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Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.
Figure 1. Purification and characterization of recombinant extracellular cadherin proteins. The extracellular domains of HE-cadherin and C-cadherin were both expressed in CHO cells as a COOH-terminal fusion proteins with the Fc part of human IgG. Recombinant proteins were purified from the media on a Protein A column. (A) Coomassie staining of HEEC1â5Fc and CEC1â5Fc run under reducing and nonreducing conditions. (B) Immunoblot analysis of the recombinant HEEC1â5Fc and CEC1â5Fc proteins using antiâhuman Fc, anti-HE cadherin, or antiâC-cadherin antibodies. (C) Bead aggregation assay: protein-Aâcoated beads coupled to HEEC1â5Fc or CEC1â5Fc were allowed to aggregate in the presence of Ca2+ or EDTA for the indicated time period.
Figure 2. C-cadherinâ or HE-cadherinâexpressing CHO cells bind equally well to either HEEC1â5Fc or CEC1â5Fc in an adhesion flow assay. (A) Adhesion flow assay using 100 μg of either HEEC1â5Fc or CEC1â5Fc as substrates. Note that the curves representing CHO cells on either substrate or any samples in the presence of EDTA are all collapsed on the X-axis. (B) Strengthening of adhesion. Cells were allowed to attach to HEEC1â5Fc (75 μg/ml) or CEC1â5Fc (50 μg/ml) substrates for either 10 or 40 min before they were subjected to increasing flow rates. (C) Different concentrations of HEEC1â5Fc substrate in the adhesion flow assay using both C-CHO and HE-CHO cells. (D) Different concentrations of CEC1â5Fc substrates in the adhesion flow assay using both C-CHO cells and HE-CHO cells.
Figure 3. Xenopus blastomeres adhere similarly well to both CEC1â5Fc and HEEC1â5Fc proteins. Blastomeres isolated from Xenopus animal cap tissue explants were allowed to adhere to different amounts of HEEC1â5Fc, CEC1â5Fc, or BSA. The results shown are the average of four independent experiments. Black bars, HEEC1â5Fc protein; white bars, CEC1â5Fc protein. Note that adherence of blastomeres to BSA alone is negligible, and therefore does not appear in the graph.
Figure 4. Characterization of CHO cells expressing different classical type I cadherins. (A) Immunofluorescence staining of different cadherin-expressing CHO cell lines, using antibodies to either the specific cadherin as indicated, β-catenin, or p120ctn. (B) Western blot analysis of expression levels of cadherins or β-catenin in the different cadherin CHO cell lines using equal micrograms of total protein. The same membrane was incubated with a β-catenin antibody and a pan-cadherin antibody (PEP-1). (C) Cell surface expression of different cadherins on CHO cells. Intact cells were biotinylated, lysed, and equal amounts of protein were immunoprecipitated with a β-catenin antiserum and immunoprecipitates were Western blotted, after which the biotinylated proteins were recognized by streptavidin-HRP.
Figure 5. XE-cadherin CHO cells and HN-cadherin CHO cells show no adhesive specificity. (A) Adhesion flow assay of XE-CHO cells attached to either CEC1â5Fc or HEEC1â5Fc substrates (100 μg/ml). (B) Adhesion flow assay of HN-CHO attached to either CEC1â5Fc or HEEC1â5 (100 μg/ml). (C) Different concentrations of HEEC1â5Fc substrate in the adhesion flow assay using both HN-CHO and HE-CHO cells. (D) Different concentrations of CEC1â5Fc substrates in the adhesion flow assay using both HN-CHO and HE-CHO cells.
Figure 6. Variable sorting specificities for different cadherin-expressing CHO cells. Cell aggregation assays using cells labeled with fluorescent dyes, either diI (red) or diO (green). Cells were allowed to aggregate for 3 h. For AâC, examples of fluorescence are shown in top panels, with quantification of sorting versus mixing shown in the graphs below. (A) diI-labeled C-CHO cells mix completely with diO-labeled C-CHO cells, and diI-labeled HE-CHO cells also mix completely with diO-labeled HE-CHO cells, showing that the fluorescent label does not cause cells to sort artifactually. Middle, C-CHO cells (red) also mix to a large extent with HE-CHO cells (green). (B) XE-CHO cells (red in all cases) mix with XE-CHO cells (green) or with C-CHO cells (green), but sort out from HE-CHO cells (green). (C) HN-CHO cells (red in all cases) mix with HN-CHO cells (green) or C-CHO cells (green), but sort out from HE-CHO cells (green). Total number of counted aggregates is set at 100%. Black bars, diI labeled aggregates (red); striped bars, diI- and diO-labeled mixed aggregates; white bars, diO-labeled aggregates (green).
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