Limits...
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.

Show MeSH

Related in: MedlinePlus

mAb AA5 enhances the C-cadherin–mediated adhesion of activin-induced animal cap blastomeres. (A) Blastomere  aggregation assay. Dissociated blastomeres were treated with or  without activin (5 ng/ml). Activin-treated blastomeres were incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse  IgG Fab (1 μg/ml) in the presence of calcium, and then allowed  to aggregate with constant shaking for 15 min. (B) Blastomere  adhesion to CEC1-5. Dissociated blastomeres were treated with  or without activin. Activin-treated blastomeres were incubated  with either mAb AA5 Fab or nonimmune mouse IgG Fab, and  then allowed to attach to spots of CEC1-5 protein coated on a tissue culture plate in the presence of calcium for 30 min. The plates  were then shaken continuously for 2 min on a horizontal shaker  and the numbers of blastomeres per unit area remaining attached  were counted. The graph shows the results of three different experiments, each performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132887&req=5

Figure 2: mAb AA5 enhances the C-cadherin–mediated adhesion of activin-induced animal cap blastomeres. (A) Blastomere aggregation assay. Dissociated blastomeres were treated with or without activin (5 ng/ml). Activin-treated blastomeres were incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse IgG Fab (1 μg/ml) in the presence of calcium, and then allowed to aggregate with constant shaking for 15 min. (B) Blastomere adhesion to CEC1-5. Dissociated blastomeres were treated with or without activin. Activin-treated blastomeres were incubated with either mAb AA5 Fab or nonimmune mouse IgG Fab, and then allowed to attach to spots of CEC1-5 protein coated on a tissue culture plate in the presence of calcium for 30 min. The plates were then shaken continuously for 2 min on a horizontal shaker and the numbers of blastomeres per unit area remaining attached were counted. The graph shows the results of three different experiments, each performed in duplicate.

Mentions: Although mAb AA5 stimulates CEC1-5–coated bead binding to blastomeres, we wished to determine whether it stimulated blastomere adhesion using more standard types of adhesion assays. Furthermore, to rule out artifactual adhesion due to crosslinking by the bivalent antibody, we also tested monovalent Fab fragments of mAb AA5. Fab fragments of mAb AA5 also stimulated CEC1-5–coated beads binding to blastomeres (data not shown). In all further experiments described, purified Fab fragments of mAb AA5 were used (although still referred to as mAb AA5). Activin treatment reduces the rate of Ca2+-dependent aggregation of animal cap blastomeres that had been dissociated by Ca2+ removal (Brieher and Gumbiner, 1994) (Fig. 2 A, left and center). Inclusion of mAb AA5 in the aggregation assay stimulated the rate of blastomere aggregation of activin-treated blastomeres, producing larger aggregates after the same time interval (Fig. 2 A, right). We also examined the activity of mAb AA5 in a cell attachment assay (Fig. 2 B). Attachment of dissociated blastomeres to surfaces coated with CEC1-5 was assayed. No attachment to BSA-coated surfaces was observed (data not shown). Activin treatment reduced the number of blastomeres that adhered to CEC1-5 under gentle shaking conditions, and mAb AA5 significantly increased the number of activin-treated blastomeres that adhered to CEC1-5. Thus, monovalent Fab fragments of mAb AA5 stimulated C-cadherin–mediated adhesion of activin-treated blastomeres to near the level of adhesion of untreated blastomeres.


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

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

mAb AA5 enhances the C-cadherin–mediated adhesion of activin-induced animal cap blastomeres. (A) Blastomere  aggregation assay. Dissociated blastomeres were treated with or  without activin (5 ng/ml). Activin-treated blastomeres were incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse  IgG Fab (1 μg/ml) in the presence of calcium, and then allowed  to aggregate with constant shaking for 15 min. (B) Blastomere  adhesion to CEC1-5. Dissociated blastomeres were treated with  or without activin. Activin-treated blastomeres were incubated  with either mAb AA5 Fab or nonimmune mouse IgG Fab, and  then allowed to attach to spots of CEC1-5 protein coated on a tissue culture plate in the presence of calcium for 30 min. The plates  were then shaken continuously for 2 min on a horizontal shaker  and the numbers of blastomeres per unit area remaining attached  were counted. The graph shows the results of three different experiments, each performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: mAb AA5 enhances the C-cadherin–mediated adhesion of activin-induced animal cap blastomeres. (A) Blastomere aggregation assay. Dissociated blastomeres were treated with or without activin (5 ng/ml). Activin-treated blastomeres were incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse IgG Fab (1 μg/ml) in the presence of calcium, and then allowed to aggregate with constant shaking for 15 min. (B) Blastomere adhesion to CEC1-5. Dissociated blastomeres were treated with or without activin. Activin-treated blastomeres were incubated with either mAb AA5 Fab or nonimmune mouse IgG Fab, and then allowed to attach to spots of CEC1-5 protein coated on a tissue culture plate in the presence of calcium for 30 min. The plates were then shaken continuously for 2 min on a horizontal shaker and the numbers of blastomeres per unit area remaining attached were counted. The graph shows the results of three different experiments, each performed in duplicate.
Mentions: Although mAb AA5 stimulates CEC1-5–coated bead binding to blastomeres, we wished to determine whether it stimulated blastomere adhesion using more standard types of adhesion assays. Furthermore, to rule out artifactual adhesion due to crosslinking by the bivalent antibody, we also tested monovalent Fab fragments of mAb AA5. Fab fragments of mAb AA5 also stimulated CEC1-5–coated beads binding to blastomeres (data not shown). In all further experiments described, purified Fab fragments of mAb AA5 were used (although still referred to as mAb AA5). Activin treatment reduces the rate of Ca2+-dependent aggregation of animal cap blastomeres that had been dissociated by Ca2+ removal (Brieher and Gumbiner, 1994) (Fig. 2 A, left and center). Inclusion of mAb AA5 in the aggregation assay stimulated the rate of blastomere aggregation of activin-treated blastomeres, producing larger aggregates after the same time interval (Fig. 2 A, right). We also examined the activity of mAb AA5 in a cell attachment assay (Fig. 2 B). Attachment of dissociated blastomeres to surfaces coated with CEC1-5 was assayed. No attachment to BSA-coated surfaces was observed (data not shown). Activin treatment reduced the number of blastomeres that adhered to CEC1-5 under gentle shaking conditions, and mAb AA5 significantly increased the number of activin-treated blastomeres that adhered to CEC1-5. Thus, monovalent Fab fragments of mAb AA5 stimulated C-cadherin–mediated adhesion of activin-treated blastomeres to near the level of adhesion of untreated blastomeres.

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