Molecular tools to study the role of Rho GTPases in the endothelium

The research presented in this thesis aimed to develop molecular tools to study the role of Rho GTPases in the endothelium. The endothelium is the inner layer of the blood vessels walls and it acts as a barrier between blood and tissue but also as a guide for the immune system. Here we focused on the Rho GTPase signaling that controls actin polymerization and membrane structures, which are both involved in transmigration of immune cells and barrier integrity. We observed membrane and actin structures in endothelial cells and then visualized and manipulated Rho GTPase activity. The visualization was achieved by optimization and characterization of location-based biosensors for endogenous Rho, Rac and Cdc42 activity. The manipulation was done with optogenetics, namely the iLID based Opto-RhoGEFs, which were optimized and applied to control Rho GTPase activity in endothelial cells and showed a great potential to study Rho GTPase activity locally and globally, and in a reversible manner. Many constructs from these studies have been made available on Addgene and have been requested by the community. Therefore, we are hopeful that the new tools will not only contribute increasing the understanding of Rho GTPase signaling in the endothelium but also in other fields.