Linking Rap to Rho: Rap GTPases control ArhGAP29 in endothelial barrier function

Small GTPases of the Rap family are involved in a plethora of actin cytoskeleton-linked biological processes. The small GTPase Rap1 does so by regulating the Rho small GTPase family, the master regulators of actin cytoskeletal dynamics (discussed in chapter 1). When and where small GTPases become activated has profound implications for the biological outcome. Accordingly, spatio-temporal control of GTPase activity is an important component of small GTPase biology. In this thesis we have investigated how active Rap1 subsequently regulates the actin cytoskeleton, by modulating Rho. Most importantly, we have identified the RhoGAP ArhGAP29 as a downstream effector of Rap1 signaling. Through ArhGAP29 Rap1 regulates Rho signaling and thus actin cytoskeletal dynamics. Chapter 2 describes the development of an siRNA screening tool, to identify downstream signaling cascades of Rap1. For this we analyzed the induction of cells spreading upon Epac1-Rap1 activation. This resulted in the identification of the Rap1-effector, Radil, as a mediator of Rap1-induced cell spreading. Furthermore, the plasma membrane-actin linker, Ezrin, was identified as a crucial component of Rap1-induced cell spreading, but not cell adhesion. The interconnectivity between these two proteins has been investigated and described in chapter 3, identifying ERM proteins as a potential scaffold for Rap1-signaling, integrating upstream signaling by binding Epac1, and downstream signaling by binding Radil. In chapter 4, we have identified a novel Rap1-effector, Rasip1, which is a homologue of Radil. We confirmed that it functions downstream of Rap1, and modulates the actin cytoskeletal driven processes of cell spreading and endothelial barrier control. Moreover, we identified that in concert with Radil, Rasip1 modulates the actin cytoskeleton through the RhoGAP ArhGAP29, thus impinging on Rho signaling. In chapter 5, we have elucidated how this signaling cascade functions mechanistically. Here we reveal that Rap1 regulates Radil, Rasip1 and ArhGAP29 by inducing their dynamic translocation to the plasma membrane, where they form a multimeric complex. Elucidating the pathway through which Rap1 induces endothelial barrier control through the modulation of Rho, together with other reports, has led us to develop a dual tension model in the control of endothelial barrier function, described in chapter 6. Finally, in chapter 7 we identify a Rap1-Radil-Rasip1-independent function of ArhGAP29. Rather than Rap1, this module incorporates Rap2 signaling, suggesting that besides a Rap1-Rho axis, there also is a Rap2-Rho axis. Integration of this axis is achieved through the tyrosine phosphatase PTPL1. Of note, chapters 3, 5 and 7 all identify potential spatial cues for Rap-Rho signaling axes in various cytoskeletal driven processes