Limits...
Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Niessen CM, Gumbiner BM - J. Cell Biol. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199232&req=5

fig4: 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.

Mentions: Both human N-cadherin and Xenopus E-cadherin were stably expressed in CHO cells. Immunofluorescence analysis of the cell lines showed that all four different cadherin lines express the cadherin at the plasma membrane, together with β-catenin and p120ctn (Fig. 4 A). It has been shown that sorting of cells can occur when cells with different levels of the same cadherin are mixed (Friedlander et al., 1989; Steinberg and Takeichi, 1994). Therefore, we used cell lines that showed similar levels of the different cadherins. Cadherin expression levels were assessed by immunoblotting with either β-catenin antibodies or a pan-cadherin antiserum that recognizes a conserved peptide in the cytoplasmic domain of cadherins (Levine et al., 1994). CHO cells express only very low levels of β-catenin, which is upregulated upon transfection of the cells with a cadherin. Since the stoichiometry between β-catenin and cadherins is 1:1 (Ozawa and Kemler, 1992; Huber et al., 2001), the cadherin levels can be directly correlated to those of β-catenin levels. Similar amounts of total β-catenin were detected by an anti–β-catenin antiserum in lysates of Xenopus E-cadherin (XE)-expressing CHO cells, human N-cadherin (HN)-expressing CHO cells, HE-CHO, and C-CHO cells (Fig. 4 B). A similar result was found with the pan-cadherin antibody, although here the difference between the C-CHO or HE-CHO cells compared with the XE- or HN-CHO cells seemed more pronounced than when compared with the β-catenin result (Fig. 4 B). Since both antibody incubations were done on the same blot, the difference cannot be explained by loading differences. Although we do not know why we see a difference in reaction intensity with the two antibodies, it might be due to differences in pan-cadherin affinity for the cadherins used. We therefore sought to also examine the cell surface expression of the cadherins, since cadherins can only function in adhesion when present on the cell surface. No major differences were found in surface expression of the different cadherins on CHO cells, as judged by accessibility to cell surface biotinylation (Fig. 4 B). Therefore, the different cadherins were all expressed at similar levels, facilitating comparison between cell lines.


Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Niessen CM, Gumbiner BM - J. Cell Biol. (2002)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199232&req=5

fig4: 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.
Mentions: Both human N-cadherin and Xenopus E-cadherin were stably expressed in CHO cells. Immunofluorescence analysis of the cell lines showed that all four different cadherin lines express the cadherin at the plasma membrane, together with β-catenin and p120ctn (Fig. 4 A). It has been shown that sorting of cells can occur when cells with different levels of the same cadherin are mixed (Friedlander et al., 1989; Steinberg and Takeichi, 1994). Therefore, we used cell lines that showed similar levels of the different cadherins. Cadherin expression levels were assessed by immunoblotting with either β-catenin antibodies or a pan-cadherin antiserum that recognizes a conserved peptide in the cytoplasmic domain of cadherins (Levine et al., 1994). CHO cells express only very low levels of β-catenin, which is upregulated upon transfection of the cells with a cadherin. Since the stoichiometry between β-catenin and cadherins is 1:1 (Ozawa and Kemler, 1992; Huber et al., 2001), the cadherin levels can be directly correlated to those of β-catenin levels. Similar amounts of total β-catenin were detected by an anti–β-catenin antiserum in lysates of Xenopus E-cadherin (XE)-expressing CHO cells, human N-cadherin (HN)-expressing CHO cells, HE-CHO, and C-CHO cells (Fig. 4 B). A similar result was found with the pan-cadherin antibody, although here the difference between the C-CHO or HE-CHO cells compared with the XE- or HN-CHO cells seemed more pronounced than when compared with the β-catenin result (Fig. 4 B). Since both antibody incubations were done on the same blot, the difference cannot be explained by loading differences. Although we do not know why we see a difference in reaction intensity with the two antibodies, it might be due to differences in pan-cadherin affinity for the cadherins used. We therefore sought to also examine the cell surface expression of the cadherins, since cadherins can only function in adhesion when present on the cell surface. No major differences were found in surface expression of the different cadherins on CHO cells, as judged by accessibility to cell surface biotinylation (Fig. 4 B). Therefore, the different cadherins were all expressed at similar levels, facilitating comparison between cell lines.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus