MYOSIN MOVEMENT IN VITRO--PHYSIOLOGICAL CHARACTER

肌球蛋白的体外运动——生理特征

• 批准号: 2391991
• 负责人: MICHAEL Patrick SHEETZ
• 金额: $ 23.3万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2391991
• 关键词: 3T3 cells; CHO cells; binding proteins; cell adhesion; cell migration; crosslink; cytoskeleton; extracellular matrix; fibronectins; fluorescence; integrins; lasers; myosins; phosphorylation; receptor binding; tissue /cell culture; western blottings

Cell migration requires the reversible linkage between the force- generating cytoskeleton and the extracellular matrix through the integrins. The integrins form a large family of transmembrane glycoproteins which bind matrix ligands and the cytoskeleton which are pivotal for cell migration. We propose to study the molecular and physical basis for fibronectin binding and linkage to the cytoskeleton through the alpha5beta1 integrin. Using single particle tracking (SPT) and the laser tweezers, we propose to measure the time course of ligand-receptor linkage to the cytoskeleton and force generation on attached ligand. Initially, we will determine the dependence of cell region, integrin tail sequence, force, ligand aggregate size and valency on the attachment of integrin, alpha5beta1 to the cytoskeleton in 3T3 and CHO cells. Because force generation is important for migration we will follow the course of force generation by the cell on dorsally applied beads with the laser tweezers and on ventral contacts with a newly designed force-measuring chip. From biochemical studies, integrins are recruited laterally into ligand contact regions. We will determine if that recruitment is active or passive and how it depends on contact size, force, ligand density and intracellular signaling. As the cell migrates past contacts integrins must release from matrix ligands in a time or regionally dependent manner. Our preliminary evidence suggests that integrin release from both ligand and cytoskeleton is regionally dependent. On the basis of these findings we hope to understand the molecular and physical basis of fibronectin-integrin- cytoskeleton complexes in migrating cells. In the future, we can hope to better understand which steps in cell migration are modified in chemotaxis, pathfinding and with regard to alteration of specific protein function.

细胞迁移需要力之间的可逆联系 - 通过 整合素。整合素形成了大型跨膜家族 结合基质配体和细胞骨架的糖蛋白是 细胞迁移的关键。我们建议研究分子和物理 通过纤连蛋白结合并与细胞骨架连接的基础 alpha5beta1整合素。使用单个粒子跟踪（SPT）和激光器 镊子，我们建议测量配体链接的时间过程 到附着配体上的细胞骨架和力产生。最初，我们 将确定细胞区域的依赖性，整联蛋白尾序， 整合素的附着的力，配体骨料大小和价值 3T3和CHO细胞中的alpha5beta1至细胞骨架。因为力 一代对于迁移很重要，我们将遵循力量 用激光镊子在背侧施加的珠子上通过细胞产生 并与新设计的力测量芯片进行腹侧接触。 从 生化研究，整联蛋白横向募集到配体接触中 地区。我们将确定该招聘是活跃还是被动的，并且 它如何取决于接触大小，力，配体密度和细胞内 信号。当细胞迁移过去的触点时，整联蛋白必须从 矩阵配体以时间或区域依赖的方式。 我们的初步 有证据表明整联蛋白都从配体和细胞骨架上释放 是区域依赖的。根据这些发现，我们希望 了解纤连蛋白 - 整合素的分子和物理基础 - 迁移细胞中的细胞骨架复合物。将来，我们可以希望 更好地了解细胞迁移中的哪些步骤已修改 趋化性，探路和特异性蛋白质的改变 功能。