Leukocyte extravasation (diapedesis) is a critical inflammatory event in which leukocytes exist from the circulation by migrating between endothelial cells. A goal of the project is to understand the cascade of adhesive, motile and signaling events in this complex, but poorly understood process. Our hypothesis is that monocyte adhesion to endothelial cells triggers sequential activation of Cdc42, Rac and Rho, and that these activated G proteins regulate successive steps in leukocyte extravasation. We will measure the activity of Rho, Rac and Cdc42 during monocyte adhesion and extravasation. We will perturb the functions of these proteins by introducing dominant negative and constitutively active forms of the proteins. A extravasating cell must dissociated endothelial adherens junctions made by VE-cadherin. We will investigate signaling cascades that affect the strength of VE-cadherin homophilic interactions. Beads coated with the VE-cadherin ectodomain will be adhered to endothelial cells in culture and the strength of adhesion measured using laser tweezers under a variety of conditions likely to affect adherens junction integrity. Because tyrosine phosphorylation has been implicated in regulating adherens junctions, we will examine the tyrosine phosphorylation of catenins during monocyte passage through endothelial monolayers. Using in vitro assays, the effect of phosphorylation on binding of catenins to cadherin cytoplasmic domains will be studied. During inflammation, cytokines stimulate migration of fibroblasts and epithelial cells. For migration to proceed, adhesions to extracellular matrix, such as focal adhesions, must be released. We will determined whether IL-8 induced focal adhesion disassembly occurs by inhibition of cell contractility or through phosphorylation of focal adhesion components. Upon completion of the inflammatory response, migration is inhibited. We will examine whether the clustering of cadherins inhibits cell migration, using cadherin constructs that can be aggregated by addition of chemical dimerizers. The juxtamembrane region of cadherins has been implicated in regulating migration. This region binds to p120cas, leading to our hypothesis that p120cas can bind either to cadherins or to a guanine nucleotide exchange factor that regulates Rac/Cdc42 activity, thereby controlling motility. We will look for guanine nucleotide exchange activity complexed with p120cas and determine whether this activity is lost when p120cas binds to aggregated cadherins.
Showing the most recent 10 out of 70 publications
