MICROMECHANICS OF THE EXTRACELLULAR MATRIX

细胞外基质的微观力学

• 批准号: 6694409
• 负责人: Julio M Fernandez
• 金额: $ 40.61万
• 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/6694409
• 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：（从申请人的摘要中）细胞外矩阵（ECM）是 确定弹性和拉伸强度的机械支架 器官和组织，通过控制细胞来细节调节其发育 粘附和迁移。 ECM由模块化蛋白和 多糖通过自组装和与 各种细胞类型的细胞粘附受体。机械力发挥 在ECM组装和功能中的重要作用。 ECM原纤维被预割伤 到它们的休息长度的四倍，被认为可以翻译机械 通过暴露的神秘结合位点进入生物信号的力 通过机械展开。但是，关于分子基础一无所知 分子的机械可扩展性和机械信号传导 组成ECM。该提议的长期目的是确定力量 驱动的构象变化使ECM分子在 施加力并将这种力变成细胞信号。朝向这个目标 将结合细胞和分子生物学技术与状态 艺术力光谱（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，我们建议专注于 纤连蛋白和肝素，丰富的分子被认为发挥着重要的作用 ECM中的机械作用，并具有一般动物的中心功能 生理和病理学。我们将使用力光谱检查 天然纤连蛋白和选定纤连蛋白模块的机械展开 已知在矩阵组件中扮演重要的机械角色。我们将 工程师重组纤连蛋白蛋白设计的特定机械 然后将其转染到纤连蛋白的CHO细胞中的特性 分泌和矩阵组件。我们将使用新型基于GFP的能量转移 探针以测量每个分子的静息力并确定是否是否 展开发生在体内。我们还将使用力光谱检测力 基质糖胺聚糖，特别是肝素的驱动构象。我们 将使用GFP探针检查纤连蛋白模块与肝素的结合 在拉伸力下。机械力在ECM中起关键作用 组装和功能。提出的实验将研究第一个 时间，基质力学的分子基础。发现可能很棒 对器官和组织工程和伤口修复的重要性。