Actin microdomains on endothelial cells: association with CD44, ERM proteins, and signaling molecules during quiescence and wound healing

Abstract

During studies of the actin cytoskeleton in cultured endothelial cells we have observed that the luminal side of many cells contains F-actin microdomains that are rich in the hyaluronan receptor CD44 and in ezrin-radixin-moesin (ERM) proteins. A small subpopulation of the domains are also enriched in tyrosine phosphorylated proteins and signaling molecules. Confocal microscopy of rat aortic endothelial cells in situ demonstrated that similar microdomains occur in vivo. During healing of endothelial wounds, characteristic alterations of the actin cytoskeleton occurred. Thus, in many cells close to the wound, focal F-actin branching points appeared. The branching points were similar to the microdomains in that they colocalized with CD44 and ERM proteins, but, in addition, they formed centers for actin filament branching and were associated with phosphorylated protein kinase C alpha/betaII. These colocalization data are consonant with the view that activated PKC is responsible for activating ERM-mediated crosslinking between CD44 and the actin cytoskeleton. Importantly, inhibition of PKC activity decreased staining for phosphorylated ERM proteins, decreased the frequency of F-actin branching points, and inhibited monolayer wound healing. Together, our data show that endothelial cells contain a novel actin cytoskeletal structure, the F-actin microdomain, and suggest that during wound healing such structures become associated with activated signaling molecules and thereby enhance actin cytoskeletal remodeling.