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Spatiotemporal regulation of Rho-family GTPases

Rho 家族 GTP 酶的时空调控

基本信息

批准号：
16390078
负责人：
MATSUDA Michiyuki
金额：
$ 9.73万
依托单位：
Kyoto University (2006)Osaka University (2004-2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390078/
关键词：
Rho Rac Cdc42 GFP FRET TC10 全反射蛍光顕微鏡小胞輸送 FRETプローブ上皮細胞増殖因子

项目摘要

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.

在信号转导领域，研究人员一直致力于识别信号分子以及它们之间的联系。下一步，我们需要知道信号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做出贡献。本研究以Rho家族GTP酶为研究对象，开发了荧光共振能量转移探针，用于Rho家族GTP酶活性的可视化研究。此外，为了提高探针的灵敏度，我们将我们的FRET探针应用于全内反射荧光显微镜（TIRF）。通过使用这些技术，我们已经表征了Rho家族的GTdR TC 10。已经表明，TC 10在胰岛素诱导的GLUT 4从细胞质到质膜的易位中起关键作用;然而，其确切作用仍然难以捉摸。我们开发了TC 10的FRET探针，并在TRIF技术的帮助下，我们发现TC 10在胞吐囊泡上的活性在囊泡融合到质膜时迅速下降。此外，通过使用显性失活突变体和siRNA技术，我们发现TC 10活性的降低是胞外囊泡与质膜融合所必需的。目前，我们已经开发了一系列涵盖Ras家族和Rho家族GTP酶的FRET探针。这些FRET探针将是非常有价值的，在未来的信号网络的研究，也是在硅片上运行的动力学模拟模型的发展。