Bioengineering to map stress propagation in cytoskeleton

描述(申请人提供)：机械转导-细胞对机械应力的反应-被认为发生在细胞质中，对许多基本细胞功能至关重要。然而，施加的压力如何在细胞质内传播并转化为细胞反应尚不清楚。在这一应用中，我们建议在细胞骨架中定位负荷引起的位移和应力，细胞骨架是细胞质中假定的应力承载网络。初步数据表明，我们可以使用同步检测方法追踪标记有绿色荧光蛋白的细胞内细胞骨架结构。我们还可以测量这些结构的位移的空间分布，并计算细胞内的应力，这些应力是由外部施加在细胞上的微小局部机械变形引起的。我们惊讶地发现，细胞内应变和应力的诱导场并没有像目前所有的细胞力学模型所预测的那样在空间中迅速衰减，而是显示出集中的应力在长距离传播。在这里，我们提出了四个具体的目标：目标1是进一步发展细胞骨架中三维细胞内位移场和应力场的量化技术。第二个目的是验证预应力介导长距离应力传播的假设，并确定细胞骨架中应力和应变集中的来源。目标3是绘制细胞骨架结构在局部振荡载荷作用下的三维动态特征图。目的4确定波形蛋白、细胞骨架交联蛋白plectin、粘着斑蛋白vinculin和talin在细胞骨架应激传播中的作用。拟议的生物工程研究将收缩状态的新机械测量与细胞变形的数学分析结合在一起。实验方法是以高空间和时间分辨率测量局部机械载荷响应的细胞内变形场，并表征特定干预过程中的力学状态和细胞骨架结构。本研究可能对阐明细胞质深处的机械转导的特定位点和结构途径具有重要意义。

DESCRIPTION (provided by applicant): Mechanotransduction - the cellular response to mechanical stress - is thought to occur in the cytoplasm and is vital to many fundamental cell functions. However, how applied stresses are propagated within the cytoplasm and transduced into cellular responses is unknown. In this application we propose to map load-induced displacements and stresses in the cytoskeleton, the putative stress-bearing network in the cytoplasm. Preliminary data establish that we can track intracellular cytoskeletal structures marked with green fluorescent protein using a synchronous detection method. We can also measure the spatial distribution of displacements of these structures and compute intracellular stresses that arise in response to a small localized mechanical deformation imposed on the cell from the outside. We were surprised to find that the induced fields of intracellular strain and stress did not decay rapidly in space, as would be predicted from all current models of cell mechanics, but rather exhibited focused stress propagation over long distances. Here we propose four Specific Aims: Aim 1 is to further develop the technology to quantify intracellular displacement and stress fields in three dimensions in the cytoskeleton. Aim 2 is to test the hypothesis that the prestress mediates long distance stress propagation and to identify the origin of stress and strain concentration in the cytoskeleton. Aim 3 is to map the dynamic features of the cytoskeletal structures in three dimensions in response to localized oscillatory loads. Aim 4 is to determine the roles of vimentin, cytoskeletal crosslinking protein plectin, and focal adhesion proteins vinculin and talin in cytoskeletal stress propagation. The proposed bioengineering research combines novel mechanical measurements of the contractile state with mathematical analysis of cell deformation. The experimental method is to measure with high spatial and temporal resolution the intracellular deformation field in response to localized mechanical loading, and to characterize the mechanical state and cytoskeletal structure during specific interventions. The current poject may have implications in elucidating specific loci and structural pathways for mechanotranduction at sites deep in the cytoplasm.