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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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Apical proteins accumulate in an Hrs-positive endocytotic compartment in Cdc42-compromised embryos. (A and B) Wild-type (A and A′) and Cdc42-DN (B and B′) embryos labeled with Crb and the endosome marker Hrs. (C and D) Wild-type (C and C′) and Cdc42-DN (D and D′) embryos labeled for Crb and the early endosome marker Rab5. (E and F) Wild-type (E and E′) and Cdc42-DN (F and F′) embryos labeled for Crb and the early endosome marker Avl. (G and H) Wild-type (G and G′) and Cdc42-DN (H and H′) embryos labeled for Crb and the recycling endosome marker Rab11. (I and J) Embryos expressing the late endosome marker UAS-YFP-Rab7 under the control of da-Gal4 (I and I′) and embryos expressing both UAS-Cdc42-DN and UAS-YFP-Rab7 under the control of da-Gal4 (J and J′) labeled for Crb and GFP. (K and L) Wild-type (K and K′) and Cdc42-DN (L and L′) embryos labeled for Crb and the Golgi marker Lva. (M and N) Wild-type (M and M′) and Cdc42-DN (N and N′) embryos labeled for Crb and the ER marker KDEL. Bars: (A–N) 10 μm; (A′–N′) 5 μm.
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fig5: Apical proteins accumulate in an Hrs-positive endocytotic compartment in Cdc42-compromised embryos. (A and B) Wild-type (A and A′) and Cdc42-DN (B and B′) embryos labeled with Crb and the endosome marker Hrs. (C and D) Wild-type (C and C′) and Cdc42-DN (D and D′) embryos labeled for Crb and the early endosome marker Rab5. (E and F) Wild-type (E and E′) and Cdc42-DN (F and F′) embryos labeled for Crb and the early endosome marker Avl. (G and H) Wild-type (G and G′) and Cdc42-DN (H and H′) embryos labeled for Crb and the recycling endosome marker Rab11. (I and J) Embryos expressing the late endosome marker UAS-YFP-Rab7 under the control of da-Gal4 (I and I′) and embryos expressing both UAS-Cdc42-DN and UAS-YFP-Rab7 under the control of da-Gal4 (J and J′) labeled for Crb and GFP. (K and L) Wild-type (K and K′) and Cdc42-DN (L and L′) embryos labeled for Crb and the Golgi marker Lva. (M and N) Wild-type (M and M′) and Cdc42-DN (N and N′) embryos labeled for Crb and the ER marker KDEL. Bars: (A–N) 10 μm; (A′–N′) 5 μm.

Mentions: To identify the compartment that accumulates apical proteins in da>Cdc42-DN embryos, we used several markers that highlight different stages in the endocytotic pathway. Double labeling of da>Cdc42-DN embryos with Crb and either Avalanche (Avl), a syntaxin associated with early endosome (Lu and Bilder, 2005), the early endosome marker Rab5 (Wucherpfennig et al., 2003), the recycling endosome marker Rab11 (Dollar et al., 2002; Pelissier et al., 2003), the multivesicular body/late endosome marker Rab7 (Entchev et al., 2000), or Hrs, which marks an early transitory stage between early and late endosomes (Lloyd et al., 2002; Kanwar and Fortini, 2008), showed that only Hrs colocalized with Crb (Fig. 5, A–J) and other apical transmembrane proteins (not depicted). Hrs labels a large number of cytoplasmic puncta. However, colocalization with Crb, Cdc42, and other apical proteins was only seen in approximately one large vesicle per cell in da>Cdc42-DN embryos. These double-labeling experiments were also performed in cdc42 mutant embryos with similar results (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200807020/DC1; and not depicted). Hrs-positive vesicles of similar size were not seen in wild-type control embryos, suggesting that loss of Cdc42 function changes the morphology of endosomes, enlarging them likely by blocking the processing of apical proteins from early to late endosomes. Previous work has found that Cdc42 is associated with the Golgi and implicated Cdc42 in the regulation of exocytosis in some mammalian cells (Kroschewski et al., 1999; Müsch et al., 2001). We did not detect Cdc42 in association with the Golgi, nor did we detect any abnormal accumulation of the seven membrane proteins we examined (Crb, Notch, Delta, Cad87A, DEcad, Ed, and Nrt) in the secretory pathway using markers for the ER (KDEL [Lys-Asp-Glu-Leu]), the Golgi (Lava lamp [Lva]), or the recycling endosome (Rab11), which serves as an exocytotic conduit (Fig. 5, G, H, and K–N; Lock and Stow, 2005; Blankenship et al., 2007). Together, these findings suggest that Cdc42 regulates endocytosis but not exocytosis in Drosophila embryonic epithelia.


