Mechanism of Atheroprone Mechanotransduction Studied By Single Cell Imaging

项目摘要 血管内皮细胞对血流动力的反应在血管内皮细胞对血流动力学的调节中起着重要作用。 血管动态平衡。活体研究表明，动脉树分支点的内皮细胞是 暴露于干扰流(DF)，并表达促炎和促动脉粥样硬化表型。相比之下， 动脉树直段的内皮细胞暴露在层流剪切流(LF)中，通常不受影响 死于动脉粥样硬化。我们假设动脉粥样硬化和动脉粥样硬化保护性血流通过不同的方式激活内皮细胞。 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 决议。我们还将研究跨质膜、相邻细胞之间的信号传递。 作为细胞内的胞浆-核的转变来理解细胞的时空动力学 机械转导。为了实现生物传感器研究的有效性，我们将纳入新的 MOrange2-mCherry FRET对和CFP-YFP对一起，同时监测两个不同的 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 同时显示跨质膜、细胞之间的多个分子信号 在细胞体内部，使用我们实验室开发的相关FRET成像显微镜(CFIm)。三 提出了具体的目标：1)可视化跨等离子体的时空力学转导 膜：细胞外剪应力(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 不同的流程，以阐明微域和分子元素在质膜上的作用。2)至 剖析TRPC6在不同流动条件下对黏附连接(AJ)的调节作用：An-catenin 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)破译MCP-1基因的膜-胞浆-核ERK信号 调节：胞质和细胞核ERK FRET生物传感器的不同流动调节将被确定为 构建ERK与单核细胞趋化蛋白-1基因表达关系的时空激活图谱。结果是 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 动脉粥样硬化的力学机制，这是心血管疾病中的一个主要病理生理事件。

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.