RhoA is required for monocyte tail retraction during transendothelial migration

Transendothelial migration of monocytes is the process by which monocytes l eave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration re quires coordination of alterations in cell shape and adhesive properties th at are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the e ndothelium nor subsequent spreading of the monocyte on the endothelial surf ace. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolaye r and were able to invade between neighboring endothelial cells. However, R hoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase e ffector of RhoA, is both necessary and sufficient for RhoA-mediated tail re traction. Finally, we find that p160ROCK signaling negatively regulates int egrin adhesions and that inhibition of RhoA results in an accumulation of b eta2 integrin in the unretracted tails.