Wang Yingxiao, Chien Shu
Department of Bioengineering and Molecular & Integrative Physiology, Neuroscience Program, Center for Biophysics and Computational Biology, Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, USA.
Methods Enzymol. 2007;426:177-201. doi: 10.1016/S0076-6879(07)26009-4.
Fluorescence resonance energy transfer (FRET) has been proven to be a powerful tool to visualize and quantify the signaling cascades in live cells with high spatiotemporal resolutions. Here we describe the development of the genetically encoded and FRET-based biosensors for imaging of integrin-related signaling cascades. The construction of a FRET biosensor for Src kinase, an important tyrosine kinase involved in integrin-related signaling pathways, is used as an example to illustrate the construction procedure and the pitfalls involved. The design strategies and considerations on improvements of sensitivity and specificity are also discussed. The FRET-based biosensors provide a complementary approach to traditional biochemical assays for the analysis of the functions of integrins and their associated signaling molecules. The dynamic and subcellular visualization enabled by FRET can shed new light on the molecular mechanisms regulating integrin signaling and advance our knowledge in the understanding of integrin-related pathophysiological processes.
荧光共振能量转移（FRET）已被证明是一种强大的工具，可在高时空分辨率下可视化和量化活细胞中的信号级联反应。在此，我们描述了用于整合素相关信号级联成像的基于基因编码和FRET的生物传感器的开发。以构建参与整合素相关信号通路的重要酪氨酸激酶Src激酶的FRET生物传感器为例，说明构建过程及其中涉及的陷阱。还讨论了提高灵敏度和特异性的设计策略及注意事项。基于FRET的生物传感器为传统生化分析提供了一种补充方法，用于分析整合素及其相关信号分子的功能。FRET实现的动态和亚细胞可视化可为调节整合素信号的分子机制提供新的线索，并推动我们对整合素相关病理生理过程的理解。
