Mechanism of Atheroprone Mechanotransduction Studied By Single Cell Imaging

描述（由申请人提供）：血管内皮细胞（EC）对血流动力学力的反应在血管稳态调节中起重要作用。体内研究表明，动脉树的分支点中的EC暴露于扰动流（DF）并表达促炎性和促动脉粥样硬化表型。相比之下，动脉树的直线部分中的EC暴露于层流剪切流（LF），并且通常免于动脉粥样硬化。我们假设atheroprone和atheroprotective流量激活内皮细胞在亚细胞水平的差异时空特征，触发不同的细胞反应。我们建议使用基于荧光蛋白（FP）和荧光共振能量转移（FRET）的遗传编码生物传感器，以前所未有的时空分辨率可视化单个活细胞中的分子活动。我们将 研究跨质膜，相邻细胞之间的信号中继，以及细胞内的胞质溶胶-核转换，以了解机械转导的时间和空间动力学。为了实现生物传感器研究的有效性，我们将结合一个新的mOrange 2-mCherry FRET对与CFP-YFP对一起，以同时监测同一活细胞中的两个不同的分子事件。我们将进一步整合荧光寿命成像显微镜（FLIM），以同时可视化跨质膜，细胞之间和细胞体内部的多个分子信号，使用我们实验室开发的相关FRET成像显微镜（CCLO）。提出了三个具体的目标：1）可视化跨质膜的时空机械转导：在不同流量下同时监测细胞外剪切应力（剪切传感器）和细胞内分子信号（跨膜TRPC 6和Src在不同膜微区的活性），以阐明微区和分子元件在质膜上的作用。2)为了剖析TRPC 6在不同流量下调节粘附连接（AJs）中的作用：将使用连环蛋白生物传感器来监测AJs处的机械张力及其与细胞外/细胞间钙离子浓度的相互作用。3)为了破译MCP-1基因调控的膜-胞质-核ERK信号传导：将确定胞质和核ERK FRET生物传感器的差异流动调节以重建与MCP-1基因表达相关的ERK时空激活图。从这些研究中获得的结果将使我们能够生成分子转导/相互作用的时空相关图，并评估膜微区/元件在调节这些事件中的作用。这些发现将提供新的理解的时空基础的分子和力学机制的动脉粥样硬化，在心血管疾病的主要病理生理事件。

DESCRIPTION (provided by applicant): 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.