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Binding site for p120/delta-catenin is not required for Drosophila E-cadherin function in vivo.

Pacquelet A, Lin L, Rorth P - J. Cell Biol. (2003)

Bottom Line: As expected, DE-cadherin-Delta beta did not substitute for DE-cadherin in these processes, although it retained some residual activity.Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations.Thus, interaction with p120/delta-catenin does not appear to be required for DE-cadherin function in vivo.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

ABSTRACT
Homophilic cell adhesion mediated by classical cadherins is important for many developmental processes. Proteins that interact with the cytoplasmic domain of cadherin, in particular the catenins, are thought to regulate the strength and possibly the dynamics of adhesion. beta-catenin links cadherin to the actin cytoskeleton via alpha-catenin. The role of p120/delta-catenin proteins in regulating cadherin function is less clear. Both beta-catenin and p120/delta-catenin are conserved in Drosophila. Here, we address the importance of cadherin-catenin interactions in vivo, using mutant variants of Drosophila epithelial cadherin (DE-cadherin) that are selectively defective in p120ctn (DE-cadherin-AAA) or beta-catenin-armadillo (DE-cadherin-Delta beta) interactions. We have analyzed the ability of these proteins to substitute for endogenous DE-cadherin activity in multiple cadherin-dependent processes during Drosophila development and oogenesis; epithelial integrity, follicle cell sorting, oocyte positioning, as well as the dynamic adhesion required for border cell migration. As expected, DE-cadherin-Delta beta did not substitute for DE-cadherin in these processes, although it retained some residual activity. Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations. Thus, interaction with p120/delta-catenin does not appear to be required for DE-cadherin function in vivo.

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DE-cadherin-wt and DE-cadherin-AAA but not DE-cadherin-Δβ transgenes can rescue shg phenotypes. (A–E) Phalloidin (red) stains the actin cytoskeleton of egg chambers and non-GFP (non-green) cells are wild type (A–E) or shg mutant (A'–E') clones. (A and B) Border cell migration. In stage 10 wild type egg chambers, border cells (arrow) have reached the oocyte (A and B). Lack of DE-cadherin in border cells (A') or in nurse cells (B') prevent border cells from penetrating between the nurse cells. (A'' and B'') Migration defects in shg mutant border cells (A'') and germ line (B'') clones and in the presence of the indicated transgenes (15 ≤ n ≤ 73). White, full migration; gray, incomplete migration; black, no migration. Bars, ∼20 μm. (C and D) Oocyte mispositioning. The oocyte (asterisk) is located at the posterior of a wild type egg chamber (C and D). shg mutant clones often causes a mislocalization of the oocyte (C' and D', follicular cells and germ line clones, respectively). All ovarioles are represented anterior (younger egg chambers) to the left. (C'' and D'') Oocyte mispositioning in shg mutant follicular cells (C'') and germ line (D'') clones and in the presence of the indicated transgenes (17 ≤ n ≤ 57). White, oocyte posteriorly located; black, mispositioned oocyte. Bars, ∼15 μm. (E) Follicular cell sorting. Wild type follicular clones intermingle (E), whereas shg mutant clones sort (E'). (E'') Sorting in shg mutant clones and in the presence of the indicated transgenes (27 ≤ n ≤ 68). White, no sorting; black, sorting. Bar, ∼20 μm. (F) Cuticle phenotype. F is a wild-type cuticle and F' a cuticle of a homozygous shg mutant embryo. (F'') Cuticle defects in homozygous shg mutant embryos and in the presence of the indicated transgenes (32 ≤ n ≤ 232). White, wild type cuticle; black, cuticle with shg phenotype. Bar, ∼50 μm. (G) Hatching of homozygous shg mutant embryos and in the presence of the indicated transgenes (n ≥ 278). White, hatching; black, no hatching.
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fig4: DE-cadherin-wt and DE-cadherin-AAA but not DE-cadherin-Δβ transgenes can rescue shg phenotypes. (A–E) Phalloidin (red) stains the actin cytoskeleton of egg chambers and non-GFP (non-green) cells are wild type (A–E) or shg mutant (A'–E') clones. (A and B) Border cell migration. In stage 10 wild type egg chambers, border cells (arrow) have reached the oocyte (A and B). Lack of DE-cadherin in border cells (A') or in nurse cells (B') prevent border cells from penetrating between the nurse cells. (A'' and B'') Migration defects in shg mutant border cells (A'') and germ line (B'') clones and in the presence of the indicated transgenes (15 ≤ n ≤ 73). White, full migration; gray, incomplete migration; black, no migration. Bars, ∼20 μm. (C and D) Oocyte mispositioning. The oocyte (asterisk) is located at the posterior of a wild type egg chamber (C and D). shg mutant clones often causes a mislocalization of the oocyte (C' and D', follicular cells and germ line clones, respectively). All ovarioles are represented anterior (younger egg chambers) to the left. (C'' and D'') Oocyte mispositioning in shg mutant follicular cells (C'') and germ line (D'') clones and in the presence of the indicated transgenes (17 ≤ n ≤ 57). White, oocyte posteriorly located; black, mispositioned oocyte. Bars, ∼15 μm. (E) Follicular cell sorting. Wild type follicular clones intermingle (E), whereas shg mutant clones sort (E'). (E'') Sorting in shg mutant clones and in the presence of the indicated transgenes (27 ≤ n ≤ 68). White, no sorting; black, sorting. Bar, ∼20 μm. (F) Cuticle phenotype. F is a wild-type cuticle and F' a cuticle of a homozygous shg mutant embryo. (F'') Cuticle defects in homozygous shg mutant embryos and in the presence of the indicated transgenes (32 ≤ n ≤ 232). White, wild type cuticle; black, cuticle with shg phenotype. Bar, ∼50 μm. (G) Hatching of homozygous shg mutant embryos and in the presence of the indicated transgenes (n ≥ 278). White, hatching; black, no hatching.

