MICROMECHANICS OF THE EXTRACELLULAR MATRIX

细胞外基质的微观力学

• 批准号: 6832212
• 负责人: Julio M Fernandez
• 金额: $ 41.59万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6832212
• 关键词: CHO cells; atomic force microscopy; biomechanics; conformation; elasticity; extracellular matrix; fibronectins; fluorescence resonance energy transfer; green fluorescent proteins; heparin; molecular assembly /self assembly; molecular dynamics; mucopolysaccharides; nanotechnology; protein engineering; protein folding; recombinant proteins; transfection

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.

描述：（来自申请人的摘要）细胞外基质（ECM）是 机械支架，决定弹性和拉伸强度 并通过控制细胞来精细地调节它们的发育 粘附和迁移。ECM由模块化蛋白质形成， 多糖通过自组装和与 多种细胞类型的细胞粘附受体。机械力作用 在ECM组装和功能中的重要作用。ECM纤维被预先拉伸 四倍于它们静止时的长度， 通过暴露的隐蔽结合位点， 通过机械展开。然而，我们还不知道 分子的机械延展性和机械信号 组成ECM。该提案的长期目标是确定力量 驱动的构象变化，允许ECM分子在一个 并将这种力转化为细胞信号。为了这个目标，我们 将联合收割机结合细胞和分子生物学技术， 现有技术的力光谱（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，我们建议重点关注 纤维连接蛋白和肝素，这些丰富的分子被认为在 在ECM中的机械作用，并在一般动物中具有中枢功能 生理学和病理学。我们将使用力谱来检查 天然纤连蛋白和所选纤连蛋白模块的机械解折叠 已知其在基体组装中起重要的机械作用。我们将 用特异性机械设计的工程重组纤连蛋白 然后将其转染到CHO细胞中用于纤连蛋白的性质 分泌和基质组装。我们将使用新的基于GFP的能量转移 探针，以测量每个分子的静止力，并确定 解折叠发生在体内。我们还将使用力谱来检测力 基质糖胺聚糖中的驱动构象，特别是肝素。我们 将使用GFP探针来检查纤连蛋白模块与肝素的结合 在拉伸力的作用下机械力在电解加工中起着关键作用 装配和功能。拟议的实验将调查，首先， 时间，矩阵力学的分子基础。这些发现可能对 对器官和组织工程以及伤口修复的重要性。