Zhong Y et al. (1999), Analysis of C-cadherin regulation during tissue...

XB-ART-13596

J Cell Biol 1999 Jan 25;1442:351-9. doi: 10.1083/jcb.144.2.351.

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

Zhong Y , Brieher WM , Gumbiner BM .

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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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Species referenced: Xenopus
Genes referenced: cdh3 fn1
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	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.
	Figure 3. Inhibition of activin-induced morphogenetic movement of animal cap explants by mAb AA5. (A) Inhibition of the elongation of activin-induced animal caps by mAb AA5. Xenopus animal caps were incubated with or without activin and treated with mAb AA5 Fab (1 μg/ml), mAb 6B6 Fab (1 μg/ml), or nonimmune mouse IgG Fab (1 μg/ml). (B) Frequency of inhibition of elongation by mAb AA5. Activin-induced elongation was plotted as a percentage of the total. n = total animal caps analyzed. Any explant exhibiting a discernible protrusion was scored as elongated. (C) mAb AA5 did not inhibit the induction of expression of mesodermal gene markers in response to activin. Animal caps were incubated with or without activin and incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse IgG Fab (1 μg/ml) until gastrula stage 10.5. Total RNA was harvested and mRNA was analyzed by RT-PCR for the presence of the indicated transcripts. RNA from whole embryos (E) provides a positive control. The −RT lane is identical to the embryo lane, except reverse transcriptase was omitted and serves as a negative control. EF-1, ubiquitously expressed, is a loading control. Brachyury is a marker of general mesoderm. Goosecoid is a marker of dorsal mesoderm.
	Figure 4. Analysis of mAb AA5 activity on cell lines and in vitro. (A) Effect of mAb AA5 on C-cadherin–mediated adhesion of CHO cells. C-CHO cells (expressing wild-type C-cadherin) were harvested in the presence of calcium and then allowed to attach to CEC1-5–coated capillary tube in the presence of either mAb AA5 Fab or nonimmune mouse Fab for 30 min. Adhesive strength was measured as the resistance of cell detachment to progressively increasing flow rates. The experiment was performed in triplicate and the percentage of cells remaining ± SE was plotted as a function of flow rate. (B) Effect of mAb AA5 on the adhesive function of a cytoplasmic tail truncated C-cadherin expressed in CHO cells (CT-CHO). Adhesion of CT-CHO cells was assayed in the presence of either mAb AA5 Fab or nonimmune mouse Fab using the flow assay described in A. The effect of inhibitory mAb 6B6 is shown for comparison. The experiment was performed in triplicate and the percentage of cells remaining ± SE was plotted as a function of time. (C) Effect of mAb AA5 on the aggregation of CEC1-5–coated FluoSpheres. Dispersed CEC1-5–coated FluoSpheres were incubated either with mAb AA5 Fab or with nonimmune mouse IgG Fab in the presence of calcium for various time periods. As a negative control, samples were also incubated with the presence of EDTA. The number of aggregated FluoSpheres (superthreshold particles) were counted using a Coulter counter. The experiment was performed in triplicate and the number of superthreshold particles ± SE was plotted as a function of time.
	Figure 5. Activin regulation of blastomere attachment to anti– C-cadherin mAbs. (A) Attachment of uninduced, activin-induced, or activin-induced and mAb AA5-treated blastomeres to substrates coated with CEC1-5, anti–C-cadherin mAb 6B6, or anti–C-cadherin mAb 5G5. (B) Uninduced and activin-induced blastomere attachment to fibronectin. (C) Attachment of uninduced and activin-induced blastomeres expressing an IL-2β receptor–C-cadherin cytoplasmic tail fusion protein to substrates coated with anti–IL-2β receptor mAbs.
	Figure 6. mAb AA5 activates cell-associated C-cadherin only. The ability of mAb AA5 to activate C-cadherin adhesion was determined after its addition to different components of the cell attachment assay. It was added either to the complete attachment assay (as in Fig. 2), to the CEC1-5–coated substrate alone (rinsed), or to the blastomeres alone (rinsed).
	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.

References [+] :

Behrens, Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. 1993, Pubmed