MYOSIN MOVEMENT IN VITRO--PHYSIOLOGICAL CHARACTER

肌球蛋白的体外运动——生理特征

• 批准号: 2684815
• 负责人: MICHAEL Patrick SHEETZ
• 金额: $ 23.98万
• 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/2684815
• 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 通过生成细胞骨架和细胞外基质 整合素。整合素形成跨膜大家族 结合基质配体和细胞骨架的糖蛋白 对于细胞迁移至关重要。我们建议研究分子和物理 纤连蛋白通过以下途径与细胞骨架结合和连接的基础 α5β1 整合素。使用单粒子跟踪 (SPT) 和激光 镊子，我们建议测量配体-受体连接的时间过程 细胞骨架和附着配体上的力产生。最初，我们 将确定细胞区域、整合素尾序列的依赖性， 整合素附着上的力、配体聚集体大小和化合价， 3T3 和 CHO 细胞中细胞骨架的 alpha5beta1。因为力 世代对移民很重要，我们将遵循武力路线 用激光镊子在背侧应用的珠子上产生细胞 以及与新设计的测力芯片的腹侧接触。 从 生化研究，整合素被横向招募到配体接触中 地区。我们将确定该招聘是主动还是被动，并且 它如何取决于接触尺寸、力、配体密度和细胞内 发信号。当细胞迁移经过接触点时，整合素必须从 基质配体以时间或区域依赖的方式。 我们的初步 有证据表明整合素从配体和细胞骨架中释放 具有地域依赖性。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。未来，我们可以希望 更好地了解细胞迁移的哪些步骤被修改 趋化性、寻路以及特定蛋白质的改变 功能。