Spatiotemporal regulation of Rho-family GTPases

在信号转导领域，研究人员一直致力于信号分子的识别和它们之间的联系。下一步，我们需要知道信号实际上是如何通过这些信号分子和网络传输的。基于荧光共振能量转移原理的探针有望为此作出贡献。在本研究项目中，我们专注于rho家族GTPases，并开发了FRET探针来可视化rho家族GTPases的活性。此外，为了提高探针的灵敏度，我们将FRET探针应用于全内反射荧光显微镜（TIRF）。通过使用这些技术，我们表征了rho家族GTPase TC10。研究表明，TC10在胰岛素诱导的GLUT4从细胞质到质膜的易位中起关键作用；然而，它的确切作用仍然难以捉摸。我们开发了一种针对TC10的FRET探针，利用TRIF技术，我们发现TC10在囊泡融合到质膜时对囊泡的活性迅速下降。此外，通过使用显性负突变体和siRNA技术，我们发现TC10活性的降低是胞囊与质膜融合所必需的。现在，我们已经开发了一系列FRET探针，涵盖ras家族和rho家族的gtp酶。这些FRET探针将在未来的信号网络研究和在计算机上运行的动力学模拟模型的发展中具有重要的价值。

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.