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Analysis of C-cadherin regulation during tissue morphogenesis with an activating antibody.

Zhong Y, Brieher WM, Gumbiner BM - J. Cell Biol. (1999)

Bottom Line: Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation.It does not work when added to CEC1-5 on the substrate.Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule. Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin-mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519-527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation. Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti-C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

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Detection of anti–C-cadherin activating mAb AA5 in a  functional screen. (A) mAb AA5 restores high C-cadherin–mediated adhesion to activin-treated Xenopus blastomeres. Dissociated Xenopus animal cap blastomeres were treated with or without activin (5 ng/ml). The activin-induced blastomeres were  treated with either mAb AA5 hybridoma supernatant or nonimmune mouse IgG. Blastomere samples were then incubated with  CEC1-5–coated FluoSpheres in the presence of calcium for 60  min. (B, C, and D) Western blot analysis demonstrating mAb  AA5 binds to domain 5 of C-cadherin. CEC1-5 protein (B),  CEC1-5 expressing CHO cell extracts (C), and CEC1-4 expressing CHO cells extracts (D) were probed with anti–C-cadherin  6B6 mAb, AA5 mAb, or nonimmune mouse IgG.
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Figure 1: Detection of anti–C-cadherin activating mAb AA5 in a functional screen. (A) mAb AA5 restores high C-cadherin–mediated adhesion to activin-treated Xenopus blastomeres. Dissociated Xenopus animal cap blastomeres were treated with or without activin (5 ng/ml). The activin-induced blastomeres were treated with either mAb AA5 hybridoma supernatant or nonimmune mouse IgG. Blastomere samples were then incubated with CEC1-5–coated FluoSpheres in the presence of calcium for 60 min. (B, C, and D) Western blot analysis demonstrating mAb AA5 binds to domain 5 of C-cadherin. CEC1-5 protein (B), CEC1-5 expressing CHO cell extracts (C), and CEC1-4 expressing CHO cells extracts (D) were probed with anti–C-cadherin 6B6 mAb, AA5 mAb, or nonimmune mouse IgG.

Mentions: The screening assay was performed at room temperature. The dissociated blastomeres were incubated with 5 ng/ml activin for 1 h and then incubated with either the anti–CEC1-5 monoclonal hybridoma supernatant or RPMI medium (with 10% FCS) for 1 h. Calcium (final concentration = 2 mM) and CEC1-5–coated FluoSpheres (20 μl for each experiment) were added, and the mixture of blastomeres and FluoSpheres was agitated horizontally for 30 min. Specimens were examined with a Zeiss Axioskop equipped with plan-APOCHROMATx40 objective. Hybridoma supernatants were screened for the ability to stimulate the binding of CEC1-5 protein–coated FluoSpheres to activin-induced blastomeres (Fig. 1).


Analysis of C-cadherin regulation during tissue morphogenesis with an activating antibody.

Zhong Y, Brieher WM, Gumbiner BM - J. Cell Biol. (1999)

Detection of anti–C-cadherin activating mAb AA5 in a  functional screen. (A) mAb AA5 restores high C-cadherin–mediated adhesion to activin-treated Xenopus blastomeres. Dissociated Xenopus animal cap blastomeres were treated with or without activin (5 ng/ml). The activin-induced blastomeres were  treated with either mAb AA5 hybridoma supernatant or nonimmune mouse IgG. Blastomere samples were then incubated with  CEC1-5–coated FluoSpheres in the presence of calcium for 60  min. (B, C, and D) Western blot analysis demonstrating mAb  AA5 binds to domain 5 of C-cadherin. CEC1-5 protein (B),  CEC1-5 expressing CHO cell extracts (C), and CEC1-4 expressing CHO cells extracts (D) were probed with anti–C-cadherin  6B6 mAb, AA5 mAb, or nonimmune mouse IgG.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132887&req=5

Figure 1: Detection of anti–C-cadherin activating mAb AA5 in a functional screen. (A) mAb AA5 restores high C-cadherin–mediated adhesion to activin-treated Xenopus blastomeres. Dissociated Xenopus animal cap blastomeres were treated with or without activin (5 ng/ml). The activin-induced blastomeres were treated with either mAb AA5 hybridoma supernatant or nonimmune mouse IgG. Blastomere samples were then incubated with CEC1-5–coated FluoSpheres in the presence of calcium for 60 min. (B, C, and D) Western blot analysis demonstrating mAb AA5 binds to domain 5 of C-cadherin. CEC1-5 protein (B), CEC1-5 expressing CHO cell extracts (C), and CEC1-4 expressing CHO cells extracts (D) were probed with anti–C-cadherin 6B6 mAb, AA5 mAb, or nonimmune mouse IgG.
Mentions: The screening assay was performed at room temperature. The dissociated blastomeres were incubated with 5 ng/ml activin for 1 h and then incubated with either the anti–CEC1-5 monoclonal hybridoma supernatant or RPMI medium (with 10% FCS) for 1 h. Calcium (final concentration = 2 mM) and CEC1-5–coated FluoSpheres (20 μl for each experiment) were added, and the mixture of blastomeres and FluoSpheres was agitated horizontally for 30 min. Specimens were examined with a Zeiss Axioskop equipped with plan-APOCHROMATx40 objective. Hybridoma supernatants were screened for the ability to stimulate the binding of CEC1-5 protein–coated FluoSpheres to activin-induced blastomeres (Fig. 1).

Bottom Line: Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation.It does not work when added to CEC1-5 on the substrate.Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule. Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin-mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519-527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation. Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti-C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

Show MeSH
Related in: MedlinePlus