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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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Inhibition of apical endocytosis rescues defects in Cdc42-compromised embryos. (A–C) Wild-type embryo (A), Cdc42-DN embryo (B), and embryo expressing both Cdc42-DN and Rab5-DN (C) labeled for DEcad. (D–F) Wild-type (D), Cdc42-DN (E), and Cdc42-DN, Rab5-DN (F and F′) embryos labeled for Crb. Arrows point to enlarged endosomes. (G) The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). For Cdc42-DN Rab5-DN embryos, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). (H) Ventral cuticle of Cdc42-DN, Rab5-DN embryo. M, ventral midline; VNE, ventral neuroectoderm; DE, dorsal ectoderm. Bars: (A–F) 20 μm; (F′) 10 μm; (H) 100 μm.
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fig6: Inhibition of apical endocytosis rescues defects in Cdc42-compromised embryos. (A–C) Wild-type embryo (A), Cdc42-DN embryo (B), and embryo expressing both Cdc42-DN and Rab5-DN (C) labeled for DEcad. (D–F) Wild-type (D), Cdc42-DN (E), and Cdc42-DN, Rab5-DN (F and F′) embryos labeled for Crb. Arrows point to enlarged endosomes. (G) The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). For Cdc42-DN Rab5-DN embryos, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). (H) Ventral cuticle of Cdc42-DN, Rab5-DN embryo. M, ventral midline; VNE, ventral neuroectoderm; DE, dorsal ectoderm. Bars: (A–F) 20 μm; (F′) 10 μm; (H) 100 μm.

Mentions: To further support the hypothesis that Cdc42 activity negatively regulates apical endocytosis, we asked whether a reduction in endocytosis can ameliorate the phenotype of da>Cdc42-DN embryos and reconstitute apical protein localization in neuroectodermal cells. We coexpressed Rab5-DN with Cdc42-DN. Rab5 is a Rab GTPase critical for early steps in endocytosis, mediating the fusion of endocytotic vesicles with the early endosome. da>Rab5-DN embryos are lethal and displayed a defective cuticle, but no ventral holes were observed (Fig. 6 G). Neuroectodermal cells of da>Cdc42-DN da>Rab5-DN embryos showed a substantial improvement of normal localization of apical proteins, including Crb and DEcad (Fig. 6, A–F). Corresponding to the normalization of apical polarity during neurogenesis, the terminal phenotype of da>Cdc42-DN is significantly ameliorated by coexpression of Rab5-DN (Fig. 6, G and H). Furthermore, we note that expression of Rab5-DN does not prevent the formation of abnormally enlarged endosomes that accumulate apical proteins in da>Cdc42-DN embryos (Fig. 6 F). This, together with the observation that enlarged apical endosomes are seen in tissues that have not lost apical polarity, again suggests that Cdc42 acts independently in two steps of the apical endocytotic pathway: as a negative regulator of apical endocytosis and as a positive regulator of early to late endosomal processing.


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)

Inhibition of apical endocytosis rescues defects in Cdc42-compromised embryos. (A–C) Wild-type embryo (A), Cdc42-DN embryo (B), and embryo expressing both Cdc42-DN and Rab5-DN (C) labeled for DEcad. (D–F) Wild-type (D), Cdc42-DN (E), and Cdc42-DN, Rab5-DN (F and F′) embryos labeled for Crb. Arrows point to enlarged endosomes. (G) The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). For Cdc42-DN Rab5-DN embryos, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). (H) Ventral cuticle of Cdc42-DN, Rab5-DN embryo. M, ventral midline; VNE, ventral neuroectoderm; DE, dorsal ectoderm. Bars: (A–F) 20 μm; (F′) 10 μm; (H) 100 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600741&req=5

fig6: Inhibition of apical endocytosis rescues defects in Cdc42-compromised embryos. (A–C) Wild-type embryo (A), Cdc42-DN embryo (B), and embryo expressing both Cdc42-DN and Rab5-DN (C) labeled for DEcad. (D–F) Wild-type (D), Cdc42-DN (E), and Cdc42-DN, Rab5-DN (F and F′) embryos labeled for Crb. Arrows point to enlarged endosomes. (G) The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). For Cdc42-DN Rab5-DN embryos, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). (H) Ventral cuticle of Cdc42-DN, Rab5-DN embryo. M, ventral midline; VNE, ventral neuroectoderm; DE, dorsal ectoderm. Bars: (A–F) 20 μm; (F′) 10 μm; (H) 100 μm.
Mentions: To further support the hypothesis that Cdc42 activity negatively regulates apical endocytosis, we asked whether a reduction in endocytosis can ameliorate the phenotype of da>Cdc42-DN embryos and reconstitute apical protein localization in neuroectodermal cells. We coexpressed Rab5-DN with Cdc42-DN. Rab5 is a Rab GTPase critical for early steps in endocytosis, mediating the fusion of endocytotic vesicles with the early endosome. da>Rab5-DN embryos are lethal and displayed a defective cuticle, but no ventral holes were observed (Fig. 6 G). Neuroectodermal cells of da>Cdc42-DN da>Rab5-DN embryos showed a substantial improvement of normal localization of apical proteins, including Crb and DEcad (Fig. 6, A–F). Corresponding to the normalization of apical polarity during neurogenesis, the terminal phenotype of da>Cdc42-DN is significantly ameliorated by coexpression of Rab5-DN (Fig. 6, G and H). Furthermore, we note that expression of Rab5-DN does not prevent the formation of abnormally enlarged endosomes that accumulate apical proteins in da>Cdc42-DN embryos (Fig. 6 F). This, together with the observation that enlarged apical endosomes are seen in tissues that have not lost apical polarity, again suggests that Cdc42 acts independently in two steps of the apical endocytotic pathway: as a negative regulator of apical endocytosis and as a positive regulator of early to late endosomal processing.

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