Mentions: We were particularly interested in how DE-cadherin might be regulated during border cell migration, which occurs during stage 9 of oogenesis. Border cells are a group of 6 to 10 somatic follicle cells that delaminate from the anterior tip of the egg chamber, invade the germ line cluster, and migrate to the oocyte. Border cells migrate in between the nurse cells and apparently use these cells as substratum. Absence of DE-cadherin from border cells or from nurse cells results in total lack of invasive migration, suggesting that border cells adhere to the nurse cell substratum through homophilic DE-cadherin interaction (Fig. 4, A and B; Niewiadomska et al., 1999). Adhesion to a substratum must in some way be dynamic for cells to move productively across it. Relative to the well-characterized function of cadherin in epithelial cell adhesion, DE-cadherin function in border cell migration might therefore require additional regulatory mechanisms. In addition, border cells normally express higher levels of DE-cadherin than other follicle cells, and migration is quite sensitive to reduction of DE-cadherin level; even weak shg alleles cause detectable delays in migration (Niewiadomska et al., 1999). For these reasons, it seemed likely that border cell migration would be sensitive to perturbations of cadherin–catenin interactions. To test this, we analyzed the activity of mutant DE-cadherin proteins in border cells as well as in the substratum, the nurse cells. In both border cells and nurse cells, expression of DE-cadherin-wt as well as DE-cadherin-AAA-#6 completely rescued the migration (Fig. 4, A′′ and B′′). Thus, DE-cadherin–p120ctn interaction does not appear to be required for the migration. DE-cadherin-AAA-#7 gave full rescue in nurse cells (Fig. 4 B′′), but incomplete rescue in border cells (Fig. 4 A′′). In border cells, DE-cadherin-AAA-#6 was expressed at levels similar to endogenous DE-cadherin, whereas DE-cadherin-AAA-#7 was expressed at lower levels (Fig. 3, E and F). Given that border cell migration is sensitive to cadherin expression level, this can explain the incomplete rescue by cadherin-AAA-#7. DE-cadherin-Δβ expression in border cells did not rescue the shg phenotype at all, suggesting that DE-cadherin must be anchored to the actin cytoskeleton via β-catenin to function in the migrating cells. DE-cadherin-Δβ could partially compensate for endogenous DE-cadherin in nurse cells (Fig. 4 B′′). Strong linkage to the cytoskeleton via β-catenin may be less critical for cadherin to function as substratum than in the actively migrating cell itself.


