Revealing Cardiovascular Stress Regulation beyond the Diffraction Limit

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物的定位和不同细胞区室在正常刺激或侮辱下的动态变化。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纳米的尺度，同时保持微观全细胞尺度在20-100微米范围内。在多学科工程和生物科学的努力下，我们将开发和应用这种“纳米显微镜”用于心血管研究，特别是心力衰竭的压力过载模型。总体假设认为，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在压力诱导下的结构重排对心力衰竭的发生和进展有不同的影响。我们展示了一种新型反射纳米显微镜设计的令人兴奋的初步进展，该显微镜实现了~100 nm的横向分辨率的全宽半最大值（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相互作用。1.1. 为了进一步改进新型反射共聚焦纳米显微镜，构建了具有FRET的活细胞快速采集多色反射共聚焦，并发展了提高其分辨率的理论。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，用于蛋白质共定位成像。目标2。通过确定以下因素，应用新型纳米显微镜观察心力衰竭压力过载模型中调节心脏和血管信号的大分子复合物的静态和动态变化：心肌细胞在压力和心力衰竭下局部应激信号（p38激酶信号体）和ec偶联缺陷的结构基础2.2. 正常、应激和受保护（如雌激素信号）心肌中蛋白酶体亚基的时空重构及其组装。2.3. 压力诱导的主动脉GPCR-Src酪氨酸激酶信号复合物的动态/重塑加剧心力衰竭。纳米成像将辅以最先进的分子操作，生化和蛋白质组学方法。这些研究将为在纳米水平上揭示亚细胞水平上蛋白质复合物的结构图谱、它们在心血管疾病中的定位和动态相互作用奠定基础。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提供机会，以减轻心血管疾病，这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.