VEGF-mediated actin dynamics in endothelial cells
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The angiogenic growth factor Vascular Endothelial Growth Factor (VEGF)-mediated endothelial cell migration is an essential process in tumor angiogenesis. Actin reorganization is necessary for cell migration. My research focuses on the study of signaling pathways involved in VEGF-mediated endothelial cell migration.;Actin reorganization involves both actin polymerization and depolymerization, and in the first part of my thesis study we demonstrate that VEGF-treatment regulates both of these activities. In regards to actin polymerization, our results indicate that the SH2/SH3 domain-containing adaptor Nck complexes with the actin nucleation factor neuronal Wiskott-Aldrich syndrome protein (N-WASP) and mediates VEGF-induced actin polymerization in endothelial cells. In regards to actin depolymerization, VEGF-treatment led to the rapid phosphorylation of the actin depolymerization factor (ADF) cofilin, and its upstream activator, LIM-Kinase (LIMK). The PAK dominant negative did not effect VEGF-induced actin reorganization. Instead, we found that phosphoinositide-3 kinase (PI3-K) and the Rho-activated kinase (ROCK) are involved the LIMK phosphorylation regulation.;Cofilin is a key regulator of actin dynamics. P-cofilin has recently been shown to be a substrate for the slingshot (SSH) family of phosphatases. To complete the study, the second part of my thesis focuses on the functional and structural study of SSH in endothelial cell migration. Expression of the SSH1L in porcine aortic endothelial cells blocked cofilin phosphorylation, and led to significantly fewer stress fibers than control cells. The expression of the conserved amino-terminus of SSH1L (N-SSH) resulted in a significant increase in the number of F-actin stress fibers, mimicking the expression of either a catalytically inactive SSH1L, or LIM-K on F-actin organization. SSH1L localizes to the periphery of cells, but N-SSH expression blocks this. We conclude that N-SSH's dominant negative effect is due to its preventing normal SSH1L cellular localization. Compared to controls, cells overexpressing SSH1L contained an abnormal number of large flat lamellipodia-like protrusions. We draw two major conclusions from these results. First, SSH1L participates in endothelial cell migration by modulating the dynamics of cell protrusions. Second, the amino terminal portion of SSH1L plays an important role in facilitating the protein's cellular localization.