Binding site for p120/delta-catenin is not required for Drosophila E-cadherin function in vivo.

Pacquelet A, Lin L, Rorth P - J. Cell Biol. (2003)

DE-cadherin-wt and DE-cadherin-AAA but not DE-cadherin-Δβ transgenes can rescue shg phenotypes. (A–E) Phalloidin (red) stains the actin cytoskeleton of egg chambers and non-GFP (non-green) cells are wild type (A–E) or shg mutant (A'–E') clones. (A and B) Border cell migration. In stage 10 wild type egg chambers, border cells (arrow) have reached the oocyte (A and B). Lack of DE-cadherin in border cells (A') or in nurse cells (B') prevent border cells from penetrating between the nurse cells. (A'' and B'') Migration defects in shg mutant border cells (A'') and germ line (B'') clones and in the presence of the indicated transgenes (15 ≤ n ≤ 73). White, full migration; gray, incomplete migration; black, no migration. Bars, ∼20 μm. (C and D) Oocyte mispositioning. The oocyte (asterisk) is located at the posterior of a wild type egg chamber (C and D). shg mutant clones often causes a mislocalization of the oocyte (C' and D', follicular cells and germ line clones, respectively). All ovarioles are represented anterior (younger egg chambers) to the left. (C'' and D'') Oocyte mispositioning in shg mutant follicular cells (C'') and germ line (D'') clones and in the presence of the indicated transgenes (17 ≤ n ≤ 57). White, oocyte posteriorly located; black, mispositioned oocyte. Bars, ∼15 μm. (E) Follicular cell sorting. Wild type follicular clones intermingle (E), whereas shg mutant clones sort (E'). (E'') Sorting in shg mutant clones and in the presence of the indicated transgenes (27 ≤ n ≤ 68). White, no sorting; black, sorting. Bar, ∼20 μm. (F) Cuticle phenotype. F is a wild-type cuticle and F' a cuticle of a homozygous shg mutant embryo. (F'') Cuticle defects in homozygous shg mutant embryos and in the presence of the indicated transgenes (32 ≤ n ≤ 232). White, wild type cuticle; black, cuticle with shg phenotype. Bar, ∼50 μm. (G) Hatching of homozygous shg mutant embryos and in the presence of the indicated transgenes (n ≥ 278). White, hatching; black, no hatching.
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Related In: Results  -  Collection

