Subcellular Response to Local Forces

描述（由申请人提供）：本项目重点研究细胞与细胞外基质黏附部位的亚细胞力在调节血管平滑肌细胞组织和功能中的作用。细胞在空间上分解和响应这种局部力的适应性过程对粘连重塑至关重要，细胞在增强特定粘连的同时破坏其他粘连，并且与许多机械介导的疾病过程相关，例如高血压诱导的血管平滑肌细胞的超增殖反应加剧了血管疾病。该领域先前的研究表明，局部黏附会对施加在它们身上的力做出反应，但由于黏附到细胞外基质上也会诱发触发细胞整体收缩的生化信号，因此尚不清楚施加在特定黏附上的力是否会导致与分布在整个细胞中的剩余黏附之间的任何机械串扰。该项目将结合两位研究人员的专业知识，采用一种新技术，同时允许对细胞粘附表面进行局部机械刺激，并对整个细胞响应这种刺激而产生的局部力场进行空间分辨测量。我们提出，局部刺激和整体机械反应之间的关系对于细胞骨架内的机械协调至关重要，这是将力转导成有意义的反应所必需的。两位研究人员最近开发了一种技术，其中微制造桩阵列的偏转报告了附着在阵列上的细胞产生的细胞骨架张力和局部力场，并且嵌入在单个桩中的纳米工程磁性材料用于对附着的细胞施加可调的亚细胞机械应力。因此，细胞可以在一个哨所受到局部干扰，而周围的哨所同时测量这种刺激的效果。该项目的具体目标1将是在纳米牛顿水平上对细胞中的单个粘附应用明确定义的力，并根据收缩性和局部粘附结构的变化来测量细胞的整体反应。具体目标2将是确定粘附信号在机械力响应中的作用。具体目标3将是研究细胞骨架张力的变化在调节焦点粘附反应力中的作用。这项新技术使实验成为可能，将导致对机械应力如何被细胞转导为适应性的、协调的细胞骨架反应的新理解，并将为对炎症、增殖和组织发育至关重要的机械转导机制开辟新的见解。公共卫生相关性：本研究将应用磁微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。当血管细胞受到异常应激时，如高血压动脉硬化，它们会表现出异常的物理和生化反应，从而进一步加剧疾病的进展。这项研究将在细胞水平上对这些过程提供新的见解，并将有可能对血管疾病的理解做出重大贡献。

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.