Subcellular Response to Local Forces

描述（由申请人提供）：该项目的重点是在细胞与其细胞外基质之间的粘附部位所经历的亚细胞力在调节血管平滑肌细胞组织和功能中的作用。细胞在空间上分辨和响应这种局部力的适应性过程对于粘附重塑是至关重要的，其中细胞在分解其他粘附的同时加强特定的粘附，并且与许多机械介导的疾病过程相关，例如加剧血管疾病的高血压诱导的血管平滑肌细胞的过度增殖反应。在这一领域的先前工作已经表明，粘着斑组装响应于施加到它们的力，但由于粘附到细胞外基质也诱导生化信号，触发全球细胞收缩性，它仍然不清楚是否施加到一个特定的粘附力的结果与分布在整个细胞的剩余的粘附任何机械串扰。该项目将联合收割机的专业知识的两名研究人员采用一种新的技术，同时允许局部机械刺激的粘附表面的细胞和空间分辨测量的局部力场产生的整个细胞在响应于这种刺激。有人提出，局部刺激和全球的机械响应之间的关系是至关重要的细胞骨架内的力转换成有意义的反应所需的机械协调。这两位研究人员最近开发了一种技术，其中微制造柱阵列的偏转报告了细胞骨架张力和由附着在阵列上的细胞产生的局部力场，并且嵌入在单个柱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，细胞可以在一个柱处局部扰动，而周围的柱同时测量该刺激的效果。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连施加明确的力，并测量细胞在收缩性和局灶性粘连结构变化方面的整体反应。具体目标2将是确定粘附信号在力的机械响应中的作用。具体目标3将是研究细胞骨架张力的变化在调节粘着斑对力的反应中的作用。通过这种新技术进行的实验将导致对细胞如何将机械应力转换为适应性，协调的细胞骨架反应的新理解，并将开辟一条通往对炎症，增殖和组织发育至关重要的机械转导机制的新见解的途径。公共卫生相关性：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，例如发生在高血压动脉硬化中，它们显示出异常的物理和生化反应，这可以进一步促进疾病的进展。这项研究将在细胞水平上为这些过程提供新的见解，并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.