Revealing Cardiovascular Stress Regulation beyond the Diffraction Limit

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和不同细胞区室中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”，以测量细胞内的结构及其动态，其3D空间分辨率低至20-40 nm，同时保持微观整个细胞尺度在20-100 um范围内。在多学科的工程和生物科学的努力，我们将开发和应用这样的“纳米显微镜”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，心脏和血管中关键信号蛋白复合物的应激诱导的结构重排（在亚细胞位置和相互作用中）对心力衰竭的发生和进展有不同的贡献。我们展示了令人兴奋的初步进展，在设计一种新的反射纳米显微镜，实现了全宽半峰（FWHM）的横向分辨率为100 nm。具体目标是：目标1。开发新的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.通过构建用于活细胞的快速采集多色反射共焦与FRET，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.将联合收割机STED与4Pi显微镜相结合，实现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。目标2.应用新型纳米显微镜观察在压力超负荷心脏衰竭模型中调节心脏和血管信号传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（p38激酶信号体）和EC偶联缺陷的结构基础。2.2.蛋白酶体亚单位的时空重构及其在正常、应激和保护（如雌激素信号）心肌中的组装。2.3.加重心力衰竭的主动脉GPCR-Src酪氨酸激酶信号复合物的应激诱导动力学/重塑纳米成像将得到最先进的分子操作，生物化学和蛋白质组学方法的补充。这些研究将是在纳米级水平上揭示蛋白质复合物在亚细胞水平上的结构图、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。

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.