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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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Ventral ectodermal defects in embryos overexpressing Cdc42-DN and Cdc42 loss-of-function embryos. (A–C) Wild-type embryo (A), embryo overexpressing Cdc42-DN under the control of da-Gal4 (Cdc42-DN; B), and embryo produced by a Cdc423/Cdc426 female (Cdc42 lof; C) labeled for the AJ marker DEcad. (D–F) Ventral cuticle of wild-type embryo (D), Cdc42-DN embryo (E), and Cdc42 loss-of-function embryo (F). Arrows indicate holes on the ventral surface. (G and H) Cdc42-DN embryos labeled for DEcad at stages 11 (G) and 14 (H). (I) Illustration of neuroblast ingression. As a neuroblast (purple) begins to ingress from the ventral neuroectoderm, existing AJs (dark gray) at the neuroblast/epidermal progenitor (blue) boundaries resolve. New AJs form between neighboring epidermal cells. (J and K) DEcad stain (J) and ventral cuticle (K) of embryos expressing both Cdc42-DN and Nintra under the control of da-Gal4. (L) The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). The difference in the number of intact belts is highly significant (P < 0.001) for Cdc42-DN embryos versus wild type, Cdc42 loss-of-function embryos versus wild type, and Cdc42-DN Nintra embryos versus Cdc42-DN embryos. Coexpression of GFP with Cdc42-DN under the control of da-Gal4 did not affect the severity of ventral cuticle defects caused by Cdc42-DN. M, ventral midline; VNE, ventral neuroectoderm; DE, dorsal ectoderm. Bars: (A–C and J) 20 μm; (D–F and K) 100 μm; (G and H) 50 μm.
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fig1: Ventral ectodermal defects in embryos overexpressing Cdc42-DN and Cdc42 loss-of-function embryos. (A–C) Wild-type embryo (A), embryo overexpressing Cdc42-DN under the control of da-Gal4 (Cdc42-DN; B), and embryo produced by a Cdc423/Cdc426 female (Cdc42 lof; C) labeled for the AJ marker DEcad. (D–F) Ventral cuticle of wild-type embryo (D), Cdc42-DN embryo (E), and Cdc42 loss-of-function embryo (F). Arrows indicate holes on the ventral surface. (G and H) Cdc42-DN embryos labeled for DEcad at stages 11 (G) and 14 (H). (I) Illustration of neuroblast ingression. As a neuroblast (purple) begins to ingress from the ventral neuroectoderm, existing AJs (dark gray) at the neuroblast/epidermal progenitor (blue) boundaries resolve. New AJs form between neighboring epidermal cells. (J and K) DEcad stain (J) and ventral cuticle (K) of embryos expressing both Cdc42-DN and Nintra under the control of da-Gal4. (L) The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). The difference in the number of intact belts is highly significant (P < 0.001) for Cdc42-DN embryos versus wild type, Cdc42 loss-of-function embryos versus wild type, and Cdc42-DN Nintra embryos versus Cdc42-DN embryos. Coexpression of GFP with Cdc42-DN under the control of da-Gal4 did not affect the severity of ventral cuticle defects caused by Cdc42-DN. M, ventral midline; VNE, ventral neuroectoderm; DE, dorsal ectoderm. Bars: (A–C and J) 20 μm; (D–F and K) 100 μm; (G and H) 50 μm.

Mentions: To address the function of Rho GTPases in AJ formation and maintenance in vivo, we have expressed dominant-negative (DN) forms of Rho1, Rac1, and Cdc42 in the Drosophila embryo using the Gal4/upstream activation sequence (UAS) system and the ubiquitous driver line da-Gal4. We found that expression of Rho1-DN led to a rather general disruption of AJs, confirming the previous work of others (Bloor and Kiehart, 2002). Rac1-DN showed only minor defects in AJ integrity, which were confined to the vicinity of the ventral midline (unpublished data). Interestingly, expression of Cdc42-DN showed a strong disruption of AJs in the ventral ectoderm. In contrast, AJs in other parts of the ectoderm, the ventral midline, and the dorsal ectoderm remained largely unaffected in these embryos (Fig. 1, A and B). We also expressed constitutively active (CA) forms of all three GTPases in embryos and found that AJs became severely disrupted throughout the ectoderm where they were organized in irregular large clusters (unpublished data). Because of the intriguing differential requirement of Cdc42 in maintaining AJs, we concentrated our further analysis on this GTPase.


