surface view. Apical Baz (red) next to centrosomes (γ-tubulin, green). (F) Schematic of apical Baz in proximity to cytoskeletal cues. Bars, 5 μm.CC BY-NC-SA · PubMed Central
Figure 4. 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.
Figure 4. 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.CC BY-NC-SA · PubMed Central
Figure 5. Baz positioning involves basal-to-apical transport and dynein. (A) BazGFP puncta moving apically at late cellularization (4 s intervals; Video S3). Velocity distribution in histogram (right). (B) Basal BazGFP puncta (green) overlap with DIC (red; arrows). Insets show overlap after CD treatment. (C) Cuticles. Most baz zygotic mutants have one cuticle hole (left, arrow). dhc64C mutations enhance the baz phenotype, producing larger holes (right, arrows), and overall loss of cuticle (right, outlined). Quantification below. Classes with >20% of dead embryos in bold. Bars, 5 μm.
Figure 5. Baz positioning involves basal-to-apical transport and dynein. (A) BazGFP puncta moving apically at late cellularization (4 s intervals; Video S3). Velocity distribution in histogram (right). (B) Basal BazGFP puncta (green) overlap with DIC (red; arrows). Insets show overlap after CD treatment. (C) Cuticles. Most baz zygotic mutants have one cuticle hole (left, arrow). dhc64C mutations enhance the baz phenotype, producing larger holes (right, arrows), and overall loss of cuticle (right, outlined). Quantification below. Classes with >20% of dead embryos in bold. Bars, 5 μm.CC BY-NC-SA · PubMed Central
Figure 6. A specific role for dynein in positioning Baz and AJs. dhc64C m/z mutants. (A–C) Cellularization. (A) Baz (red) and DE-Cad (green) mislocalize basally but often colocalize. WT, right. (B) aPKC (red) enriched apically. DE-Cad mislocalized basally (green). (C) PAR-6 (green) localizes as in WT. (D–G) Gastrulation. (D) Baz (red) and DE-Cad (green) are apical and colocalize. (E) aPKC apical. (F) PAR-6 apical. (G) Crb apical. (H and I) PMGIs. (H) aPKC (red) apical to DE-Cad (green) and Dlg (blue) segregates basally. (I) PAR-6 (green) above Baz (red) and Dlg (blue) basal. Bars, 5 μm.
Figure 6. A specific role for dynein in positioning Baz and AJs. dhc64C m/z mutants. (A–C) Cellularization. (A) Baz (red) and DE-Cad (green) mislocalize basally but often colocalize. WT, right. (B) aPKC (red) enriched apically. DE-Cad mislocalized basally (green). (C) PAR-6 (green) localizes as in WT. (D–G) Gastrulation. (D) Baz (red) and DE-Cad (green) are apical and colocalize. (E) aPKC apical. (F) PAR-6 apical. (G) Crb apical. (H and I) PMGIs. (H) aPKC (red) apical to DE-Cad (green) and Dlg (blue) segregates basally. (I) PAR-6 (green) above Baz (red) and Dlg (blue) basal. Bars, 5 μm.CC BY-NC-SA · PubMed Central
Figure 7. 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.
Figure 7. 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.CC BY-NC-SA · PubMed Central
Figure 8. 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.
Figure 8. 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.CC BY-NC-SA · PubMed Central
Figure 9. A model of apical domain assembly during cellularization and gastrulation. See Discussion for details.
Figure 9. A model of apical domain assembly during cellularization and gastrulation. See Discussion for details.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Harris, T. J. C., & Peifer, M.
Affiliations
University of North Carolina at Chapel Hill (US)
Corresponding author
Peifer, Mark
ORCID
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2005-08-29
Volume
170
Issue
5
Pages
813-823
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.200505127
PMC
PMC2171335
OpenAlex
W2089839677
Cited by
295
References
44
Citations by year
2026: 5 · 2025: 5 · 2024: 5 · 2023: 5 · 2022: 13 · 2021: 12 · 2020: 13 · 2019: 16 · 2018: 12 · 2017: 20 · 2016: 13 · 2015: 16 · 2014: 10 · 2013: 13 · 2012: 24
FWCI
6.80
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Cell Biology (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion)
  • UNC Libraries
Funders
  • National Institutes of Health
  • Canadian Institutes of Health Research
  • Natural Sciences and Engineering Research Council of Canada
  • National Institute of General Medical Sciences (GM-47857, R01GM047857)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hippo pathway signaling and YAP/TAZ
Keywords
epithelial polarity, Bazooka, adherens junctions, apical domain, atypical protein kinase C, Par6, protein segregation
MeSH terms
Actins, Animals, Cytoskeleton, Drosophila melanogaster, Dyneins, Epithelial Cells, Membrane Proteins, Microtubules, Protein Kinase C, Proteins, Recombinant Fusion Proteins, Body Patterning, Protein Transport, Adherens Junctions, Drosophila Proteins, Intracellular Signaling Peptides and Proteins

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