Mechanism of Atheroprone Mechanotransduction Studied By Single Cell Imaging

项目概要 血管内皮细胞（EC）对血流动力学的反应在调节中起着重要作用 血管稳态。体内研究表明，动脉树分支点的 ECs 暴露于血流紊乱（DF）并表达促炎和促动脉粥样硬化表型。相比之下， 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 来自动脉粥样硬化。我们假设动脉粥样硬化易发性和动脉粥样硬化保护性血流以不同的方式激活 EC 亚细胞水平的时空特征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。我们还将研究跨质膜、相邻细胞之间的信号传递 作为细胞内细胞质-细胞核转变，以了解细胞质的时间和空间动态 力传导。为了实现生物传感器研究的有效性，我们将采用新的 mOrange2-mCherry FRET 对与 CFP-YFP 对一起同时监测两个不同的 同一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）同时可视化跨质膜、细胞之间的多个分子信号，以及 使用我们实验室开发的相关 FRET 成像显微镜 (CFIM) 来观察细胞体内的情况。三 提出了具体目标：1）可视化跨等离子体的时空机械转导 膜：细胞外剪切应力（剪切传感器）和细胞内分子信号（跨膜） TRPC6和Src在不同膜微域的活性将在以下条件下同时监测 不同的流动来阐明质膜上微域和分子元素的作用。 2) 至 剖析 TRPC6 在不同流下调节粘附连接 (AJ) 中的作用：¿-连环蛋白 生物传感器将用于监测 AJ 处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) 破译MCP-1基因的膜-细胞质-细胞核ERK信号传导 调节：细胞质和核酸 ERK FRET 生物传感器的差异流量调节将被确定为 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结果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域/元件在调节这些过程中的作用 事件。这些发现将为分子和分子的时空基础提供新的理解。 动脉粥样硬化的机械机制，心血管疾病的主要病理生理事件。

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.