FRET imaging of protein-protein interactions inside living cells

细胞生物光子学部分的主要目的是使用成像和光谱技术，如双光子显微镜，光谱成像，荧光寿命显微镜，荧光相关光谱（FCS）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。这种方法具有很大的潜力，研究蛋白质的相互作用，因为它是敏感的变化，在1-10纳米尺度上分离两个荧光团的距离。FRET成像结合绿色荧光蛋白（GFP）的光谱变体的发展提供了机会，以遗传标记感兴趣的突触蛋白质和监测其相互作用与其他标记的蛋白质在真实的时间。 目前，我们在实验室有三个项目。第一个项目是建立一个专门用于研究活细胞中蛋白质复合物的双光子显微镜。我们正在组装的显微镜将能够同时测量时间分辨的荧光各向异性，以及荧光强度的波动，然后可以通过荧光相关光谱（FCS）进行分析。我们的第二个项目使用各向异性寿命衰减分析和FCS分析，以监测凸轮激酶-II的多聚体结构的变化。在大脑中，这种丰富的突触酶被认为是钙峰频率检测器，并已被证明在学习和记忆中发挥关键作用。在心脏中，CaMKII活性与几种心脏病有关。我们的研究结果表明，与CaM激酶-II激活相关的结构变化可以使用各向异性成像和FCS检测。我们现在希望在活细胞中对这种蛋白质复合物的活化进行成像。我们的第三个项目旨在应用我们实验室与Anne Kenworthy和Eric Long博士合作开发的FRET和FCS方法。

The principal aim of the Section on Cellular Biophotonics is to use imaging and spectroscopy techniques, such as two-photon microscopy, spectral imaging, fluorescence lifetime microscopy, fluorescence correlation spectroscopy (FCS), 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 3 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. We now wish to image the activation of this protein complex in living cells. Our third project is aimed at applying the FRET and FCS methodologies developed in our lab in collaboration with Drs. Anne Kenworthy and Eric Long.