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体如何调节活细胞的突触功能。最近，我们集中精力利用Forster共振能量转移(FRET)来监测蛋白质之间的相互作用。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1-10 nm尺度上两个荧光团之间的距离变化很敏感。FRET成像结合绿色荧光蛋白(GFP)光谱变体的开发，为从基因上标记感兴趣的突触蛋白并实时监测它们与其他标记蛋白的相互作用提供了机会。 目前，我们在实验室中有3个项目。第一个项目是建造一台专门为研究活细胞中的蛋白质复合体而设计的双光子显微镜。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，然后可以用荧光相关光谱(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分析来监测CaM Kinase-II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尖峰频率的探测器，并已被证明在学习和记忆中发挥关键作用。在心脏中，CaMKII活性与几种形式的心脏病有关。我们的结果表明，可以使用各向异性成像和FCS检测到与CaM Kinase-II激活相关的结构变化，现在我们希望成像这种蛋白质复合体在活的斑马鱼心脏中的激活情况。我们的第三个项目旨在应用我们实验室与安妮·肯沃西博士和理查德·尤尔博士合作开发的FRET和FCS方法来研究泛素连接酶激活前后的结构。

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 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,and we now wish to image the activation of this protein complex in living zebrafish hearts. Our third project is aimed at applying the FRET and FCS methodologies developed in our lab in collaboration with Dr. Anne Kenworthy and Dr. Richard Youle to study the structure of ubiquitin ligases before and after activation.