Revealing Cardiovascular Stress Regulation beyond the Diffraction Limit

揭示超越衍射极限的心血管压力调节

• 批准号: 7788195
• 负责人: ENRICO STEFANI
• 金额: $ 98.6万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 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/7788195
• 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; Location; MAPK14 gene; Macromolecular Complexes; Maps; Measurement; Measures; Microscope; Microscopic; Microscopy; Modeling; Molecular; Molecular Medicine; Muscle; Myocardium; Phosphotransferases; Physiology; Property; Protein Dynamics; Proteins; Proteomics; Public Health; Publications; RNA; Recycling; Regulation; Reporting; Research; Research Personnel; Resolution; Signal Pathway; Signal Transduction; Signaling Protein; 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; nano; nanoimaging; nanoscale; new therapeutic target; novel; pressure; programs; protein complex; protein protein interaction; prototype; response; spatiotemporal; src-Family Kinases; theories; user-friendly

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)：为了更好地了解细胞在健康和疾病中的功能，我们需要可视化蛋白质复合体的定位和不同细胞隔间对正常刺激或侮辱的反应的动态变化。为此，我们将开发用于荧光成像的“纳米显微镜”，以测量细胞内的结构及其动力学，其3D空间分辨率降至20-40纳米，同时将整个细胞的微观尺度保持在20-100微米的范围内。在一个多学科的工程学和生物科学的努力中，我们将开发和应用这种“纳米显微镜”到心血管研究，特别是心力衰竭的压力超负荷模型。总体假说认为，应激诱导的心脏和血管中关键信号蛋白复合体在亚细胞位置和相互作用中的结构重排对心力衰竭的发生和发展起着不同的作用。我们展示了一种新型的反射式纳米显微镜的设计方面令人振奋的初步进展，该显微镜实现了约100 nm横向分辨率的全宽半高(FWHM)。其具体目标是：目的1.开发新型纳米显微显微镜，用于测量蛋白质之间的静态和动态相互作用。1.1.为进一步完善新型Refleion共焦纳米显微镜，构建一种用于活细胞的快速获取的多色Refleion共焦显微镜，并发展其理论以提高其分辨率。1.2.将STED和4PI显微镜相结合，达到10-20 nm的3D分辨率，并扩展到两个荧光波长，用于蛋白质共定位成像。目的2.应用新型纳米显微镜观察心力衰竭压力超负荷模型中调节心脏和血管信号的大分子复合体的静态和动态变化，测定：2.1。应激和心力衰竭下心肌细胞局部应激信号(p38激酶信号体)和EC偶联缺陷的结构基础。2.2.正常、应激和保护(如雌激素信号)心肌中蛋白酶体亚单位及其组装的时空重构。2.3.应激诱导的主动脉GPCR-Src酪氨酸激酶信号复合体的动力学/重构加剧心力衰竭。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。这些研究将是在纳米级别上解开亚细胞水平上蛋白质复合体的结构图、它们在心血管疾病中的定位和动态相互作用的基础。识别细胞信号通路/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。