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，各向异性成像可以检测到与CaM Kinase-II激活相关的结构变化，我们现在希望成像这种蛋白质复合体在活的斑马鱼心脏和培养的海马棘中的激活情况。最后，我们的第三个项目是揭示脱铁蛋白的功能，这是一种钙结合蛋白，负责两种形式的肌肉营养不良。Jain基金会慷慨地设立了一项捐赠基金来支持这一项目。脱铁蛋白是一种已知与LGMD2B/Miyoshi肌营养不良症有关的蛋白质。我们的实验表明，向发育中的海胆胚胎注射反义吗啉抑制细胞分裂和伤口愈合。因此，我们扩展了这项研究，以调查去铁蛋白在钙信号转导中的作用。我们已经发现，在创伤后，细胞通过一种涉及Agatoxin敏感的电压门控钙通道的机制去极化并分泌ATP。相邻细胞对分泌的ATP的反应机制被认为涉及P2X受体和电压门控钙通道。反义脱铁蛋白阻断三磷酸腺苷的分泌，支持脱铁蛋白可能通过介导钙离子引起的胞吐起作用的假说。

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 we now wish to image the activation of this protein complex in living zebrafish hearts, and in hippocampal spines in culture. Finally our third project is involved in revealing the function of dysferlin, a calcium binding protein responsible for two forms of muscular dystrophy. The Jain foundation has generously established a gift fund in support of this project. Dysferlin is a protein that is known to be responsible for LGMD2B/Miyoshi muscular dystrophy. Our experiments indicated that anti-sense morpholinos against Dysferlin injected into developing sea urchin embryos inhibit cell division and wound healing. Accordingly, we expanded this study to investigate the role of Dysferlin in calcium signaling. We have found that upon wounding, cells depolarize and secrete ATP by a mechanism involving agatoxin sensitive voltage-gated calcium channels. Neighboring cells respond to the secreted ATP by a mechanism thought to involve P2X receptors as well as voltage-gated calcium channels. Anti-sense morpholinos against Dysferlin block the secretion of ATP supporting the hypothesis that Dysferlin might act by mediating calcium triggered exocytosis.