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)

Ventral ectodermal defects in embryos overexpressing Cdc42-DN and Cdc42 loss-of-function embryos. (A–C) Wild-type embryo (A), embryo overexpressing Cdc42-DN under the control of da-Gal4 (Cdc42-DN; B), and embryo produced by a Cdc423/Cdc426 female (Cdc42 lof; C) labeled for the AJ marker DEcad. (D–F) Ventral cuticle of wild-type embryo (D), Cdc42-DN embryo (E), and Cdc42 loss-of-function embryo (F). Arrows indicate holes on the ventral surface. (G and H) Cdc42-DN embryos labeled for DEcad at stages 11 (G) and 14 (H). (I) Illustration of neuroblast ingression. As a neuroblast (purple) begins to ingress from the ventral neuroectoderm, existing AJs (dark gray) at the neuroblast/epidermal progenitor (blue) boundaries resolve. New AJs form between neighboring epidermal cells. (J and K) DEcad stain (J) and ventral cuticle (K) of embryos expressing both Cdc42-DN and Nintra under the control of da-Gal4. (L) The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). The difference in the number of intact belts is highly significant (P < 0.001) for Cdc42-DN embryos versus wild type, Cdc42 loss-of-function embryos versus wild type, and Cdc42-DN Nintra embryos versus Cdc42-DN embryos. Coexpression of GFP with Cdc42-DN under the control of da-Gal4 did not affect the severity of ventral cuticle defects caused by Cdc42-DN. M, ventral midline; VNE, ventral neuroectoderm; DE, dorsal ectoderm. Bars: (A–C and J) 20 μm; (D–F and K) 100 μm; (G and H) 50 μm.
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Related In: Results  -  Collection

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fig1: Ventral ectodermal defects in embryos overexpressing Cdc42-DN and Cdc42 loss-of-function embryos. (A–C) Wild-type embryo (A), embryo overexpressing Cdc42-DN under the control of da-Gal4 (Cdc42-DN; B), and embryo produced by a Cdc423/Cdc426 female (Cdc42 lof; C) labeled for the AJ marker DEcad. (D–F) Ventral cuticle of wild-type embryo (D), Cdc42-DN embryo (E), and Cdc42 loss-of-function embryo (F). Arrows indicate holes on the ventral surface. (G and H) Cdc42-DN embryos labeled for DEcad at stages 11 (G) and 14 (H). (I) Illustration of neuroblast ingression. As a neuroblast (purple) begins to ingress from the ventral neuroectoderm, existing AJs (dark gray) at the neuroblast/epidermal progenitor (blue) boundaries resolve. New AJs form between neighboring epidermal cells. (J and K) DEcad stain (J) and ventral cuticle (K) of embryos expressing both Cdc42-DN and Nintra under the control of da-Gal4. (L) The extent of ventral cuticle defects was quantified by counting the number of intact abdominal denticle belts (mean ± SEM [error bars]). The difference in the number of intact belts is highly significant (P < 0.001) for Cdc42-DN embryos versus wild type, Cdc42 loss-of-function embryos versus wild type, and Cdc42-DN Nintra embryos versus Cdc42-DN embryos. Coexpression of GFP with Cdc42-DN under the control of da-Gal4 did not affect the severity of ventral cuticle defects caused by Cdc42-DN. M, ventral midline; VNE, ventral neuroectoderm; DE, dorsal ectoderm. Bars: (A–C and J) 20 μm; (D–F and K) 100 μm; (G and H) 50 μm.
Mentions: To address the function of Rho GTPases in AJ formation and maintenance in vivo, we have expressed dominant-negative (DN) forms of Rho1, Rac1, and Cdc42 in the Drosophila embryo using the Gal4/upstream activation sequence (UAS) system and the ubiquitous driver line da-Gal4. We found that expression of Rho1-DN led to a rather general disruption of AJs, confirming the previous work of others (Bloor and Kiehart, 2002). Rac1-DN showed only minor defects in AJ integrity, which were confined to the vicinity of the ventral midline (unpublished data). Interestingly, expression of Cdc42-DN showed a strong disruption of AJs in the ventral ectoderm. In contrast, AJs in other parts of the ectoderm, the ventral midline, and the dorsal ectoderm remained largely unaffected in these embryos (Fig. 1, A and B). We also expressed constitutively active (CA) forms of all three GTPases in embryos and found that AJs became severely disrupted throughout the ectoderm where they were organized in irregular large clusters (unpublished data). Because of the intriguing differential requirement of Cdc42 in maintaining AJs, we concentrated our further analysis on this GTPase.

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