Subcellular Response to Local Forces

描述(申请人提供)：本项目重点研究细胞与其细胞外基质之间的粘附点所经历的亚细胞力在调节血管平滑肌细胞组织和功能中所起的作用。细胞在空间上分解和响应这种局部作用力的适应性过程对黏附重塑至关重要，在黏附重塑过程中，细胞加强特定的黏附，同时分解其他黏附，并与许多机械介导的疾病过程有关，如高血压诱导的血管平滑肌细胞过度增殖反应，从而加剧血管疾病。该领域以前的工作表明，局部粘连是对施加的力做出反应的，但由于与细胞外基质的粘连也会诱导生物化学信号，从而触发细胞整体收缩，因此目前尚不清楚施加在特定粘附力上的力是否会导致任何机械串扰，其余粘连分布在整个细胞中。该项目将结合两名研究人员的专业知识，采用一种新技术，同时允许对细胞附着表面进行局部机械刺激，并对整个细胞产生的局部力场进行空间分辨测量，以响应这种刺激。有人提出，局部刺激和整体机械反应之间的关系对于将力转换为有意义的反应所需的细胞骨架内的机械协调至关重要。这两位研究人员最近开发了一种技术，其中微制桩阵列的偏转报告了连接到阵列上的细胞产生的细胞骨架张力和局部力场，嵌入到单个桩中的纳米工程磁性材料被用来向连接的细胞施加可调的亚细胞机械应力。因此，细胞可以在一个柱子上受到局部扰动，而周围的柱子同时测量这种刺激的效果。该项目的具体目标1将是在纳米级对细胞中的单个粘连施加明确的力，并根据细胞收缩能力和局部粘连结构的变化来测量细胞的整体反应。具体目标2将是确定粘着信号在力的机械反应中的作用。具体目标3将是研究细胞骨架张力的变化在调节局部黏附反应中的作用。由这项新技术实现的实验将导致对细胞如何将机械应力转换为适应的、协调的细胞骨架反应的新理解，并将开辟一条新的途径，以深入了解对炎症、增殖和组织发育至关重要的机械转导机制。与公共健康相关：这项研究将应用磁性微米和纳米技术的最新进展来研究血管细胞对力和机械应力的反应。当血管中的细胞受到异常压力时，例如在高血压动脉硬化时，它们会表现出异常的物理和生化反应，这可能会进一步加剧疾病的发展。这项研究将在细胞水平上提供对这些过程的新见解，并将有可能对理解血管疾病做出重大贡献。

DESCRIPTION (provided by applicant): This project focuses on the role that sub-cellular forces experienced at sites of adhesion between cells and their extracellular matrix play in regulating vascular smooth muscle cell organization and function. The adaptive process by which cells spatially resolve and respond to such localized forces is critical to adhesion remodeling, where cells reinforce specific adhesions while disassembling others, and is relevant to many mechanically-mediated disease processes such as the hypertension-induced hyperproliferative response of vascular smooth muscle cells that exacerbates vascular disease. Previous work in this area has shown that focal adhesions assemble in response to forces applied to them, but because adhesion to extracellular matrix also induces biochemical signals that trigger global cell contractility, it remains unclear whether forces applied to a specific adhesion result in any mechanical crosstalk with remaining adhesions distributed throughout the cell. This project will combine the expertise of two investigators to employ a new technique that simultaneously allows local mechanical stimulation of the adherent surface of a cell and spatially-resolved measurement of the local force fields generated throughout the cell in response to this stimulation. It is proposed that the relationship between local stimulation and global mechanical response is critical to the mechanical coordination within the cytoskeleton required for transduction of force into a meaningful response. The two investigators have recently developed a technique wherein deflections of an array of microfabricated posts report the cytoskeletal tension and local force fields generated by a cell attached to the array, and nanoengineered magnetic material embedded in individual posts is used to exerting tunable sub-cellular mechanical stresses to attached cells. Thus, cells can be locally perturbed at one post while the surrounding posts simultaneously measure the effects of this stimulation. Specific Aim 1 of the project will be to apply well- defined forces at the nanonewton level to individual adhesions in cells, and to measure the global response of a cell in terms of changes in contractility and the structure of focal adhesions. Specific Aim 2 will be to determine the role of adhesion signaling in the mechanical response to forces. Specific Aim 3 will be to investigate the role of the changes in cytoskeletal tension in regulating the focal adhesion response to forces. The experiments made possible by this novel technique will lead to new understanding of how mechanical stresses are transduced by cells into an adaptive, coordinated cytoskeletal response, and will open a pathway toward new insights into the mechanisms of mechanotransduction critical for inflammation, proliferation, and tissue development. PUBLIC HEALTH RELEVANCE: This research will apply recent advances in magnetic micro- and nanotechnology to study the response of vascular cells to force and mechanical stress. When cells in blood vessels are subject to abnormal stresses, such as occur in hypertensive arteriosclerosis, they display abnormal physical and biochemical responses that can further the progression of the disease. This research will provide new insight into these processes at the cellular level, and will have the potential to contribute significantly to the understanding of vascular disease.