Flow-Induced Cytoskeletal Mechanics in Endothelial Cells

内皮细胞中流动诱导的细胞骨架力学

• 批准号: 6999297
• 负责人: BRIAN P HELMKE
• 金额: $ 25.33万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6999297
• 关键词: actins; biological signal transduction; biomechanics; cell adhesion; cytoskeleton; green fluorescent proteins; hemodynamics; intermediate filaments; light microscopy; mechanical stress; microfilaments; paxillin; shear stress; three dimensional imaging /topography; time resolved data; vascular endothelium; vimentin

DESCRIPTION (provided by applicant): Endothelial cell (EC) adaptation to the complex local hemodynamic environment plays a critical role in both the physiological and pathological regulation of vessel wall biology, but mechanisms by which ECs transduce fluid mechanical forces into biochemical signals remains poorly understood. This proposal will address two key questions related to the initiation of mechanotransduction: is extracellular applied fluid force transmitted to the interior of the cell where biochemical signaling molecules are located, and is flow-induced intracellular deformation concentrated at discrete locations in the cell at a magnitude that mediates structural protein interactions? High-resolution 4-D microscopy imaging of green fluorescent protein fused to vimentin, actin, and paxillin will enable measurements to test the hypothesis that changes in extracellular applied fluid shear stress induce spatially focused mechanical responses in the cytoskeleton near intracellular locations where structural proteins are involved in rapid mechanochemical signal transduction. This hypothesis suggests that strain focusing by local cytoskeletal deformation provides the spatial organization of structural proteins necessary to trigger specific biochemical signaling networks in response to changes in the hemodynamic environment. The specific aims are (1) to determine the spatiotemporal distribution of strain focusing in the actin microfilament network in living ECs during a change in shear stress, (2) to determine the relative contributions of microfilament and intermediate filament networks in focusing cytoskeletal strain during onset of shear stress, and (3) to determine whether focusing of cytoskeletal strain in response to shear stress occurs near sites of focal adhesion to the extracellular matrix and initiates spatial redistribution of focal adhesion proteins. Since focal adhesion proteins are rapidly phosphorylated by onset of shear stress, signaling at these locations may be initiated by mechanical interactions with the cytoskeleton. A novel measurement of interaction strain will be defined to indicate the degree of structural rigidity of mechanical connections between the cytoskeleton and focal adhesion sites. This proposal will measure for the first time spatial and temporal relationships between mechanical interactions in the cytoskeleton and locations involved in initiation of mechanotransduction. The long-term goal of this research program is to define biomechanical mechanisms contributing to cell and tissue function in order to develop innovative approaches for treating endothelial dysfunction in vascular pathology and artificial graft design.

描述（由申请人提供）： 内皮细胞（EC）对复杂的局部血流动力学环境的适应在血管壁生物学的生理和病理调节中起着关键作用，但EC将流体机械力转化为生化信号的机制仍然知之甚少。该提案将解决两个关键问题有关的启动mechanotransduction：是细胞外施加的流体力传递到细胞内部的生化信号分子的位置，是流动诱导的细胞内变形集中在离散的位置在细胞中的一个幅度，介导结构蛋白质相互作用？高分辨率的4-D显微成像的绿色荧光蛋白融合的波形蛋白，肌动蛋白，桩蛋白将使测量测试的假设，即细胞外施加的流体剪切应力的变化诱导空间集中的机械反应在细胞内的位置附近的细胞骨架中的结构蛋白参与快速机械化学信号转导。这一假设表明，应变集中的局部细胞骨架变形提供了必要的结构蛋白的空间组织触发特定的生化信号网络，以响应血流动力学环境的变化。具体目的是（1）确定在剪切应力变化期间活EC中肌动蛋白微丝网络中应变聚焦的时空分布，（2）确定在剪切应力开始期间微丝和中间丝网络在聚焦细胞骨架应变中的相对贡献，和（3）确定响应剪切应力的细胞骨架应变的集中是否发生在与细胞外基质的粘着斑位点附近，并启动粘着斑蛋白的空间重新分布。由于粘着斑蛋白在剪切应力作用下迅速磷酸化，这些位置的信号传导可能是通过与细胞骨架的机械相互作用而启动的。一种新的测量相互作用应变将被定义为指示的程度的机械连接的细胞骨架和粘着斑网站的结构刚度。该建议将首次测量细胞骨架中的机械相互作用与机械转导启动所涉及的位置之间的空间和时间关系。该研究项目的长期目标是确定有助于细胞和组织功能的生物力学机制，以开发治疗血管病理学和人工移植物设计中内皮功能障碍的创新方法。