Leukocyte migration across the endothelium is a critical event in inflammation. The goals of this project are[unreadable] to understand the signals initiated by leukocyte adhesion to endothelial cells that promote passage of[unreadable] leukocytes across the endothelium. We will focus on two aspects of this process: the generation of[unreadable] leukocyte-induced cup-like structures that form on the surfaces of endothelial cells, and on leukocyte[unreadable] passage through endothelial cell-cell junctions. In the first aim, we will test the hypothesis that leukocyte[unreadable] adhesion to endothelial cells activates specific Rho GTPases and that these contribute both to formation of[unreadable] cups and to the disassembly of endothelial cell-cell junctions. We will explore the pathways by which[unreadable] leukocyte adhesion regulates these GTPases, using techniques to identify relevant guanine nucleotide[unreadable] exchange factors (GEFs) and GTPase activating proteins (GAPs). Particular attention will be paid to SGEF,[unreadable] which co-localizes with ICAM-1 in cups. SGEF activates RhoG, a Rho protein which induces dorsal[unreadable] membrane ruffles. We will also investigate the pathways downstream from RhoA and Rac1 that promote[unreadable] junctional disassembly. In the second aim, the hypothesis that endothelial junctions are regulated by Rap1[unreadable] activity in response to leukocyte adhesion will be examined. We will use mouse models of inflammation to[unreadable] investigate the roles of Rap isoforms in endothelial cells in mice that are null for Rapla or Raplb. In[unreadable] preliminary work, we have shown that leukocyte adhesion stimulates the tyrosine phosphorylation of[unreadable] endothelial junctional components. In the third aim, we will investigate the pathway by which this occurs,[unreadable] whether it is in response to the activation of Rac1 and generation of reactive oxygen species. We will look[unreadable] for the tyrosine kinases and phosphatases involved and determine whether the tyrosine phosphorylation of[unreadable] VE-cadherin leads to its removal from junctions by endocytosis. Several receptor tyrosine phosphatases[unreadable] reside in endothelial junctions. We will test the hypothesis that these may interact with and be inhibited by[unreadable] extravasating leukocytes so as to elevate levels of phosphotyrosine in junctions. Because leukocyte[unreadable] migration across the endothelial barrier lining blood vessels is a critical step in inflammation, the elucidation[unreadable] of signaling pathways that regulate this process may reveal novel targets for the development of therapies to[unreadable] control inflammation and inflammatory diseases.[unreadable]