FRET Imaging of Molecular Hierarchy at Subcellular Levels in Mechanotransduction

描述(由申请人提供)： 机械转导中亚细胞水平分子层次的FRET成像动脉粥样硬化是美国和大多数其他发达国家的主要死亡原因。动脉粥样硬化主要发生在血管弯曲和分支部位，这些部位的血管壁暴露于扰动的血流，而不是在以层流为主的血管的直段。与层流相比，扰流也会导致动脉粥样硬化后球囊损伤后伤口愈合过程变慢。有证据表明，血流引起的剪应力在调节血管内皮细胞(ECs)以及随后的内皮通透性和动脉粥样硬化、伤口愈合和再狭窄方面发挥着至关重要的作用。然而，关于内皮细胞如何感知这些机械刺激的时空特征以及如何在亚细胞水平上协调分子层次以确定病理生理后果，目前仍不清楚。荧光共振能量转移(FRET)技术和基因编码生物传感器为在活细胞中可视化具有高时空分辨率的活性分子事件提供了强大的工具。在这个方案中，我们将应用多色FRET生物传感器来可视化在不同流动下在亚细胞水平上参与Src信号通路的分子层次。SRC可以被剪切力激活，并在各种细胞过程中发挥核心作用。我们假设不同的流可以诱导具有不同时空模式的RhoA/肌动蛋白依赖的Src激活。激活的Src可以增强MLCK介导的肌球蛋白收缩能力，从而调节黏附连接(AJs)，从而影响内皮通透性，从而导致动脉粥样硬化。流动激活的Src还可以控制p130Cas-RAC通路，导致调控EC突起、运动、伤口愈合过程和潜在的再狭窄。为了验证我们的假设，我们提出了三个具体的目标：(1)可视化不同流动模式下亚细胞膜室中Src的时空激活模式，并研究RhoA和肌动蛋白细胞骨架在调节这些Src激活中的作用；(2)剖析Src和MLCK在调节不同流动下EC收缩和AJs中的作用；(3)研究不同流动下Src在调节p130Cas、RAC和EC突起中的作用。具有不同颜色的FRET生物传感器将被开发出来，以允许在单个活细胞中同时显示亚细胞水平的多个分子活动。药理抑制剂、siRNAs和遗传突变体将被用来评估不同信号分子的作用。多色FRET生物传感器的集成可同时显示多个分子事件，这将极大地提高我们对不同血流影响心血管疾病(如动脉粥样硬化和再狭窄)的分子机制的系统理解。新开发的生物传感器还将为检测心血管疾病以及治疗性抑制剂的疗效提供强大的工具。 公共卫生相关性： 简介：内皮细胞(ECs)不断地暴露在流动及其产生的剪应力中。已知切应力在调节血管内皮细胞功能和随后的病理生理过程中起重要作用，如动脉粥样硬化和再狭窄。然而，关于细胞如何感知剪切力和协调分子功能尚不清楚。该方案将结合先进的FRET技术和新型荧光蛋白来可视化活体内皮细胞中具有高时空分辨率的多个活性分子事件。这一结果将有助于我们更系统地理解机械转导的分子机制以及随后的病理生理过程。因此，拟议项目的成功将对改善公共卫生产生重大影响。

DESCRIPTION (provided by applicant): FRET Imaging of Molecular Hierarchy at Subcellular Levels in Mechanotransduction Atherosclerosis is the leading cause of death in the United States and most other developed countries. Atherosclerosis occurs preferentially at vascular curvature and branch sites where the vessel walls are exposed to disturbed flow, but not at the straight parts of vessels where laminar flow dominates. Disturbed flows, in comparison to laminar flows, can also cause slower wound healing process following the balloon injury after atherosclerosis. Evidence has shown that the shear stresses resulting from the flows play crucial roles in regulating vascular endothelial cells (ECs), and subsequently endothelium permeability and atherosclerosis, wound healing and restenosis. It remains unclear, however, on how ECs sense the spatiotemporal characteristics of these mechanical stimuli and coordinate the molecular hierarchy at sub-cellular levels to determine patho-physiological consequences. Fluorescence resonance energy transfer (FRET) technology and genetically encoded biosensors have provided powerful tools for visualizing active molecular events with high spatiotemporal resolutions in live cells. In this proposal, we will apply multi-color FRET biosensors for the visualization of molecular hierarchies participating in Src signaling pathways at sub-cellular levels under different flows. Src can be activated by shear stress and plays central roles in a variety of cellular processes. We hypothesized that different flows can induce a RhoA/actin-dependent activation of Src with distinct spatiotemporal patterns. The activated Src can enhance the MLCK-mediated actomyosin contractility to modulate adherens junctions (AJs), which can affect endothelium permeability and consequently atherosclerosis. The flow-activated Src can also control the p130cas-Rac pathway, leading to the modulated EC protrusion, motility, wound healing process, and potentially restenosis. To test our hypothesis, three specific aims are proposed: (1) To visualize the spatiotemporal activation patterns of Src at sub-cellular membrane compartments under different flow patterns and examine the roles of RhoA and actin cytoskeleton in mediating these Src activations; (2) To dissect the roles of Src and MLCK in mediating the modulation of EC contractility and AJs in response to different flows; (3) To investigate the role of Src in regulating the p130cas, Rac, and EC protrusion under different flows. FRET biosensors with distinct colors will be developed to allow the simultaneous visualization of multiple molecular activities at sub-cellular levels in a single live cell. Pharmacological inhibitors, siRNAs, and genetic mutants will be employed to assess the roles of different signaling molecules. The Integration of multi-color FRET biosensors for the simultaneous visualization of multiple molecular events will significantly advance our systematic understanding of the molecular mechanism by which different flows affect cardiovascular diseases, such as atherosclerosis and restenosis. The newly developed biosensors will also provide powerful tools for detecting cardiovascular diseases as well as the efficacy of therapeutic inhibitors. PUBLIC HEALTH RELEVANCE: Narrative: Endothelial cells (ECs) are continuously exposed to flow and its resultant shear stress. It has been known that shear stress plays crucial roles in regulating EC function and the ensuing patho-physiological processes, such as atherosclerosis and restenosis. It is not clear, however, on how the cells perceive shear stress and coordinate molecular functions. This proposal will integrate the cutting-edge FRET technology and novel fluorescence proteins to visualize multiple active molecular events with high spatiotemporal resolutions in live ECs. The results should shed new light and advance our systematic understanding of the molecular mechanism of mechanotransduction and the ensuing pathophysiological processes. Therefore, the success of the proposed project will have significant impact on improving public health.