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

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.

Rapid Molecular and Morphological Responses of Prostate Cell Lines to Cell–Cell Contact

“…The overall cytoskeleton and cell shape remodeling results presented here for both cell lines are consistent with the recent report of Chu et al (2004). These authors showed that (i) after 30 min, F-actin had strongly accumulated at the linear interface of doublets of sarcoma 180 cells formed in suspension and (ii) that the force required to separate cells in a doublet formed by bringing cells, each supported by a micropipette, into contact for a period of 60 min increased rapidly over the first 30 min and more slowly thereafter.…”

“…This force was primarily determined by the amount of E-cadherin expressed at the cell surface. A calculation, by the present authors, of the mean value (n = 3) of θ of the cell-cell 'chord' of E-cadherin-expressing cells in the work of Chu et al (2004) was on average 91 • (n = 3), in agreement with the value reported above in Figure 2ii for the highly E-cadherin expressing PZ-HPV-7 cells.…”

“…The rapid molecular responses of cells to contact have recently been examined using confluent monolayers of keratinocytes growing on a substratum (Zhang et al 2005), S180 sarcoma cell doublets held together by micropipettes (Chu et al 2004) and neural cell (Bazou et al 2005a) and chondrocyte (Bazou et al 2006) monolayers levitated in suspension in an ultrasound trap. In the latter two cases, cell-cell adhesion was examined on bringing the cells together synchronously, rapidly (within 30 s) and non-intrusively (Bazou et al 2005b), free of the influences of solid substrata.…”

Endothelial basement membrane laminin 511 is essential for shear stress response

“…To determine whether cell–cell adhesion strengths were affected by endothelial cell adhesion to different laminins, a dual pipette assay was performed (Chu et al , 2004; Martinez‐Rico et al , 2010). Polystyrene macrobeads (15 μm, Polysciences, Inc.) were coated overnight at 4°C with 20 μM purified laminin 511, laminin 411 or laminin 111, added to a cell suspension of HUAECs in HEPES‐buffered cell culture medium at a ratio of 1:2 and incubated at 37°C, 5% CO 2 for 1 h to permit cell adhesion to the coated beads.…”

La mécanique de l’adhésion cellulaire dans la gastrulation du poisson zèbre

“…Pour comprendre le rôle de la résistance mécanique des molé-cules d'adhésion dans la formation du contact, nous avons étudié leur capacité à résister aux forces de séparation [7]. Pour cela, nous avons utilisé des micropipettes pour séparer des doublets de Quel rôle mécanique pour les molécules d'adhésion ?…”