| The ability of endothelial cells to respond to changes in their microenvironment contributes significantly to vascular homeostasis. Weibel-Palade bodies are endothelial cell specific organelles that contain several components like von Willebrand factor (vWF), P-selectin and interleukine 8 (IL-8). High molecular weight polymers of vWF that originate from Weibel-Palade bodies play an important role in the adhesion of platelets to a damaged vessel wall. Both P-selectin and IL-8 have been implicated in inflammatory responses. Small GTP-binding proteins function as molecular switches by alternating between a GTP-bound and GDP-bound state. Previously, we have shown that thrombin-induced exocytosis of Weibel-Palade bodies is dependent on the activation of Ral, a member of the family of small GTP binding proteins. The mechanism by which Ral regulates exocytosis of Weibel-Palade bodies will be defined in this study. A GTPase activating protein (GAP) for Cdc42 has been implicated as a target for GTP-Ral which suggests a link between exocytosis of Weibel-Palade bodies and cytoskeleton dynamics. Involvement of a subgroup of the Rho-family of small GTPases (Cdc42, Rac1 and RhoA) in exocytosis of Weibel-Palade bodies will be investigated. A fusion of green fluorescent protein and vWF (GFP-vWF) will be used to visualize regulated exocytosis of Weibel-Palade bodies in living endothelial cells. Following exocytosis of Weibel-Palade bodies, P-selectin is rapidly internalized by endothelial cells. Ral has recently been implicated in endocytosis. Therefore, we will also investigate the role of Ral and effectors of Ral in endocytosis of P-selectin. Endocytosis of P-selectin will be monitored using a fusion protein of the red-shifted fluorescent protein RFP and P-selectin. Together our findings may provide new insights into the role of Ral in the dynamics of trafficking of endothelial cell-specific storage compartments. |
