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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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Characterization of Ecad cell adhesion. (A) SF measurements for Ecad cells held in contact for 0.5–60 min. (B) SF required to separate 60-min doublets (white bar) and preexisting doublets (black bars), selected as described in Materials and methods. (C) Dose-response curve of force measurements for 4-min doublets in various concentrations of calcium. (D) The effect of a control or anti–E-cadherin antibody on SF in Ecad or S180 cells. (E) FACS analysis of E-cadherin expression on the surface of Ecad cells treated with 10 μM BFA (black peaks) for 4 and 12 h or untreated (white peaks). (F) The mean SFs measured for 4- or 30-min Ecad doublets treated with 10 μM BFA (black bars) for 1 h or untreated doublets (white bars).
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fig3: Characterization of Ecad cell adhesion. (A) SF measurements for Ecad cells held in contact for 0.5–60 min. (B) SF required to separate 60-min doublets (white bar) and preexisting doublets (black bars), selected as described in Materials and methods. (C) Dose-response curve of force measurements for 4-min doublets in various concentrations of calcium. (D) The effect of a control or anti–E-cadherin antibody on SF in Ecad or S180 cells. (E) FACS analysis of E-cadherin expression on the surface of Ecad cells treated with 10 μM BFA (black peaks) for 4 and 12 h or untreated (white peaks). (F) The mean SFs measured for 4- or 30-min Ecad doublets treated with 10 μM BFA (black bars) for 1 h or untreated doublets (white bars).

Mentions: We measured SF for pairs of Ecad cells after different times of contact (Fig. 3 A). Adhesion was initiated rapidly, with cells adhering to each other after only a few seconds of contact (not depicted), but measurements for contact periods of <30 s were not reproducible. At 30 s of contact, a mean force of 20 nN was required to separate adherent cells. From 30 s to 30 min of contact (30-s doublets and 30-min doublets, respectively), the force required to separate the cells increased rapidly. It stabilized at ∼200 nN after 1 h of contact (60-min doublets). Anti–E-cadherin significantly reduced the SF of 4-min doublets (Fig. 3 D), and S180 cells lacking cadherins displayed no detectable adhesion after 4 min (Fig. 3 D) or 30 min of contact (not depicted), both results clearly indicating that the doublet formation was E-cadherin dependent.


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)

Characterization of Ecad cell adhesion. (A) SF measurements for Ecad cells held in contact for 0.5–60 min. (B) SF required to separate 60-min doublets (white bar) and preexisting doublets (black bars), selected as described in Materials and methods. (C) Dose-response curve of force measurements for 4-min doublets in various concentrations of calcium. (D) The effect of a control or anti–E-cadherin antibody on SF in Ecad or S180 cells. (E) FACS analysis of E-cadherin expression on the surface of Ecad cells treated with 10 μM BFA (black peaks) for 4 and 12 h or untreated (white peaks). (F) The mean SFs measured for 4- or 30-min Ecad doublets treated with 10 μM BFA (black bars) for 1 h or untreated doublets (white bars).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172605&req=5

fig3: Characterization of Ecad cell adhesion. (A) SF measurements for Ecad cells held in contact for 0.5–60 min. (B) SF required to separate 60-min doublets (white bar) and preexisting doublets (black bars), selected as described in Materials and methods. (C) Dose-response curve of force measurements for 4-min doublets in various concentrations of calcium. (D) The effect of a control or anti–E-cadherin antibody on SF in Ecad or S180 cells. (E) FACS analysis of E-cadherin expression on the surface of Ecad cells treated with 10 μM BFA (black peaks) for 4 and 12 h or untreated (white peaks). (F) The mean SFs measured for 4- or 30-min Ecad doublets treated with 10 μM BFA (black bars) for 1 h or untreated doublets (white bars).
Mentions: We measured SF for pairs of Ecad cells after different times of contact (Fig. 3 A). Adhesion was initiated rapidly, with cells adhering to each other after only a few seconds of contact (not depicted), but measurements for contact periods of <30 s were not reproducible. At 30 s of contact, a mean force of 20 nN was required to separate adherent cells. From 30 s to 30 min of contact (30-s doublets and 30-min doublets, respectively), the force required to separate the cells increased rapidly. It stabilized at ∼200 nN after 1 h of contact (60-min doublets). Anti–E-cadherin significantly reduced the SF of 4-min doublets (Fig. 3 D), and S180 cells lacking cadherins displayed no detectable adhesion after 4 min (Fig. 3 D) or 30 min of contact (not depicted), both results clearly indicating that the doublet formation was E-cadherin dependent.

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