Mechanism of Atheroprone Mechanotransduction Studied By Single Cell Imaging

项目摘要 血管内皮细胞对血流动力学的反应在调节血管内皮细胞功能中起重要作用。 血管稳态体内研究表明，动脉树的分支点中的EC 暴露于扰动流（DF）并表达促炎和促动脉粥样硬化表型。与此相反， 动脉树直线部分的EC暴露于层流剪切流（LF），通常不会受到影响 动脉粥样硬化我们假设，atheroprone和动脉粥样硬化保护流激活内皮细胞， 在亚细胞水平的时空特征，以触发不同的细胞反应。我们建议使用 基于荧光蛋白（FP）和荧光共振能量的基因编码生物传感器 转移（FRET），以前所未有的时空可视化单个活细胞中的分子活动 分辨率我们也将研究跨质膜的信号传递，以及相邻细胞之间的信号传递 作为细胞内的胞质溶胶-核过渡，以了解时间和空间的动态， 机械传导为了实现生物传感器研究的有效性，我们将采用新的 mOrange 2-mCherry FRET对与CFP-YFP对一起用于同时监测两个不同的细胞周期。 在同一个活细胞中的分子事件。我们将进一步整合荧光寿命成像显微镜 （FLIM）以同时可视化细胞之间跨质膜的多个分子信号，以及 在细胞体内部，使用我们实验室开发的相关FRET成像显微镜（CCEH）。三 具体目标是：1）可视化跨等离子体的时空力学传导 膜：细胞外剪切应力（剪切传感器）和细胞内分子信号（跨膜 在不同的膜微区的TRPC 6和Src活性）将在下式中同时监测。 不同的流量，以阐明在质膜微区和分子元素的作用。2)到 分析TRPC 6在不同流动下粘附连接（AJs）调节中的作用： 生物传感器将用于监测AJs的机械张力及其与细胞外/细胞间的相互作用 钙离子浓度。3)MCP-1基因的膜-胞浆-核ERK信号转导 调节：细胞溶质和核酸ERK FRET生物传感器的差异流动调节将被确定， 重建ERK与MCP-1基因表达相关的时空激活图。结果 从这些研究中获得的信息将使我们能够生成分子的时空相关图， 转导/相互作用，并评估膜微区/元件在调节这些 事件这些发现将提供新的理解的时空基础的分子和 动脉粥样硬化是心血管疾病中的主要病理生理事件，其机械机制。

Project Summary Responses of vascular endothelial cells (ECs) to hemodynamic forces play significant roles in the regulation of vascular homeostasis. In vivo studies have shown that the ECs in branch points of the arterial tree are exposing to disturbed flow (DF) and express pro-inflammatory and pro-atherogenic phenotypes. In contrast, ECs in the straight part of the arterial tree are exposed to laminar shear flow (LF) and are generally spared from atherosclerosis. We hypothesize that atheroprone and atheroprotective flows activate ECs with differential spatiotemporal characteristics at subcellular levels to trigger different cellular responses. We propose to use genetically encoded biosensors based on fluorescent proteins (FPs) and fluorescence resonance energy transfer (FRET) to visualize molecular activities in individual live cells with unprecedented spatiotemporal resolution. We will study the signals relays across the plasma membrane, between neighboring cells, as well as intracellular cytosol-nuclei transitions to understand the temporal and spatial dynamics of mechanotransduction. In order to achieve effectiveness of the biosensor studies, we will incorporate a new mOrange2-mCherry FRET pair together with the CFP-YFP pair to simultaneously monitor two different molecular events in the same live cell. We will further integrate fluorescence lifetime imaging microscopy (FLIM) to simultaneously visualize multiple molecular signals across the plasma membrane, between cells, and inside the cell body, with the use of correlative FRET imaging microscopy (CFIM) developed in our labs. Three specific aims are proposed: 1) To visualize the spatiotemporal mechanotransduction across the plasma membrane: the extracellular shear stress (shear sensors) and intracellular molecular signals (transmembrane TRPC6 and Src activities at different membrane microdomains) will be simultaneously monitored under different flows to elucidate the roles of microdomains and molecular elements at the plasma membrane. 2) To dissect the role of TRPC6 in the regulation of adherent junctions (AJs) under different flows: an ¿-catenin biosensor will be used to monitor the mechanical tension at AJs and its interplays with extra-/inter-cellular calcium ion concentrations. 3) To decipher the membrane-cytosol-nucleus ERK signaling for MCP-1 gene regulation: differential flow-regulations of the cytosolic and nucleic ERK FRET biosensors will be determined to reconstruct the spatiotemporal activation map of ERK in relation to MCP-1 gene expression. The results obtained from these studies will allow us to generate spatiotemporal correlation maps of molecular transductions/interactions and assess the roles of membrane microdomains/elements in regulating these events. These findings will provide novel understanding of the spatiotemporal basis of the molecular and mechanical mechanisms of atherosclerosis, a major pathophysiological event in cardiovascular diseases.