Bioengineering to map stress propagation in cytoskeleton

描述（由申请人提供）：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导成细胞反应尚不清楚。在这个应用程序中，我们建议绘制细胞骨架（细胞质中假定的应力承受网络）中载荷引起的位移和应力。初步数据表明，我们可以使用同步检测方法跟踪绿色荧光蛋白标记的细胞内细胞骨架结构。我们还可以测量这些结构位移的空间分布，并计算细胞内应力，这些应力是由外部施加在细胞上的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细胞力学模型所预测的那样在空间中迅速衰减，而是表现出长距离的集中应力传播。在此，我们提出了四个具体目标：目标1是进一步发展在细胞骨架中三维量化细胞内位移和应力场的技术。目的2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起源。目的3是绘制细胞骨架结构的动态特征在三维响应局部振荡载荷。目的4是确定vimentin、细胞骨架交联蛋白plectin、局点黏附蛋白vinculin和talin在细胞骨架胁迫繁殖中的作用。提出的生物工程研究结合了新颖的收缩状态的机械测量和细胞变形的数学分析。实验方法是用高时空分辨率测量细胞内变形场对局部机械载荷的响应，并表征特定干预过程中的机械状态和细胞骨架结构。目前的项目可能有助于阐明细胞质深处机械转导的特定位点和结构途径。

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.