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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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Related in: MedlinePlus

Dual micropipette assay. (A) Two cells in suspension (1 and 2) are held under weak aspiration by micropipettes, and placed in contact (B; Video 1). The formation of contact is checked (C) after displacement of the right pipette. (D) Second cell is held by the micropipette under strong aspiration. (E–I) First cell is held by the micropipette and the aspiration applied is increased as the right micropipette displaced, step by step, until the adherent cells are separated (I; Video 2).
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fig2: Dual micropipette assay. (A) Two cells in suspension (1 and 2) are held under weak aspiration by micropipettes, and placed in contact (B; Video 1). The formation of contact is checked (C) after displacement of the right pipette. (D) Second cell is held by the micropipette under strong aspiration. (E–I) First cell is held by the micropipette and the aspiration applied is increased as the right micropipette displaced, step by step, until the adherent cells are separated (I; Video 2).

Mentions: The micromanipulation assay was used to quantify the force required to separate pairs of adherent cells. Cadherin expressing cells held by gentle aspiration at the tips of two micropipettes (Fig. 2 A) were first brought gently into contact and held for a predetermined time (Fig. 2, B and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200403043/DC1). Fig. 2 D illustrates an example of a doublet of Ecad cells obtained after 4 min of contact (a 4-min doublet), the right pipette withdrawn to visualize the resulting adhesion (Fig. 2 C). Such a doublet was cyclically brought back into contact with the left pipette and then withdrawn to the right, each time after a step-wise increase in the strength of aspiration by the left pipette, until the cells were separated (see Materials and methods; Fig. 2, D–I; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200403043/DC1). The SF was defined as the aspiration force required to separate the doublet, such that one cell remained in each pipette when the right pipette was withdrawn (Fig. 2 I). SF was considered to be zero for pairs of cells that did not form adherent doublets in this assay.


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)

Dual micropipette assay. (A) Two cells in suspension (1 and 2) are held under weak aspiration by micropipettes, and placed in contact (B; Video 1). The formation of contact is checked (C) after displacement of the right pipette. (D) Second cell is held by the micropipette under strong aspiration. (E–I) First cell is held by the micropipette and the aspiration applied is increased as the right micropipette displaced, step by step, until the adherent cells are separated (I; Video 2).
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Dual micropipette assay. (A) Two cells in suspension (1 and 2) are held under weak aspiration by micropipettes, and placed in contact (B; Video 1). The formation of contact is checked (C) after displacement of the right pipette. (D) Second cell is held by the micropipette under strong aspiration. (E–I) First cell is held by the micropipette and the aspiration applied is increased as the right micropipette displaced, step by step, until the adherent cells are separated (I; Video 2).
Mentions: The micromanipulation assay was used to quantify the force required to separate pairs of adherent cells. Cadherin expressing cells held by gentle aspiration at the tips of two micropipettes (Fig. 2 A) were first brought gently into contact and held for a predetermined time (Fig. 2, B and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200403043/DC1). Fig. 2 D illustrates an example of a doublet of Ecad cells obtained after 4 min of contact (a 4-min doublet), the right pipette withdrawn to visualize the resulting adhesion (Fig. 2 C). Such a doublet was cyclically brought back into contact with the left pipette and then withdrawn to the right, each time after a step-wise increase in the strength of aspiration by the left pipette, until the cells were separated (see Materials and methods; Fig. 2, D–I; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200403043/DC1). The SF was defined as the aspiration force required to separate the doublet, such that one cell remained in each pipette when the right pipette was withdrawn (Fig. 2 I). SF was considered to be zero for pairs of cells that did not form adherent doublets in this assay.

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