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fig4: DE-cadherin-wt and DE-cadherin-AAA but not DE-cadherin-Δβ transgenes can rescue shg phenotypes. (A–E) Phalloidin (red) stains the actin cytoskeleton of egg chambers and non-GFP (non-green) cells are wild type (A–E) or shg mutant (A'–E') clones. (A and B) Border cell migration. In stage 10 wild type egg chambers, border cells (arrow) have reached the oocyte (A and B). Lack of DE-cadherin in border cells (A') or in nurse cells (B') prevent border cells from penetrating between the nurse cells. (A'' and B'') Migration defects in shg mutant border cells (A'') and germ line (B'') clones and in the presence of the indicated transgenes (15 ≤ n ≤ 73). White, full migration; gray, incomplete migration; black, no migration. Bars, ∼20 μm. (C and D) Oocyte mispositioning. The oocyte (asterisk) is located at the posterior of a wild type egg chamber (C and D). shg mutant clones often causes a mislocalization of the oocyte (C' and D', follicular cells and germ line clones, respectively). All ovarioles are represented anterior (younger egg chambers) to the left. (C'' and D'') Oocyte mispositioning in shg mutant follicular cells (C'') and germ line (D'') clones and in the presence of the indicated transgenes (17 ≤ n ≤ 57). White, oocyte posteriorly located; black, mispositioned oocyte. Bars, ∼15 μm. (E) Follicular cell sorting. Wild type follicular clones intermingle (E), whereas shg mutant clones sort (E'). (E'') Sorting in shg mutant clones and in the presence of the indicated transgenes (27 ≤ n ≤ 68). White, no sorting; black, sorting. Bar, ∼20 μm. (F) Cuticle phenotype. F is a wild-type cuticle and F' a cuticle of a homozygous shg mutant embryo. (F'') Cuticle defects in homozygous shg mutant embryos and in the presence of the indicated transgenes (32 ≤ n ≤ 232). White, wild type cuticle; black, cuticle with shg phenotype. Bar, ∼50 μm. (G) Hatching of homozygous shg mutant embryos and in the presence of the indicated transgenes (n ≥ 278). White, hatching; black, no hatching.
Mentions: We were particularly interested in how DE-cadherin might be regulated during border cell migration, which occurs during stage 9 of oogenesis. Border cells are a group of 6 to 10 somatic follicle cells that delaminate from the anterior tip of the egg chamber, invade the germ line cluster, and migrate to the oocyte. Border cells migrate in between the nurse cells and apparently use these cells as substratum. Absence of DE-cadherin from border cells or from nurse cells results in total lack of invasive migration, suggesting that border cells adhere to the nurse cell substratum through homophilic DE-cadherin interaction (Fig. 4, A and B; Niewiadomska et al., 1999). Adhesion to a substratum must in some way be dynamic for cells to move productively across it. Relative to the well-characterized function of cadherin in epithelial cell adhesion, DE-cadherin function in border cell migration might therefore require additional regulatory mechanisms. In addition, border cells normally express higher levels of DE-cadherin than other follicle cells, and migration is quite sensitive to reduction of DE-cadherin level; even weak shg alleles cause detectable delays in migration (Niewiadomska et al., 1999). For these reasons, it seemed likely that border cell migration would be sensitive to perturbations of cadherin–catenin interactions. To test this, we analyzed the activity of mutant DE-cadherin proteins in border cells as well as in the substratum, the nurse cells. In both border cells and nurse cells, expression of DE-cadherin-wt as well as DE-cadherin-AAA-#6 completely rescued the migration (Fig. 4, A′′ and B′′). Thus, DE-cadherin–p120ctn interaction does not appear to be required for the migration. DE-cadherin-AAA-#7 gave full rescue in nurse cells (Fig. 4 B′′), but incomplete rescue in border cells (Fig. 4 A′′). In border cells, DE-cadherin-AAA-#6 was expressed at levels similar to endogenous DE-cadherin, whereas DE-cadherin-AAA-#7 was expressed at lower levels (Fig. 3, E and F). Given that border cell migration is sensitive to cadherin expression level, this can explain the incomplete rescue by cadherin-AAA-#7. DE-cadherin-Δβ expression in border cells did not rescue the shg phenotype at all, suggesting that DE-cadherin must be anchored to the actin cytoskeleton via β-catenin to function in the migrating cells. DE-cadherin-Δβ could partially compensate for endogenous DE-cadherin in nurse cells (Fig. 4 B′′). Strong linkage to the cytoskeleton via β-catenin may be less critical for cadherin to function as substratum than in the actively migrating cell itself.

Bottom Line: As expected, DE-cadherin-Delta beta did not substitute for DE-cadherin in these processes, although it retained some residual activity.Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations.Thus, interaction with p120/delta-catenin does not appear to be required for DE-cadherin function in vivo.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

ABSTRACT
Homophilic cell adhesion mediated by classical cadherins is important for many developmental processes. Proteins that interact with the cytoplasmic domain of cadherin, in particular the catenins, are thought to regulate the strength and possibly the dynamics of adhesion. beta-catenin links cadherin to the actin cytoskeleton via alpha-catenin. The role of p120/delta-catenin proteins in regulating cadherin function is less clear. Both beta-catenin and p120/delta-catenin are conserved in Drosophila. Here, we address the importance of cadherin-catenin interactions in vivo, using mutant variants of Drosophila epithelial cadherin (DE-cadherin) that are selectively defective in p120ctn (DE-cadherin-AAA) or beta-catenin-armadillo (DE-cadherin-Delta beta) interactions. We have analyzed the ability of these proteins to substitute for endogenous DE-cadherin activity in multiple cadherin-dependent processes during Drosophila development and oogenesis; epithelial integrity, follicle cell sorting, oocyte positioning, as well as the dynamic adhesion required for border cell migration. As expected, DE-cadherin-Delta beta did not substitute for DE-cadherin in these processes, although it retained some residual activity. Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations. Thus, interaction with p120/delta-catenin does not appear to be required for DE-cadherin function in vivo.

Show MeSH
Related in: MedlinePlus