MICROMECHANICS OF THE EXTRACELLULAR MATRIX

细胞外基质的微观力学

• 批准号: 6642113
• 负责人: Julio M Fernandez
• 金额: $ 39.66万
• 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/6642113
• 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 决定弹性和拉伸强度的机械支架 通过控制细胞来精细调节器官和组织的发育 粘附和迁移。 ECM 由模块化蛋白质和 多糖通过自组装和相互作用而编织在一起 多种细胞类型的细胞粘附受体。机械力的发挥 在 ECM 组装和功能中发挥重要作用。 ECM原纤维被预拉伸 其静止长度的四倍，被认为可以转化为机械 通过暴露的神秘结合位点强制进入生物信号 通过机械展开。然而，人们对它的分子基础一无所知。 分子的机械延伸性和机械信号 组成 ECM。该提案的长期目标是确定力量 驱动构象变化，使 ECM 分子在 施加力并将该力转化为细胞信号。为了这个目标我们 将细胞和分子生物学技术与状态结合起来 Art Force 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。在我们的第一个资助期内，我们建议重点关注 纤连蛋白和肝素，丰富的分子被认为发挥着至关重要的作用 在 ECM 中发挥机械作用，并在一般动物中具有核心功能 生理学和病理学。我们将使用力谱来检查 天然纤连蛋白和选定纤连蛋白模块的机械展开 已知它们在矩阵组装中发挥重要的机械作用。我们将 工程重组纤连蛋白通过特定的机械设计 然后将其转染至 CHO 细胞中以获得纤连蛋白的特性 分泌和基质组装。我们将使用基于 GFP 的新型能量转移 探针以测量每个分子的静止力并确定是否 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（特别是肝素）中的驱动构象。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 ECM 中发挥着关键作用 组装和功能。拟议的实验将首先调查 时间，矩阵力学的分子基础。研究结果可能意义重大 对于器官和组织工程以及伤口修复具有重要意义。