Mechanism of Atheroprone Mechanotransduction Studied By Single Cell Imaging

描述(申请人提供)：血管内皮细胞(ECs)对血流动力的反应在调节血管内稳态方面起着重要作用。体内研究表明，动脉树分支点的内皮细胞暴露在扰流(DF)中，并表达促炎和促动脉粥样硬化的表型。相反，动脉树直段的内皮细胞暴露在层流剪切流(LF)中，通常不会出现动脉粥样硬化。我们假设动脉粥样硬化和动脉粥样硬化保护性血流在亚细胞水平激活具有不同时空特征的内皮细胞，从而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来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。我们会 研究跨质膜、相邻细胞之间的信号传递以及细胞内胞浆-细胞核的转换，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，我们将把新的mOrange2-mCherry fret对与CFP-YFP对结合在一起，以同时监测同一活细胞中的两个不同的分子事件。我们将进一步集成荧光寿命成像显微镜(FLIM)，通过使用我们实验室开发的相关FRET成像显微镜(CFIm)，同时显示跨越质膜、细胞之间和细胞体内部的多个分子信号。提出了三个具体的目标：1)可视化跨质膜的时空力学转导：在不同的流量下，同时监测细胞外剪应力(剪切传感器)和细胞内分子信号(不同膜微区的跨膜TRPC6和Src活性)，以阐明微域和质膜分子元素的作用。2)分析TRPC6在不同血流条件下黏附连接(AJ)调节中的作用：使用连环蛋白生物传感器监测AJ的机械张力及其与细胞外/胞内钙离子浓度的相互作用。3)为了破译膜-胞浆-核ERK信号对MCP-1基因的调控：将确定胞浆和细胞核ERK FRET生物传感器的不同流动调节，以重建与MCP-1基因表达相关的ERK的时空激活图谱。这些研究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，并评估膜微域/元件在调节这些事件中的作用。这些发现将为动脉粥样硬化的分子和力学机制提供新的时空基础，动脉粥样硬化是心血管疾病中的一种主要病理生理事件。

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.