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)

Apical proteins accumulate in an Hrs-positive endocytotic compartment in Cdc42-compromised embryos. (A and B) Wild-type (A and A′) and Cdc42-DN (B and B′) embryos labeled with Crb and the endosome marker Hrs. (C and D) Wild-type (C and C′) and Cdc42-DN (D and D′) embryos labeled for Crb and the early endosome marker Rab5. (E and F) Wild-type (E and E′) and Cdc42-DN (F and F′) embryos labeled for Crb and the early endosome marker Avl. (G and H) Wild-type (G and G′) and Cdc42-DN (H and H′) embryos labeled for Crb and the recycling endosome marker Rab11. (I and J) Embryos expressing the late endosome marker UAS-YFP-Rab7 under the control of da-Gal4 (I and I′) and embryos expressing both UAS-Cdc42-DN and UAS-YFP-Rab7 under the control of da-Gal4 (J and J′) labeled for Crb and GFP. (K and L) Wild-type (K and K′) and Cdc42-DN (L and L′) embryos labeled for Crb and the Golgi marker Lva. (M and N) Wild-type (M and M′) and Cdc42-DN (N and N′) embryos labeled for Crb and the ER marker KDEL. Bars: (A–N) 10 μm; (A′–N′) 5 μm.
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fig5: Apical proteins accumulate in an Hrs-positive endocytotic compartment in Cdc42-compromised embryos. (A and B) Wild-type (A and A′) and Cdc42-DN (B and B′) embryos labeled with Crb and the endosome marker Hrs. (C and D) Wild-type (C and C′) and Cdc42-DN (D and D′) embryos labeled for Crb and the early endosome marker Rab5. (E and F) Wild-type (E and E′) and Cdc42-DN (F and F′) embryos labeled for Crb and the early endosome marker Avl. (G and H) Wild-type (G and G′) and Cdc42-DN (H and H′) embryos labeled for Crb and the recycling endosome marker Rab11. (I and J) Embryos expressing the late endosome marker UAS-YFP-Rab7 under the control of da-Gal4 (I and I′) and embryos expressing both UAS-Cdc42-DN and UAS-YFP-Rab7 under the control of da-Gal4 (J and J′) labeled for Crb and GFP. (K and L) Wild-type (K and K′) and Cdc42-DN (L and L′) embryos labeled for Crb and the Golgi marker Lva. (M and N) Wild-type (M and M′) and Cdc42-DN (N and N′) embryos labeled for Crb and the ER marker KDEL. Bars: (A–N) 10 μm; (A′–N′) 5 μm.
Mentions: To identify the compartment that accumulates apical proteins in da>Cdc42-DN embryos, we used several markers that highlight different stages in the endocytotic pathway. Double labeling of da>Cdc42-DN embryos with Crb and either Avalanche (Avl), a syntaxin associated with early endosome (Lu and Bilder, 2005), the early endosome marker Rab5 (Wucherpfennig et al., 2003), the recycling endosome marker Rab11 (Dollar et al., 2002; Pelissier et al., 2003), the multivesicular body/late endosome marker Rab7 (Entchev et al., 2000), or Hrs, which marks an early transitory stage between early and late endosomes (Lloyd et al., 2002; Kanwar and Fortini, 2008), showed that only Hrs colocalized with Crb (Fig. 5, A–J) and other apical transmembrane proteins (not depicted). Hrs labels a large number of cytoplasmic puncta. However, colocalization with Crb, Cdc42, and other apical proteins was only seen in approximately one large vesicle per cell in da>Cdc42-DN embryos. These double-labeling experiments were also performed in cdc42 mutant embryos with similar results (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200807020/DC1; and not depicted). Hrs-positive vesicles of similar size were not seen in wild-type control embryos, suggesting that loss of Cdc42 function changes the morphology of endosomes, enlarging them likely by blocking the processing of apical proteins from early to late endosomes. Previous work has found that Cdc42 is associated with the Golgi and implicated Cdc42 in the regulation of exocytosis in some mammalian cells (Kroschewski et al., 1999; Müsch et al., 2001). We did not detect Cdc42 in association with the Golgi, nor did we detect any abnormal accumulation of the seven membrane proteins we examined (Crb, Notch, Delta, Cad87A, DEcad, Ed, and Nrt) in the secretory pathway using markers for the ER (KDEL [Lys-Asp-Glu-Leu]), the Golgi (Lava lamp [Lva]), or the recycling endosome (Rab11), which serves as an exocytotic conduit (Fig. 5, G, H, and K–N; Lock and Stow, 2005; Blankenship et al., 2007). Together, these findings suggest that Cdc42 regulates endocytosis but not exocytosis in Drosophila embryonic epithelia.

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