Flow-Induced Cytoskeletal Mechanics in Endothelial Cells

描述(由申请人提供)： 血管内皮细胞(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控中起着至关重要的作用，但ECs将流体机械力转化为生化信号的机制尚不清楚。这一提议将解决与机械转导的启动有关的两个关键问题：细胞外施加的流体力是否被传递到生化信号分子所在的细胞内部，以及流动诱导的细胞内变形是否集中在细胞内离散的位置，其程度与结构蛋白相互作用有关？融合到波形蛋白、肌动蛋白和帕西林的绿色荧光蛋白的高分辨率4-D显微镜成像将使测量能够验证这样的假设，即细胞外施加的流体剪应力的变化在细胞内位置附近的细胞骨架中诱导空间聚焦的机械反应，在这些位置结构蛋白参与快速机械化学信号转导。这一假说表明，通过局部细胞骨架变形聚焦的应变提供了结构蛋白的空间组织，这些结构蛋白是触发特定的生化信号网络以响应血液动力学环境变化所必需的。其具体目的是(1)确定在剪应力变化期间内皮细胞肌动蛋白微丝网络中应变聚焦的时空分布，(2)确定在剪应力开始时微丝和中间丝网络在聚焦细胞骨架应变中的相对贡献，以及(3)确定细胞骨架应变在剪应力响应下的聚焦是否发生在粘着焦点与细胞外基质的位置附近，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被磷酸化，这些位置的信号可能是通过与细胞骨架的机械相互作用而启动的。将定义一种新的相互作用应变的测量来指示细胞骨架和局部粘连部位之间的机械连接的结构刚性程度。这项提议将首次测量细胞骨架中的机械相互作用和参与机械转导启动的位置之间的空间和时间关系。该研究计划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，以开发血管病理学和人工移植物设计中治疗内皮功能障碍的创新方法。

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.