The connexin 43/ZO-1 complex regulates cerebral endothelial F-actin architecture and migration

Endothelial cell migration is a fundamental process during angiogenesis and, therefore, a point of intervention for therapeutic strategies aimed at controlling pathologies involving blood vessel growth. We sought to determine the role of the gap junction protein connexin 43 (Cx43) in key features of angiogenesis in the central nervous system. We used an in vitro model to test the hypothesis that a complex of interacting proteins, including Cx43 and zonula occludens-1 (ZO-1), regulates the migratory behavior of cerebral endothelium. With knockdown and overexpression experiments, we demonstrate that the rate of healing following scrape-wounding of endothelium is regulated by the level of Cx43 protein expression. The effects on cell motility and proliferation were independent of gap junction communication as cells were sensitive to altered Cx43 expression in single plated cells. Coupling of Cx43/ZO-1 critically regulates this process as demonstrated with the use of a Cx43 alpha-carboxy terminus 1 peptide mimetic (alpha CT1) and overexpression of a mutant ZO-1 with the Cx43-binding PDZ2 domain deleted. Disrupting the Cx43/ZO-1 complex with these treatments resulted in collapse of the organized F-actin cytoskeleton and the appearance of actin nodes. Preincubation with the myosin 2 inhibitors blebbistatin or Y-27632 disrupted the Cx43/ZO-1 complex and inhibited cell spreading at the leading edge of migration. Cells studied individually in time-lapse open field locomotion assays wandered less when Cx43/ZO-1 interaction was disrupted without significant change in speed, suggesting that faster wound healing is a product of linearized migration. In contrast to the breakdown of F-actin architecture, microtubule architecture was not obviously affected by treatments. This study provides new insight into the fundamental regulatory mechanisms of cerebral endothelial cell locomotion. Cx43 tethers the F-actin cytoskeleton through a ZO-1 linker and supports cell spreading and exploration during locomotion. Here, we demonstrate that releasing this actin-coupled tether shifts the balance of directional migration control to a more linear movement that enhances the rate of wound healing.