Mechanism of Atheroprone Mechanotransduction Studied By Single Cell Imaging

单细胞成像研究动脉粥样硬化的机械传导机制

基本信息

批准号：
8787794
负责人：
SHU CHIEN
金额：
$ 59.28万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-12-20 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8787794
关键词：
Adherens Junction Atherosclerosis Biochemical Biological Assay Biosensor Blood Blood Vessels Calcium ion Cardiovascular Diseases Cell Adhesion Molecules Cell Count Cell Nucleus Cell membrane Cells Characteristics Code Color Coupling Cytosol Deposition Effectiveness Elements Endothelial Cells Event Feedback Fluorescence Fluorescence Resonance Energy Transfer Functional disorder Gene Expression Gene Expression Regulation Health Homeostasis Image Immune Indium Individual Inflammatory Knowledge Lead Libraries Life Low-Density Lipoproteins Maps Measures Mechanics Mediating Membrane Membrane Microdomains Microscopy Modeling Molecular Monitor Monocyte Chemoattractant Protein-1 Mutation Outcome Pathway interactions Permeability Phenotype Physiological Play Process Production Proteins Recruitment Activity Regulation Resolution Role Sensitivity and Specificity Signal Transduction Site Surface TRP channel Time Trees Vascular Endothelial Cell adherent junction atherogenesis atheroprotective base cellular imaging chemokine design directed evolution disorder prevention extracellular hemodynamics in vivo macromolecule meetings monocyte monolayer neuronal cell body novel response screening sensor shear stress spatiotemporal

项目摘要

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.

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的调节中发挥着重要作用。体内研究表明，动脉树分支点的 EC 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，动脉树直部分的 EC 暴露于层流剪切流 (LF)，并且通常呈层流剪切流 (LF) 状态。我们追求动脉粥样硬化和动脉粥样硬化流动在亚细胞水平上激活具有差异时空特征的EC，以触发不同的细胞反应，我们建议使用基于荧光蛋白（FP）和荧光共振能量转移（FRET）的基因编码生物传感器来可视化。我们将在单个活细胞中以前所未有的时空分辨率进行分子活动。研究跨质膜、相邻细胞之间的信号传递以及细胞内细胞质-细胞核转变，以了解机械转导的时间和空间动态。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。与 CFP-YFP 对一起使用，可同时监测同一活细胞中的两个不同分子事件。我们将进一步集成荧光寿命成像显微镜 (FLIM)，以同时可视化血浆中的多个分子信号。使用我们实验室开发的相关 FRET 成像显微镜（CFIM），我们提出了三个具体目标：1）可视化跨质膜的时空机械转导：细胞外剪切应力（剪切传感器）和细胞内分子信号（不同膜微域的跨膜TRPC6和Src活性）将在不同流量下同时监测，以阐明微域的作用2) 剖析 TRPC6 在不同流量下调节粘附连接 (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/细胞间钙离子浓度的相互作用 3) 破译 MCP-1 基因调节的膜-细胞质-细胞核 ERK 信号传导：差异流量调节。将确定细胞质和核酸 ERK FRET 生物传感器的变化，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机械机制的时空基础提供新的理解。心血管疾病的一个主要病理生理事件。