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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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crb and shg enhance the Cdc42-DN phenotype. Quantification of ventral cuticle defects in wild-type; Cdc42-DN; Cdc42-DN, crb/+; Cdc42-DN, shg/+; and Cdc42-DN, Notch/+ embryos. Embryo collections of double-mutant genotypes contained a predicted 50% of double mutants and 50% of Cdc42-DN embryos. (A) The number of intact abdominal denticle belts is presented as mean ± SEM (error bars). For Cdc42-DN, crb/+ and Cdc42-DN, shg/+ embryos, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). For Cdc42-DN, Notch/+ embryos, there is no significant difference relative to Cdc42-DN embryos. (B) Distribution of ventral cuticle phenotypes in a population of Cdc42-DN embryos. (C) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, crb11a22/+. (C′) Ventral cuticle of a Cdc42-DN embryo. Arrows indicate holes in the ventral surface. (C′′) Ventral cuticle of an embryo from a Cdc42-DN, crb/+ population with severely enhanced ventral defects (indicated by bracket). (D) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, shgR69/+. (E) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, N55e11/+. Bar, 100 μm.
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fig7: crb and shg enhance the Cdc42-DN phenotype. Quantification of ventral cuticle defects in wild-type; Cdc42-DN; Cdc42-DN, crb/+; Cdc42-DN, shg/+; and Cdc42-DN, Notch/+ embryos. Embryo collections of double-mutant genotypes contained a predicted 50% of double mutants and 50% of Cdc42-DN embryos. (A) The number of intact abdominal denticle belts is presented as mean ± SEM (error bars). For Cdc42-DN, crb/+ and Cdc42-DN, shg/+ embryos, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). For Cdc42-DN, Notch/+ embryos, there is no significant difference relative to Cdc42-DN embryos. (B) Distribution of ventral cuticle phenotypes in a population of Cdc42-DN embryos. (C) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, crb11a22/+. (C′) Ventral cuticle of a Cdc42-DN embryo. Arrows indicate holes in the ventral surface. (C′′) Ventral cuticle of an embryo from a Cdc42-DN, crb/+ population with severely enhanced ventral defects (indicated by bracket). (D) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, shgR69/+. (E) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, N55e11/+. Bar, 100 μm.

Mentions: Mammalian Rab5 has been associated with both apical and basolateral endocytosis in epithelial cells (Bucci et al., 1994). In Drosophila epithelial cells, however, Rab5 appears to have an essential function only in apical endocytosis, as Rab5- epithelial cells accumulate apical markers such as Crb and Notch at the plasma membrane but not basolateral markers such as DEcad (Lu and Bilder, 2005). This would imply that the endocytosis of apical proteins, which is counteracted by Rab5-DN, is the primary effect of the loss of Cdc42 function and that the failure to maintain AJs is secondary to the loss of apical polarity regulators such as Crb (Fig. 2, F and G) or the Par proteins (see following section), which are known to control AJ assembly and stability (Tepass et al., 2001; Knust and Bossinger, 2002). To further test this model, we studied functional interactions between Cdc42 and Crb, DEcad, or Notch by reducing the gene copy of crb, shotgun (shg; the gene encoding DEcad), and Notch from two to one in a da>Cdc42-DN background. The interactions were evaluated and quantified by examining the degree to which the ventral cuticle was disrupted (Fig. 7). Removal of a single copy of crb, shg, or Notch in a wild-type background does not cause lethality or defects in the embryonic epidermis. We did not find interactions with Notch (Fig. 7, A and E), which is consistent with the fact that Cdc42-compromised embryos did not show a Notch-like phenotype (Genova et al., 2000). In contrast, loss of one copy of shg in da>Cdc42-DN embryos led to a significant enhancement of the da>Cdc42-DN phenotype, and the loss of a single copy of crb caused a dramatic enhancement of the da>Cdc42-DN phenotype (Fig. 7, A–D). Fig. 7 A compares the mean phenotypic strength of different genotypes. This representation underestimates the actual strength of the phenotypic enhancement, as only an estimated 50% of embryos evaluated for each genotype are double mutants, whereas the other 50% are da>Cdc42-DN embryos. Therefore, we also present our data by listing the number of embryos in each phenotypic class (Fig. 7, B–E; 0 = no ventral epidermis; 8 = normal ventral epidermis). The majority of da>Cdc42-DN, crb/+ embryos apparently lack abdominal denticle bands and in fact lack most of their ventral epidermis (Fig. 7 C). The strong interaction between Cdc42 and crb compared with the weak interaction between Cdc42 and shg is consistent with the model that the loss of Crb from the membrane is the key event in response to Cdc42 down-regulation that compromises the ability of the neuroectoderm to maintain AJs. Collectively, our findings suggest that Cdc42 negatively regulates the frequency of apical endocytosis to promote apical polarity and, as a consequence, the stability of AJs during cell rearrangement.


