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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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Adhesive properties of Ecad cells. Immunodetection of β-cat (A and D), E-cadherin (B), and actin (C) in S180 cells (A) and Ecad cells (B–D). E, FACS analysis on isolated Ecad cells in suspension, after TC treatment, with an antibody directed against the extracellular domain of E-cadherin. Immunodetection of E-cadherin (F and J), β-cat (G and K), and actin (H and L) in doublets formed in suspension for 4- (F–I) or 30-min (J–M). Merged images are shown in I and M. Bars: (A) 20 μm; (F and J) 10 μm.
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fig1: Adhesive properties of Ecad cells. Immunodetection of β-cat (A and D), E-cadherin (B), and actin (C) in S180 cells (A) and Ecad cells (B–D). E, FACS analysis on isolated Ecad cells in suspension, after TC treatment, with an antibody directed against the extracellular domain of E-cadherin. Immunodetection of E-cadherin (F and J), β-cat (G and K), and actin (H and L) in doublets formed in suspension for 4- (F–I) or 30-min (J–M). Merged images are shown in I and M. Bars: (A) 20 μm; (F and J) 10 μm.

Mentions: S180 cells contain no detectable β-cat (Fig. 1 A) or cadherins (not depicted) and display minimal cell–cell adhesion in tissue culture (Friendlander et al., 1989; Dufour et al., 1999). By contrast, S180 cells stably transfected to express E-cadherin (Ecad clone) displayed characteristic intercellular adhesion in culture, with E-cadherin, β-cat and actin all detected concentrated at sites of cell–cell adhesion (Fig. 1, B–D). Ecad cells that had been dissociated by trypsin-calcium (TC) treatment (see Materials and methods) expressed E-cadherin on the cell surface (Fig. 1 E) and readily formed doublets or aggregates in suspension. Cell adhesion sites matured over time, becoming enriched in E-cadherin, β-cat and actin, and increasing in area (Fig. 1, F–I vs. J–M). In doublets of S180 cells transiently transfected with pEcad-GFP, E-cadherin–GFP molecules were concentrated at cell–cell interface (Video 3, frames 1–5, available at http://www.jcb.org/cgi/content/full/jcb.200403043/DC1) but were redistributed uniformly in the membrane after separation of the adherent cells (see next paragraph; Video 3, frames 9–12).


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)

Adhesive properties of Ecad cells. Immunodetection of β-cat (A and D), E-cadherin (B), and actin (C) in S180 cells (A) and Ecad cells (B–D). E, FACS analysis on isolated Ecad cells in suspension, after TC treatment, with an antibody directed against the extracellular domain of E-cadherin. Immunodetection of E-cadherin (F and J), β-cat (G and K), and actin (H and L) in doublets formed in suspension for 4- (F–I) or 30-min (J–M). Merged images are shown in I and M. Bars: (A) 20 μm; (F and J) 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172605&req=5

fig1: Adhesive properties of Ecad cells. Immunodetection of β-cat (A and D), E-cadherin (B), and actin (C) in S180 cells (A) and Ecad cells (B–D). E, FACS analysis on isolated Ecad cells in suspension, after TC treatment, with an antibody directed against the extracellular domain of E-cadherin. Immunodetection of E-cadherin (F and J), β-cat (G and K), and actin (H and L) in doublets formed in suspension for 4- (F–I) or 30-min (J–M). Merged images are shown in I and M. Bars: (A) 20 μm; (F and J) 10 μm.
Mentions: S180 cells contain no detectable β-cat (Fig. 1 A) or cadherins (not depicted) and display minimal cell–cell adhesion in tissue culture (Friendlander et al., 1989; Dufour et al., 1999). By contrast, S180 cells stably transfected to express E-cadherin (Ecad clone) displayed characteristic intercellular adhesion in culture, with E-cadherin, β-cat and actin all detected concentrated at sites of cell–cell adhesion (Fig. 1, B–D). Ecad cells that had been dissociated by trypsin-calcium (TC) treatment (see Materials and methods) expressed E-cadherin on the cell surface (Fig. 1 E) and readily formed doublets or aggregates in suspension. Cell adhesion sites matured over time, becoming enriched in E-cadherin, β-cat and actin, and increasing in area (Fig. 1, F–I vs. J–M). In doublets of S180 cells transiently transfected with pEcad-GFP, E-cadherin–GFP molecules were concentrated at cell–cell interface (Video 3, frames 1–5, available at http://www.jcb.org/cgi/content/full/jcb.200403043/DC1) but were redistributed uniformly in the membrane after separation of the adherent cells (see next paragraph; Video 3, frames 9–12).

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