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Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis.

Harris KP, Tepass U - J. Cell Biol. (2008)

Bottom Line: Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability.The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins.This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.

ABSTRACT
Cell rearrangements require dynamic changes in cell-cell contacts to maintain tissue integrity. We investigated the function of Cdc42 in maintaining adherens junctions (AJs) and apical polarity in the Drosophila melanogaster neuroectodermal epithelium. About one third of cells exit the epithelium through ingression and become neuroblasts. Cdc42-compromised embryos lost AJs in the neuroectoderm during neuroblast ingression. In contrast, when neuroblast formation was suppressed, AJs were maintained despite the loss of Cdc42 function. Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability. In addition, Cdc42 has a second function in regulating endocytotic trafficking, as it is required for the progression of apical cargo from the early to the late endosome. The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins. This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.

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Cdc42 interacts with the Par complex to regulate apical endocytosis. (A and B) Wild-type (A) and Cdc42-DN (B) embryos labeled for Baz and Cdc42. (C and D) Wild-type (C) and Cdc42-DN (D) embryos labeled for Par6 and Cdc42. (E and F) Wild-type (E) and Cdc42-DN (F) embryos labeled for aPKC and Cdc42. (G) Cdc42-DN ventral defects are enhanced by the loss of zygotic aPKC, baz, or par6. The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). For all double-mutant combinations, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). (H and I) baz mutant embryos (H and H′) and par6 mutant embryos (I and I′) labeled for Crb and Cdc42. Bars: (A–F, H′, and I′) 5 μm; (H and I) 10 μm.
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fig8: Cdc42 interacts with the Par complex to regulate apical endocytosis. (A and B) Wild-type (A) and Cdc42-DN (B) embryos labeled for Baz and Cdc42. (C and D) Wild-type (C) and Cdc42-DN (D) embryos labeled for Par6 and Cdc42. (E and F) Wild-type (E) and Cdc42-DN (F) embryos labeled for aPKC and Cdc42. (G) Cdc42-DN ventral defects are enhanced by the loss of zygotic aPKC, baz, or par6. The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). For all double-mutant combinations, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). (H and I) baz mutant embryos (H and H′) and par6 mutant embryos (I and I′) labeled for Crb and Cdc42. Bars: (A–F, H′, and I′) 5 μm; (H and I) 10 μm.

Mentions: Cdc42 can contribute to endocytosis through regulation of its downstream effector Wasp and actin polymerization (for reviews see Cerione, 2004; Ridley 2006). Also, a second Cdc42 effector, the Par complex, was recently implicated in the regulation of endocytosis in C. elegans and human HeLa cells (Balklava et al., 2007). This study suggested that Cdc42 and the Par proteins Par3, Par6, and aPKC are required to promote endocytosis. However, the function of Cdc42 and Par proteins in endocytosis in epithelial cells was not investigated. To address the question whether Drosophila Par proteins cooperate with Cdc42 in the regulation of endocytosis in neuroectodermal cells, we first examined the distribution of Bazooka (Baz; Drosophila Par3), Par6, and aPKC in da>Cdc42-DN embryos. We found that all three Par proteins are strongly depleted from the apical membrane of neuroectodermal cells and colocalized with Cdc42 at enlarged apical endosomes similar to apical transmembrane proteins such as Crb (Fig. 8, A–F). Normal apical localization but abnormal accumulation of Par proteins at apical endosomes was also observed in other ectodermal epithelial cells that displayed normal apical polarity in da>Cdc42-DN embryos. The loss of both the Crb and Par complexes, which can act redundantly in promoting epithelial polarity (Tanentzapf and Tepass, 2002), from the apical membrane explains the inability of neuroectodermal cells to maintain their AJs.


Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis.

Harris KP, Tepass U - J. Cell Biol. (2008)

Cdc42 interacts with the Par complex to regulate apical endocytosis. (A and B) Wild-type (A) and Cdc42-DN (B) embryos labeled for Baz and Cdc42. (C and D) Wild-type (C) and Cdc42-DN (D) embryos labeled for Par6 and Cdc42. (E and F) Wild-type (E) and Cdc42-DN (F) embryos labeled for aPKC and Cdc42. (G) Cdc42-DN ventral defects are enhanced by the loss of zygotic aPKC, baz, or par6. The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). For all double-mutant combinations, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). (H and I) baz mutant embryos (H and H′) and par6 mutant embryos (I and I′) labeled for Crb and Cdc42. Bars: (A–F, H′, and I′) 5 μm; (H and I) 10 μm.
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fig8: Cdc42 interacts with the Par complex to regulate apical endocytosis. (A and B) Wild-type (A) and Cdc42-DN (B) embryos labeled for Baz and Cdc42. (C and D) Wild-type (C) and Cdc42-DN (D) embryos labeled for Par6 and Cdc42. (E and F) Wild-type (E) and Cdc42-DN (F) embryos labeled for aPKC and Cdc42. (G) Cdc42-DN ventral defects are enhanced by the loss of zygotic aPKC, baz, or par6. The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). For all double-mutant combinations, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). (H and I) baz mutant embryos (H and H′) and par6 mutant embryos (I and I′) labeled for Crb and Cdc42. Bars: (A–F, H′, and I′) 5 μm; (H and I) 10 μm.
Mentions: Cdc42 can contribute to endocytosis through regulation of its downstream effector Wasp and actin polymerization (for reviews see Cerione, 2004; Ridley 2006). Also, a second Cdc42 effector, the Par complex, was recently implicated in the regulation of endocytosis in C. elegans and human HeLa cells (Balklava et al., 2007). This study suggested that Cdc42 and the Par proteins Par3, Par6, and aPKC are required to promote endocytosis. However, the function of Cdc42 and Par proteins in endocytosis in epithelial cells was not investigated. To address the question whether Drosophila Par proteins cooperate with Cdc42 in the regulation of endocytosis in neuroectodermal cells, we first examined the distribution of Bazooka (Baz; Drosophila Par3), Par6, and aPKC in da>Cdc42-DN embryos. We found that all three Par proteins are strongly depleted from the apical membrane of neuroectodermal cells and colocalized with Cdc42 at enlarged apical endosomes similar to apical transmembrane proteins such as Crb (Fig. 8, A–F). Normal apical localization but abnormal accumulation of Par proteins at apical endosomes was also observed in other ectodermal epithelial cells that displayed normal apical polarity in da>Cdc42-DN embryos. The loss of both the Crb and Par complexes, which can act redundantly in promoting epithelial polarity (Tanentzapf and Tepass, 2002), from the apical membrane explains the inability of neuroectodermal cells to maintain their AJs.

Bottom Line: Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability.The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins.This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.

ABSTRACT
Cell rearrangements require dynamic changes in cell-cell contacts to maintain tissue integrity. We investigated the function of Cdc42 in maintaining adherens junctions (AJs) and apical polarity in the Drosophila melanogaster neuroectodermal epithelium. About one third of cells exit the epithelium through ingression and become neuroblasts. Cdc42-compromised embryos lost AJs in the neuroectoderm during neuroblast ingression. In contrast, when neuroblast formation was suppressed, AJs were maintained despite the loss of Cdc42 function. Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability. In addition, Cdc42 has a second function in regulating endocytotic trafficking, as it is required for the progression of apical cargo from the early to the late endosome. The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins. This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.

Show MeSH
Related in: MedlinePlus