FRET imaging of protein-protein interactions inside living cells

细胞生物光子学部分的主要目的是利用成像技术，如双光子显微镜、光谱成像、荧光寿命显微镜和荧光各向异性分析来研究蛋白质复合物如何调节活细胞中的突触功能。最近，我们集中精力利用福斯特共振能量转移（FRET）来监测蛋白质-蛋白质相互作用。这种方法在研究蛋白质相互作用方面具有很大的潜力，因为它对分离两个荧光团的距离在1-10纳米尺度上的变化很敏感。FRET成像与绿色荧光蛋白（GFP）光谱变异的发展相结合，提供了对感兴趣的突触蛋白进行遗传标记的机会，并实时监测它们与其他标记蛋白的相互作用。

The principal aim of the Section on Cellular Biophotonics is to use imaging techniques, such as two-photon microscopy, spectral imaging, fluorescence lifetime microscopy, and fluorescence anisotropy analysis to study how protein complexes regulate synaptic function in living cells. Recently, we have concentrated our efforts on utilizing Forster Resonance Energy Transfer (FRET) to monitor protein-protein interactions. This method has great potential for studying protein interactions because it is sensitive to changes in the distance separating two fluorophores on the 1-10 nm scale. FRET imaging in conjunction with the development of spectral variants of Green Fluorescent Protein (GFP) provides the opportunity to genetically tag synaptic proteins of interest and monitor their interactions with other labeled proteins in real time. Currently we have 2 projects in the lab. The first project is involved in building a two-photon microscope specifically designed to study protein complexes in living cells. The microscope we are assembling will be capable of simultaneously measuring time resolved fluorescence anisotropy, and the fluctuations in fluorescence intensity that can then be analyzed by fluorescence correlation spectroscopy (FCS). Our second project uses anisotropy lifetime decay analysis and FCS analysis to monitor changes in the multimeric structure of Cam kinase-II. In the brain this abundant synaptic enzyme is thought to be a calcium spike frequency detector, and has been shown to play a pivotal role in learning and memory. In the heart CaMKII activity has been linked to several forms of heart disease. Our results indicate that structural changes associated with CaM kinase-II activation can be detected using anisotropy imaging and FCS,and we now wish to image the activation of this protein complex in living zebrafish hearts.