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Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42.

Chu YS, Thomas WA, Eder O, Pincet F, Perez E, Thiery JP, Dufour S - J. Cell Biol. (2004)

Bottom Line: Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels.Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time.Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-Institut Curie, Paris, France.

ABSTRACT
We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell-cell adhesion. The force required to separate E-cadherin-expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin-based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.

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SF depends on cadherin expression at the surface. (A and B) Characterization of clones differing in E-cadherin expression level. (A) Western blot analysis of cell extracts with anti–E-cadherin or β-cat, paired with an anti–α-tubulin. Quantification of cadherin and β-cat in cell extracts is indicated in violet. (B) FACS analysis of E-cadherin expression on the cell surface of four different clones. (C) SF (y axis, nN) measured for 30-min doublets of various clones (x axis, relative cadherin content in %). In red, the rate of increase of SF (y axis, nN/min) varies linearly with the square of the % cadherin expression (x axis). The equation for the best fitting red line is Y = 3 × 10−4 X + 0.2661.
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fig4: SF depends on cadherin expression at the surface. (A and B) Characterization of clones differing in E-cadherin expression level. (A) Western blot analysis of cell extracts with anti–E-cadherin or β-cat, paired with an anti–α-tubulin. Quantification of cadherin and β-cat in cell extracts is indicated in violet. (B) FACS analysis of E-cadherin expression on the cell surface of four different clones. (C) SF (y axis, nN) measured for 30-min doublets of various clones (x axis, relative cadherin content in %). In red, the rate of increase of SF (y axis, nN/min) varies linearly with the square of the % cadherin expression (x axis). The equation for the best fitting red line is Y = 3 × 10−4 X + 0.2661.

Mentions: To test the effect of cadherin concentration on cell adhesion, we generated various stably transfected S180 clones differing in the amount of E-cadherin expressed at the cell surface. Clones were selected by FACS, on the basis of homogeneous cadherin expression in all cells of the population. Western blot analyses with anti–E-cadherin and anti–β-cat were used to quantify the levels of these two proteins in each clone. The highest value obtained, that of the Ecad clone, was set at 100% and was treated as the reference clone in the analysis (E100). Clones were renamed based on their E-cadherin levels on Western blots. Clones E2, E14, E38, E41, and E58 were selected for further analysis; their total cadherin levels relative to the Ecad (100%) were 2%, 14%, 38%, 41%, and 58%, respectively (Fig. 4 A). The relative levels of β-cat were similar to those of cadherins, indicating that β-cat content could be used to estimate cadherin content in cells. By contrast, α-cat and p120 levels, and the pattern of tyrosine phosphorylation, were similar in all the clones studied (unpublished data). The results obtained by flow cytometry analysis (Fig. 4 B) were similar to those obtained by Western blotting.


Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42.

Chu YS, Thomas WA, Eder O, Pincet F, Perez E, Thiery JP, Dufour S - J. Cell Biol. (2004)

SF depends on cadherin expression at the surface. (A and B) Characterization of clones differing in E-cadherin expression level. (A) Western blot analysis of cell extracts with anti–E-cadherin or β-cat, paired with an anti–α-tubulin. Quantification of cadherin and β-cat in cell extracts is indicated in violet. (B) FACS analysis of E-cadherin expression on the cell surface of four different clones. (C) SF (y axis, nN) measured for 30-min doublets of various clones (x axis, relative cadherin content in %). In red, the rate of increase of SF (y axis, nN/min) varies linearly with the square of the % cadherin expression (x axis). The equation for the best fitting red line is Y = 3 × 10−4 X + 0.2661.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172605&req=5

fig4: SF depends on cadherin expression at the surface. (A and B) Characterization of clones differing in E-cadherin expression level. (A) Western blot analysis of cell extracts with anti–E-cadherin or β-cat, paired with an anti–α-tubulin. Quantification of cadherin and β-cat in cell extracts is indicated in violet. (B) FACS analysis of E-cadherin expression on the cell surface of four different clones. (C) SF (y axis, nN) measured for 30-min doublets of various clones (x axis, relative cadherin content in %). In red, the rate of increase of SF (y axis, nN/min) varies linearly with the square of the % cadherin expression (x axis). The equation for the best fitting red line is Y = 3 × 10−4 X + 0.2661.
Mentions: To test the effect of cadherin concentration on cell adhesion, we generated various stably transfected S180 clones differing in the amount of E-cadherin expressed at the cell surface. Clones were selected by FACS, on the basis of homogeneous cadherin expression in all cells of the population. Western blot analyses with anti–E-cadherin and anti–β-cat were used to quantify the levels of these two proteins in each clone. The highest value obtained, that of the Ecad clone, was set at 100% and was treated as the reference clone in the analysis (E100). Clones were renamed based on their E-cadherin levels on Western blots. Clones E2, E14, E38, E41, and E58 were selected for further analysis; their total cadherin levels relative to the Ecad (100%) were 2%, 14%, 38%, 41%, and 58%, respectively (Fig. 4 A). The relative levels of β-cat were similar to those of cadherins, indicating that β-cat content could be used to estimate cadherin content in cells. By contrast, α-cat and p120 levels, and the pattern of tyrosine phosphorylation, were similar in all the clones studied (unpublished data). The results obtained by flow cytometry analysis (Fig. 4 B) were similar to those obtained by Western blotting.

Bottom Line: Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels.Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time.Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-Institut Curie, Paris, France.

ABSTRACT
We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell-cell adhesion. The force required to separate E-cadherin-expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin-based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.

Show MeSH
Related in: MedlinePlus