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：
8941396
负责人：
Steven S Vogel
金额：
$ 111.24万
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/8941396
关键词：
Advisory Committees Anisotropy Basal Ganglia Behavioral Binding Biological Biophotonics Brain Ca(2+)-Calmodulin Dependent Protein Kinase Calcium Cell physiology Cells Collaborations Development Energy Transfer Fiber Fiber Optics Fluorescence Fluorescence Anisotropy Fluorescence Microscopy Fluorescence Polarization Fluorescent Probes Goals Green Fluorescent Proteins Human Image Individual Institutes Label Laboratories Life Logistics Measurement Mediating Methods Microscopy Mission Molecular Monitor Mus Nanotechnology National Institute on Alcohol Abuse and Alcoholism Neurons Physiological Physiology Presynaptic Terminals Production Proteins Research Site Spectrum Analysis Structure Synapses System Techniques Time Transgenic Mice Translations Uncertainty United States National Institutes of Health Variant base fluorescence imaging fluorophore imaging modality imaging probe interest neuronal cell body programs protein complex protein protein interaction sensor spectroscopic imaging synaptic function theories tool development two-photon

项目摘要

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 4 projects in the lab: The goal of the first project is to adapt FPFA (fluorescence polarization and fluctuation analysis), a mixture of anisotropy lifetime decay analysis and FCS, to: monitoring dynamic changes in protein complex structure, to identify specific sites of protein-protein interactions, and to automate FPFA to enable the wide-scale biophysical analysis of protein-protein interactions between hundreds of potentially interacting proteins that comprise the interactome (a cells complete network of molecular interactions). Our second project is aimed at applying FPFA in collaborations with Drs. Anne Kenworthy and Eric Long. The third project is involved in developing fiber-optics based spectroscopy for use in monitoring fluorescent probes expresses in living mice. Once perfected, these fiber-based systems will be used to monitor specific changes in fluorophores expressed in specific subsets of neurons of transgenic mice as they perform behavioral tasks. Our biological goal in this third project is to understand how the basal ganglia controls voluntary action at the molecular level. Our technological goal is to adapt fiber optics and advanced spectroscopic techniques to dynamically monitor bio-sensors and/or protein-protein interaction in neurons deep within the functioning brain. Our fourth project, in collaboration with Drs. Paul Blank and Wieb van der Meer, is to develop the underlying theory to allow accurate estimates of distance between biological components based on FRET measurements despite uncertainties in fluorophore orientation.

细胞生物素化学部分的主要目的是使用成像和光谱技术，例如两光子显微镜，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如何蛋白质调节活细胞的蛋白质。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4个项目：第一个项目的目的是适应FPFA（荧光极化和波动分析），各向异性生命周期衰减分析和FC的混合物，以：监测蛋白质复杂结构的动态变化，以识别蛋白质蛋白质相互作用的特定位点，以识别FPFA相互作用的质量分析，以构成质量的质量分析，从而构成了质量分析的质量分析。相互作用组（一个细胞完整的分子相互作用网络）。我们的第二个项目旨在与DRS合作应用FPFA。安妮·肯沃西（Anne Kenworthy）和埃里克·朗（Eric Long）。第三个项目参与开发基于光纤的光谱法，用于监测活小鼠中表达的荧光探针。一旦完善，这些基于纤维的系统将用于监视转基因小鼠神经元的特定子集表达的特定变化，它们执行行为任务。我们在第三个项目中的生物学目标是了解基底神经节如何控制分子水平的自愿行动。我们的技术目标是调整光纤和高级光谱技术，以动态监测功能性大脑内部神经元中的生物传感器和/或蛋白质 - 蛋白质相互作用。我们的第四个项目与Drs合作。 Paul Blank和Wieb van der Meer是开发基本理论，以基于FRET测量值的精确估计生物组件之间的距离，尽管荧光团取向不确定性。