FRET imaging of protein-protein interactions inside living cells

活细胞内蛋白质-蛋白质相互作用的 FRET 成像

基本信息

批准号：
8559273
负责人：
Steven S Vogel
金额：
$ 120.15万
依托单位：
NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8559273
关键词：
Advisory Committees Anisotropy Biophotonics Brain Ca(2+)-Calmodulin Dependent Protein Kinase Calcium Calcium Spikes Cell physiology Cells Collaborations Development Energy Transfer Enzymes Fluorescence Fluorescence Anisotropy Fluorescence Resonance Energy Transfer Frequencies Green Fluorescent Proteins Heart Heart Diseases Human Image Imaging Techniques Individual Institutes Label Laboratories Learning Life Link Logistics Measures Mediating Memory Methodology Methods Microscope Microscopy Mission Molecular Monitor Nanotechnology National Institute on Alcohol Abuse and Alcoholism Phosphotransferases Physiological Physiology Play Presynaptic Terminals Production Proteins Research Role Site Spectrum Analysis Structure Synapses Techniques Time Translations United States National Institutes of Health Variant Zebrafish calmodulin-dependent protein kinase II design detector fluorescence imaging fluorophore imaging modality imaging probe interest neuronal cell body programs protein complex protein protein interaction spectroscopic imaging synaptic function tool development two-photon ubiquitin ligase

项目摘要

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.

细胞生物光谱学部分的主要目的是使用成像技术，例如两光子显微镜，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Forster共振能量转移（FRET）来监测蛋白质 - 蛋白质相互作用。该方法具有研究蛋白质相互作用的巨大潜力，因为它对距离的变化敏感，将两个荧光团以1-10 nm的比例分开。 FRET成像结合了绿色荧光蛋白（GFP）的光谱变体的发展提供了机会，可以实时对具有遗传标记感兴趣的突触蛋白进行遗传标记突触蛋白，并实时监测其与其他标记蛋白的相互作用。目前，我们在实验室中有3个项目。第一个项目涉及构建专为研究活细胞中蛋白质复合物而设计的两光子显微镜。我们组装的显微镜将能够同时测量时间分辨的荧光各向异性，并且荧光强度的波动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分析来监测CAM激酶-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检测器，并且已被证明在学习和记忆中起着关键作用。在心脏中，CaMKII活性与多种形式的心脏病联系在一起。我们的结果表明，可以使用各向异性成像和FC检测与CAM激酶-II激活相关的结构变化，现在我们希望在活着的斑马鱼心中对这种蛋白质复合物的激活进行想象。我们的第三个项目旨在应用FRET和FCS方法与Anne Kenworthy博士和Richard Youle博士合作开发的FCS方法，以研究激活之前和之后的泛素连接酶的结构。