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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

Activation of small GTPases during Ecad cell aggregation. Representative Western blot analysis of GTP-bound (active) and total Rac, Cdc42, and Rho on S180 and Ecad cells taken at different times of the aggregation assay in suspension (A). (B) Fold activation of the Rac (white bars), Cdc42 (gray bars) and Rho (black bars) GTPases; the activation level at time 0 serves as the reference level. Activation fold represents the mean ± SEM from three independent experiments.
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fig7: Activation of small GTPases during Ecad cell aggregation. Representative Western blot analysis of GTP-bound (active) and total Rac, Cdc42, and Rho on S180 and Ecad cells taken at different times of the aggregation assay in suspension (A). (B) Fold activation of the Rac (white bars), Cdc42 (gray bars) and Rho (black bars) GTPases; the activation level at time 0 serves as the reference level. Activation fold represents the mean ± SEM from three independent experiments.

Mentions: We used GTPase pull down assays to test the effect of E-cadherin–mediated intercellular adhesion on endogenous activity of Rho-like GTPases in Ecad cells in suspension. The levels of endogenous active and total Rho-like GTPases were monitored in S180 cells and Ecad cells at different times during a 60-min aggregation assay (Fig. 7A). In S180 cells no change was observed in the activation levels of Rac, Cdc42, and Rho during the assay. In clear contrast with this result, activation of Rac was observed in Ecad cells as soon as 5 min after the start of aggregation and reached a maximum by the end of the assay (Fig. 7 B). The kinetics of activation for Cdc42 were comparable to those described for Rac, but activation of Rho followed a very different pattern (gray, white, and black bars, respectively; Fig. 7 B). The levels of activated Rho in Ecad cells did not significantly change throughout the aggregation assay (Fig. 7 B). Results from total lysates indicated that the differences observed in band densities after precipitation were not due to variations in the total amount of protein. Each GST pull-down assay was repeated three times.


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)

Activation of small GTPases during Ecad cell aggregation. Representative Western blot analysis of GTP-bound (active) and total Rac, Cdc42, and Rho on S180 and Ecad cells taken at different times of the aggregation assay in suspension (A). (B) Fold activation of the Rac (white bars), Cdc42 (gray bars) and Rho (black bars) GTPases; the activation level at time 0 serves as the reference level. Activation fold represents the mean ± SEM from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Activation of small GTPases during Ecad cell aggregation. Representative Western blot analysis of GTP-bound (active) and total Rac, Cdc42, and Rho on S180 and Ecad cells taken at different times of the aggregation assay in suspension (A). (B) Fold activation of the Rac (white bars), Cdc42 (gray bars) and Rho (black bars) GTPases; the activation level at time 0 serves as the reference level. Activation fold represents the mean ± SEM from three independent experiments.
Mentions: We used GTPase pull down assays to test the effect of E-cadherin–mediated intercellular adhesion on endogenous activity of Rho-like GTPases in Ecad cells in suspension. The levels of endogenous active and total Rho-like GTPases were monitored in S180 cells and Ecad cells at different times during a 60-min aggregation assay (Fig. 7A). In S180 cells no change was observed in the activation levels of Rac, Cdc42, and Rho during the assay. In clear contrast with this result, activation of Rac was observed in Ecad cells as soon as 5 min after the start of aggregation and reached a maximum by the end of the assay (Fig. 7 B). The kinetics of activation for Cdc42 were comparable to those described for Rac, but activation of Rho followed a very different pattern (gray, white, and black bars, respectively; Fig. 7 B). The levels of activated Rho in Ecad cells did not significantly change throughout the aggregation assay (Fig. 7 B). Results from total lysates indicated that the differences observed in band densities after precipitation were not due to variations in the total amount of protein. Each GST pull-down assay was repeated three times.

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