Abstract
Cell-cell adhesion in simple epithelia involves the engagement of E-cadherin and nectins, and the reorganization of the actin cytoskeleton and membrane dynamics by Rho GTPases, particularly Rac1. However, it remains unclear whether E-cadherin and nectins up-regulate, maintain or suppress Rac1 activity during cell-cell adhesion. Roles for Rho GTPases are complicated by cell spreading and integrin-based adhesions to the extracellular matrix that occur concurrently with cell-cell adhesion, and which also require Rho GTPases. Here, we designed a simple approach to examine Rac1 activity upon cell-cell adhesion by MDCK epithelial cells, without cell spreading or integrin-based adhesion. Upon initiation of cell-cell contact in 3-D cell aggregates, we observed an initial peak of Rac1 activity that rapidly decreased by ∼66% within 5 minutes, and further decreased to a low baseline level after 30 minutes. Inhibition of E-cadherin engagement with DECMA-1 Fab fragments or competitive binding of soluble E-cadherin, or nectin2alpha extracellular domain completely inhibited Rac1 activity. These results indicate that cadherins and nectins cooperate to induce and then rapidly suppress Rac1 activity during initial cell-cell adhesion, which may be important in inhibiting the migratory cell phenotype and allowing the establishment of initially weak cell-cell adhesions.
Highlights
Cell-cell adhesion is essential for the development and maintenance of tissue structure and function, and is mediated by different classes of junctional membrane proteins, principally cadherins and nectins [1,2]
When Madin-darby canine kidney (MDCK) cells were allowed to adhere to collagen I-coated tissue culture plates for 3 hours and induced to form cell-cell contacts, there was a gradual increase in Rac1 activity from 0 to 15 minutes followed by a decrease to baseline levels at 30 minutes (Figure 1, D and E)
To determine whether the changes in Rac1 activity corresponded to a change in the localization of E-cadherin upon calcium switch, cells plated on collagen-coated coverslips were fixed and analyzed by immunofluorescence microscopy
Summary
Cell-cell adhesion is essential for the development and maintenance of tissue structure and function, and is mediated by different classes of junctional membrane proteins, principally cadherins and nectins [1,2]. Cadherins are the major class of calcium-dependent transmembrane proteins involved in initial cell-cell adhesion. Nectins are calciumindependent immunoglobulin-like cell-cell adhesion molecules that form homo-cis dimers and hetero-trans dimers via their extracellular domains and associate with the cytoskeleton through the F-actin binding protein, afadin [6,7]. Nectins participate in cell-cell adhesion by interacting with E-cadherin via their cytoplasmic domain-associated proteins [8], and by regulating Ecadherin trans-interactions at cell-cell contacts [9,10,11]. The Rho GTPases, RhoA, Rac and Cdc, are a class of proteins that are key regulators of the actin cytoskeleton in coordinating cell migration and cell-cell adhesion [15,16]. Analysis of Rho GTPases activity in traditional adhesion assays in 2-D has given rise to apparently contradictory conclusions
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