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：
7251721
负责人：
ENRICO STEFANI
金额：
$ 111.24万
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 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7251721
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 DNA Sequence Rearrangement Defect Disease Doctor of Philosophy Engineering Estrogens Event Failure Figs - dietary Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Functional disorder G-Protein-Coupled Receptors Gene Proteins Genes Genomics Goals Guidelines Head Health Heart Heart Hypertrophy Heart failure Human Image Imagery Intellectual Property Internet Laboratories Laboratory Research Lateral Leg Life Localized Location MAPK14 gene Macromolecular Complexes Maps Measurement Measures Microscope Microscopic Microscopy Modeling Molecular Molecular Medicine Muscle Myocardium Phosphotransferases Physiology Property Proteins Proteomics Public Health Publications RNA Range Recycling Regulation Reporting Research Research Personnel Resolution Signal Pathway Signal Transduction Signaling Protein Stimulus Stress Structure Synapses System Technology Time United States Upper arm Width base commercialization concept design fluorescence imaging fluorophore genetic manipulation improved instrument interest intracellular protein transport multicatalytic endopeptidase complex nano nanoimaging nanoscale novel novel therapeutics pressure programs protein localization location protein protein interaction prototype response spatiotemporal src-Family Kinases theories therapeutic target 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）：为了更好地理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在响应正常刺激或侮辱的不同细胞室中不同细胞室中的动态变化。为此，我们将开发用于荧光成像的“纳米显微镜”，以测量结构及其在3D空间分辨率下至20-40 nm的细胞内的结构及其动力学，同时在20-100 UM范围内保持显微镜全细胞尺度。在多学科的工程和生物学科学工作中，我们将开发并将这种“纳米显微镜”开发到心血管研究中，特别是针对心力衰竭压力超负荷模型。总体假设指出，应力引起的结构重排 - 在亚细胞位置和相互作用 - 心脏和血管中关键信号蛋白复合物的相互作用 - 有差异地促进了心力衰竭的发作和进展。我们在设计新型反射纳米显微镜的设计中展示了令人兴奋的初步进步，该反射纳米显微镜达到了〜100 nm横向分辨率的全差最大（FWHM）。具体目的是：目标1。开发新型纳米显微镜以测量静态和动态蛋白质 - 蛋白质相互作用。 1.1。为了进一步改善新型反射共焦纳米显微镜，通过与FRET构建快速获取多色反射共焦用于活细胞，并开发出该理论以增强其超出其分辨率。 1.2。与4PI显微镜结合使用以实现10-20 nm 3D分辨率，并扩展到两个荧光波长进行蛋白质共定位成像。 AIM 2。应用新型纳米显微镜来可视化大分子复合物的静态和动态变化，以通过确定：2.1的心力衰竭压力过载模型，调节心脏和血管信号传导。2.1。局部应力信号传导（p38激酶信号）和心脏衰竭下心肌细胞中的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，应力和受保护（例如雌激素信号）心肌中的组装。 2.3。应力诱导的动力学/重塑主动脉GPCR-SRC酪氨酸激酶信号传导复合物会加剧心力衰竭。纳米成像将由最先进的分子操作，生化和蛋白质组学方法补充。这些研究将是在纳米级水平上解散的基础 - 亚细胞水平的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。确定细胞信号通路/网络的结构基础将为发现新的治疗靶标的机会减轻心血管疾病，这是美国的主要死亡原因。