Revealing Cardiovascular Stress Regulation beyond the Diffraction Limit

揭示超越衍射极限的心血管压力调节

基本信息

批准号：
8065410
负责人：
ENRICO STEFANI
金额：
$ 100.55万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8065410
关键词：
Address Affect Animals Aorta Area Arts Biochemical Biological Sciences Biomedical Engineering Biomedical Research Biomedical Technology Blood Vessels Cardiac Myocytes Cardiovascular Diseases Cardiovascular Physiology Cardiovascular system Cause of Death Cell physiology Cells Cellular Structures Communities Complement Complex Coupling DNA Sequence Rearrangement Defect Disease Doctor of Philosophy Engineering Estrogens Event Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Functional disorder G-Protein-Coupled Receptors Gene Proteins Genes Genomics Goals Guidelines Head Health Heart Heart failure Human Image Imagery Intellectual Property Internet Laboratories Laboratory Research Lateral Leg Life Location MAPK14 gene Macromolecular Complexes Maps Measurement Measures Microscope Microscopic Microscopy Modeling Molecular Molecular Medicine Muscle Myocardium Pathway interactions Phosphotransferases Physiology Property Protein Dynamics Proteins Proteomics Public Health Publications RNA Recycling Regulation Reporting Research Research Personnel Resolution Signal Transduction Stimulus Stress Structure Synapses System Technology Time United States Width arm base commercialization design fluorescence imaging fluorophore genetic manipulation improved instrument interest multicatalytic endopeptidase complex nanoimaging nanoscale new therapeutic target novel pressure programs protein complex protein protein interaction prototype response spatiotemporal src-Family Kinases theories user-friendly

项目摘要

DESCRIPTION (provided by the applicant): To better understand cell function in health and disease, we need to visualize the localization of protein complexes and dynamic changes in different cellular compartments in response to normal stimuli or insult. To this end, we will develop "Nanomicroscopes" for fluorescence imaging to measure structures and their dynamics inside a cell with a 3D spatial resolution down to the scale of 20-40 nm while maintaining the microscopic whole cell scale over a 20-100 um range. In a multi-disciplinary engineering and biological sciences effort, we will develop and apply such "Nanomicroscopes" to cardiovascular research, specifically to a pressure-overload model of heart failure. The overall hypothesis states that, stress-induced structural rearrangements -in the subcellular location and interactions- of key signaling protein complexes in the heart and blood vessels differentially contribute to the onset and progression of heart failure. We show exciting preliminary advances in the design of a novel Reflexion Nanomicroscope that achieves a full-width-half- maximum (FWHM) of ~100 nm lateral resolution. The Specific Aims are: Aim 1. TO DEVELOP NOVEL NANOMICROSCOPIES TO MEASURE STATIC AND DYNAMIC PROTEIN-PROTEIN INTERACTIONS. 1.1. To further improve the novel Reflexion Confocal Nanomicroscope by constructing a fast acquisition multicolor Reflexion Confocal with FRET for living cells and develop the theory to enhance its resolution beyond. 1.2. To combine STED with 4Pi microscopy to achieve 10-20 nm 3D resolution and expand to two fluorescene wavelengths for protein colocalization imaging. Aim 2. TO APPLY THE NOVEL NANOMICROSCOPES TO VISUALIZE STATIC AND DYNAMIC CHANGES OF MACROMOLECULAR COMPLEXES REGULATING HEART AND VASCULAR SIGNALING IN A PRESSURE OVERLOAD MODEL OF HEART FAILURE BY DETERMINING: 2.1. The structural basis of local stress signaling (p38 kinase signalsome) and EC-coupling defects in cardiomyocytes under stress and heart failure. 2.2. The spatiotemporal remodeling of proteasome subunits and their assembly in normal, stressed and protected (e.g. estrogen signals) myocardium. 2.3. The stress-induced dynamics/remodeling of aortic GPCR-Src tyrosine kinase signaling complexes that exacerbate heart failure. Nano-imaging will be complemented by state-of-the-art molecular manipulations, biochemical and proteomic approaches. These studies will be the basis to unravel -at the nanoscale level- the structural map of protein complexes at the subcellular level, their localization and dynamic interactions in cardiovascular disease. Identifying the structural basis of cell signaling pathways/networks will provide opportunities to discover new therapeutic targets to alleviate cardiovascular disease, a leading cause of death in the United States.

描述（由申请人提供）：为了更好地了解健康和疾病中的细胞功能，我们需要可视化蛋白质复合物的定位以及不同细胞区室响应正常刺激或损伤的动态变化。为此，我们将开发用于荧光成像的“纳米显微镜”，以 3D 空间分辨率低至 20-40 nm 的尺度测量细胞内的结构及其动态，同时保持微观全细胞尺度在 20-100 um 范围内。在多学科工程和生物科学的努力中，我们将开发这种“纳米显微镜”并将其应用于心血管研究，特别是心力衰竭的压力过载模型。总体假设表明，压力诱导的心脏和血管中关键信号蛋白复合物的亚细胞位置和相互作用的结构重排对心力衰竭的发生和进展有不同的影响。我们展示了新型反射纳米显微镜设计中令人兴奋的初步进展，该显微镜实现了约 100 nm 横向分辨率的半高全宽 (FWHM)。具体目标是：目标 1. 开发新型纳米显微镜来测量静态和动态蛋白质-蛋白质相互作用。 1.1.通过构建具有 FRET 的活细胞快速采集多色反射共焦纳米显微镜，进一步改进新型反射共焦纳米显微镜，并发展理论以提高其分辨率。 1.2.将 STED 与 4Pi 显微镜相结合，实现 10-20 nm 3D 分辨率，并扩展到两个荧光波长以进行蛋白质共定位成像。目标 2. 通过确定以下内容，应用新型纳米显微镜来可视化在心力衰竭压力超负荷模型中调节心脏和血管信号转导的大分子复合物的静态和动态变化： 2.1。应激和心力衰竭下心肌细胞局部应激信号传导（p38 激酶信号传导）和 EC 偶联缺陷的结构基础。 2.2.蛋白酶体亚基的时空重塑及其在正常、应激和受保护（例如雌激素信号）心肌中的组装。 2.3.应激诱导的主动脉 GPCR-Src 酪氨酸激酶信号复合物的动力学/重塑会加剧心力衰竭。纳米成像将得到最先进的分子操作、生化和蛋白质组学方法的补充。这些研究将成为在纳米级水平上揭示亚细胞水平上蛋白质复合物的结构图、它们在心血管疾病中的定位和动态相互作用的基础。确定细胞信号通路/网络的结构基础将为发现新的治疗靶点以减轻心血管疾病提供机会，心血管疾病是美国的主要死亡原因。