Spatiotemporal regulation of Rho-family GTPases

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In the signal transduction field, researchers have been devoted themselves for the identification signaling molecules and connection between them. In the next step, we need to know how the signals are actually transmitted through these signaling molecules and networks. Probes based on the principle of fluorescence resonance energy transfer are expected to contribute for this purpose. In this research project, we focused on Rho-family GTPases and developed FRET probes for the visualization of the activities of Rho-family GTPases. Furthermore, to improve the sensitivity of the probes, we applied our FRET probes to total internal reflection fluorescence microscopy (TIRF). By using these techniques, we have characterized a Rho-family GTPase TC10. It has been shown that TC10 plays a critical role in the insulin-induced translocation of GLUT4 from cytoplasm to the plasma membrane ; however, its precise role has remained elusive. We developed a FRET probe for TC10 and, with the help of TRIF technique, we found that the activity of TC10 on the exocytic vesicles dropped down rapidly at the time of vesicle fusion to the plasma membrane. Furthermore, by using dominant negative mutants and siRNA techniques, we showed that this decrease in TC10 activity is required for the fusion of exocytic vesicles to the plasma membrane. Now, we have developed a series of FRET probes that cover Ras-family and Rho-family GTPases. These FRET probes will be of great value in the future studies of the signaling network and also of the development of kinetic simulation model that operates in silico.