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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.

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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 A–C, 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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fig6: 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 A–C, 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).

Mentions: Even though the different cadherin expressing CHO cells failed to reveal any specificity in the basic adhesion assay, we decided to evaluate cell sorting behaviors between the different cadherin expressing CHO cells using a coaggregation assay. Xenopus C-CHO– and HE-CHO–expressing cells were examined first. Cells were labeled with either dI or diO and allowed to aggregate for different periods of time. Labeled untransfected CHO cells were used as a control and did not show much nonspecific aggregation, even after overnight incubation (3–5 cell aggregates at the most; unpublished data). In addition, no aggregation was observed for either C-CHO or HE-CHO cells in the presence of EDTA. As expected, diI-labeled C-cadherin cells were totally intermixed with diO-labeled C-cadherin cells (Fig. 6 A), showing that the fluorescent label did not influence the formation of mixed aggregates. Similarly, the diI- and diO-labeled HE-CHO cells were mixed (Fig. 6 A). Surprisingly, diI-labeled C-cadherin cells also mixed significantly with diO-labeled HE-cadherin cells, albeit less completely (Fig. 6 A). Quantification showed that >40% of counted aggregates consisted of both C-CHO and HE-CHO cells (Fig. 6 A). Therefore, C-cadherin and human E-cadherin cells did not strongly sort out from each other.


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

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

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 A–C, 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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Related In: Results  -  Collection

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fig6: 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 A–C, 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).
Mentions: Even though the different cadherin expressing CHO cells failed to reveal any specificity in the basic adhesion assay, we decided to evaluate cell sorting behaviors between the different cadherin expressing CHO cells using a coaggregation assay. Xenopus C-CHO– and HE-CHO–expressing cells were examined first. Cells were labeled with either dI or diO and allowed to aggregate for different periods of time. Labeled untransfected CHO cells were used as a control and did not show much nonspecific aggregation, even after overnight incubation (3–5 cell aggregates at the most; unpublished data). In addition, no aggregation was observed for either C-CHO or HE-CHO cells in the presence of EDTA. As expected, diI-labeled C-cadherin cells were totally intermixed with diO-labeled C-cadherin cells (Fig. 6 A), showing that the fluorescent label did not influence the formation of mixed aggregates. Similarly, the diI- and diO-labeled HE-CHO cells were mixed (Fig. 6 A). Surprisingly, diI-labeled C-cadherin cells also mixed significantly with diO-labeled HE-cadherin cells, albeit less completely (Fig. 6 A). Quantification showed that >40% of counted aggregates consisted of both C-CHO and HE-CHO cells (Fig. 6 A). Therefore, C-cadherin and human E-cadherin cells did not strongly sort out from each other.

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