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)

crb and shg enhance the Cdc42-DN phenotype. Quantification of ventral cuticle defects in wild-type; Cdc42-DN; Cdc42-DN, crb/+; Cdc42-DN, shg/+; and Cdc42-DN, Notch/+ embryos. Embryo collections of double-mutant genotypes contained a predicted 50% of double mutants and 50% of Cdc42-DN embryos. (A) The number of intact abdominal denticle belts is presented as mean ± SEM (error bars). For Cdc42-DN, crb/+ and Cdc42-DN, shg/+ embryos, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). For Cdc42-DN, Notch/+ embryos, there is no significant difference relative to Cdc42-DN embryos. (B) Distribution of ventral cuticle phenotypes in a population of Cdc42-DN embryos. (C) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, crb11a22/+. (C′) Ventral cuticle of a Cdc42-DN embryo. Arrows indicate holes in the ventral surface. (C′′) Ventral cuticle of an embryo from a Cdc42-DN, crb/+ population with severely enhanced ventral defects (indicated by bracket). (D) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, shgR69/+. (E) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, N55e11/+. Bar, 100 μm.
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fig7: crb and shg enhance the Cdc42-DN phenotype. Quantification of ventral cuticle defects in wild-type; Cdc42-DN; Cdc42-DN, crb/+; Cdc42-DN, shg/+; and Cdc42-DN, Notch/+ embryos. Embryo collections of double-mutant genotypes contained a predicted 50% of double mutants and 50% of Cdc42-DN embryos. (A) The number of intact abdominal denticle belts is presented as mean ± SEM (error bars). For Cdc42-DN, crb/+ and Cdc42-DN, shg/+ embryos, the difference in the mean number of intact belts relative to Cdc42-DN embryos is statistically significant (P < 0.001). For Cdc42-DN, Notch/+ embryos, there is no significant difference relative to Cdc42-DN embryos. (B) Distribution of ventral cuticle phenotypes in a population of Cdc42-DN embryos. (C) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, crb11a22/+. (C′) Ventral cuticle of a Cdc42-DN embryo. Arrows indicate holes in the ventral surface. (C′′) Ventral cuticle of an embryo from a Cdc42-DN, crb/+ population with severely enhanced ventral defects (indicated by bracket). (D) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, shgR69/+. (E) Distribution of ventral cuticle phenotypes in a population in which 50% of embryos are predicted to be Cdc42-DN and 50% are predicted to be Cdc42-DN, N55e11/+. Bar, 100 μm.
Mentions: Mammalian Rab5 has been associated with both apical and basolateral endocytosis in epithelial cells (Bucci et al., 1994). In Drosophila epithelial cells, however, Rab5 appears to have an essential function only in apical endocytosis, as Rab5- epithelial cells accumulate apical markers such as Crb and Notch at the plasma membrane but not basolateral markers such as DEcad (Lu and Bilder, 2005). This would imply that the endocytosis of apical proteins, which is counteracted by Rab5-DN, is the primary effect of the loss of Cdc42 function and that the failure to maintain AJs is secondary to the loss of apical polarity regulators such as Crb (Fig. 2, F and G) or the Par proteins (see following section), which are known to control AJ assembly and stability (Tepass et al., 2001; Knust and Bossinger, 2002). To further test this model, we studied functional interactions between Cdc42 and Crb, DEcad, or Notch by reducing the gene copy of crb, shotgun (shg; the gene encoding DEcad), and Notch from two to one in a da>Cdc42-DN background. The interactions were evaluated and quantified by examining the degree to which the ventral cuticle was disrupted (Fig. 7). Removal of a single copy of crb, shg, or Notch in a wild-type background does not cause lethality or defects in the embryonic epidermis. We did not find interactions with Notch (Fig. 7, A and E), which is consistent with the fact that Cdc42-compromised embryos did not show a Notch-like phenotype (Genova et al., 2000). In contrast, loss of one copy of shg in da>Cdc42-DN embryos led to a significant enhancement of the da>Cdc42-DN phenotype, and the loss of a single copy of crb caused a dramatic enhancement of the da>Cdc42-DN phenotype (Fig. 7, A–D). Fig. 7 A compares the mean phenotypic strength of different genotypes. This representation underestimates the actual strength of the phenotypic enhancement, as only an estimated 50% of embryos evaluated for each genotype are double mutants, whereas the other 50% are da>Cdc42-DN embryos. Therefore, we also present our data by listing the number of embryos in each phenotypic class (Fig. 7, B–E; 0 = no ventral epidermis; 8 = normal ventral epidermis). The majority of da>Cdc42-DN, crb/+ embryos apparently lack abdominal denticle bands and in fact lack most of their ventral epidermis (Fig. 7 C). The strong interaction between Cdc42 and crb compared with the weak interaction between Cdc42 and shg is consistent with the model that the loss of Crb from the membrane is the key event in response to Cdc42 down-regulation that compromises the ability of the neuroectoderm to maintain AJs. Collectively, our findings suggest that Cdc42 negatively regulates the frequency of apical endocytosis to promote apical polarity and, as a consequence, the stability of AJs during cell rearrangement.

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