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1 Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA; General Surgery, University of Muenster, Muenster, NRW, Germany
2 Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA
3 Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA; Pritzker School of Medicine, University of Chicago, Chicago, Il, USA
* To whom correspondence should be addressed. E-mail: bruwer{at}uni-muenster.de.
Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJ) and adherens junctions (AJ) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1 and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. Using a tetracycline-repressible system to achieve regulated expression in MDCK epithelial cells, we used dominant-negative (DN) and constitutively-active (CA) forms of RhoA, Rac1 and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2 and JAM-1 were dramatically redistributed in the presence of CA-RhoA or CA-Cdc42, while only claudin-1 and -2 were redistributed in response to CA-Rac1. DN-Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, while DN-Cdc42 influenced only claudin-2 and DN-RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN-Rac1 induced a reduction in E-cadherin detergent solubility. All CA-GTPases increased the detergent solubility of claudins-1 and -2, but CA-RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms, and that perturbations in barrier function reflect any imbalance in active : resting GTPase levels rather than simply loss or gain of GTPase activity.
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