MICROMECHANICS OF THE EXTRACELLULAR MATRIX

描述：(摘自申请人摘要)细胞外基质(ECM)是 一种机械脚手架，它决定了 器官和组织，并通过控制细胞精细地调节它们的发育 黏附和迁移。细胞外基质是由模块化蛋白质和 通过自组装和通过与之相互作用而编织在一起的多糖 多种细胞类型的细胞黏附受体。机械力发挥作用 在ECM组装和功能中的重要作用。ECM纤维被预拉伸起来 到它们静止长度的四倍，被认为是将机械 通过暴露的隐秘结合部位转化为生物信号 通过机械展开。然而，我们对其分子基础一无所知。 分子的机械延伸性和机械信号 组成ECM。这项提议的长期目标是确定部队 驱动的构象变化，允许ECM分子在 施加的力量，并将这种力量转化为细胞信号。为了实现这一目标，我们 将细胞和分子生物学技术与国家 ART力谱(AFM)技术和基于GFP的荧光 成像技术，能够观察力驱动的构象变化 单分子。在我们的第一个赠款期间，我们建议将重点放在 纤维连接蛋白和肝素，这两种丰富的分子被认为起着至关重要的作用 机械在ECM中的作用，并在一般动物中起中心作用 生理学和病理学。我们将使用力谱学来研究 天然纤维连接蛋白和部分纤维连接蛋白模块的机械展开 它们在基质组装中起着重要的机械作用。我们会 利用特定机械设计工程重组纤维连接蛋白 然后将其导入CHO细胞以获得纤维连接蛋白 分泌和基质组装。我们将使用新的基于绿色荧光蛋白的能量转移 以测量每个分子的静止力，并确定 展开发生在活体内。我们还将使用力光谱来检测力 基质糖胺多聚糖，特别是肝素的驱动构象。我们 将使用GFP探针检查纤维连接蛋白模块与肝素的结合 在拉伸力的作用下。机械力在ECM中起着至关重要的作用 装配和功能。拟议中的实验将首次进行调查 时间，矩阵力学的分子基础。这些发现可能有很大的意义 对器官和组织工程以及伤口修复的重要性。

Description: (From the applicant's abstract) The extracellular matrix (ECM) is the mechanical scaffold that determines the elasticity and tensile strength of organs and tissues and finely regulates their development by controlling cell adhesion and migration. The ECM is formed by modular proteins and polysaccharides knitted together by self-assembly and through interactions with the cell adhesion receptors of a variety of cell types. Mechanical forces play important roles in ECM assembly and function. ECM fibrils are pre-stretched up to four times their resting length and are thought to translate mechanical forces into biological signals through cryptic binding sites that are exposed by mechanical unfolding. However, nothing is known about the molecular basis of the mechanical extensibility and mechanical signaling of the molecules composing the ECM. The long term aim of this proposal is to determine the force driven conformational changes that allow the ECM molecules to extend under an applied force and turn this force into a cellular signal. Towards this aim we will combine cellular and molecular biological techniques together with state of the art force spectroscopy (AFM) techniques and GFP based fluorescence imaging techniques, capable of observing force driven conformational changes in single molecules. During our first grant period we propose to focus on fibronectin and heparin, abundant molecules which are thought to play crucial mechanical roles in the ECM and have a central function in general animal physiology and pathology. We will use force spectroscopy to examine the mechanical unfolding of native fibronectin and of selected fibronectin modules that are known to play important mechanical roles in matrix assembly. We will engineer recombinant fibronectin proteins designed with specific mechanical properties that then will be transfected into CHO cells for fibronectin secretion and matrix assembly. We will use novel GFP based energy transfer probes in order to measure the resting force per molecule and to determine if unfolding occurs in vivo. We will also use force spectroscopy to detect force driven conformations in matrix glycosaminoglycans, in particular of heparin. We will use GFP probes to examine the binding of fibronectin modules to heparin under a stretching force. Mechanical forces play a critical role in ECM assembly and function. The proposed experiments will investigate, for the first time, the molecular basis of matrix mechanics. The findings may be of great importance for organ and tissue engineering and wound repair.