CYTOPLASMIC MYOSIN FUNCTION IN VITRO AND IN VIVO

体外和体内细胞质肌球蛋白功能

基本信息

批准号：
3283906
负责人：
DANIEL PETER KIEHART
金额：
$ 30.7万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-09-30 至 1996-08-31
项目状态：
已结题

项目摘要

Our goals are to examine the biological processes that require myosin function , identify the components and architecture of the supramolecular complex that contributes to force generation for cell shape change and investigate the molecular mechanism by which myosin functions. We focus on the function of nonmuscle (cytoplasmic) myosin in Drosophila for two reasons. First, a diverse array of interesting movements provide the structural basis for spatial differentiation of the embryo and are characteristic of various movements seen during the development of other species throughout phylogeny. They include nuclear divisions and migrations, pole cell formation and cellularization, the complex cellular migrations and shape changes of gastrulation and later stages of embryo, larval and pupal morphogenesis. Second, these movements are amenable to study by a range of powerful approaches. We used classical protein biochemical and immunological methods to purify and characterize cytoplasmic myosin from Drosophila cells in culture, then showed that in developing embryos that myosin is localized in a pattern consistent with its role in cellularization and cell sheet movements. The real advantage of Drosophila is its accessibility to genetic, molecular biological and modem molecular genetic manipulation, so we cloned the genes that encode its 3 polypeptide subunits. We used reverse genetic methods to recover mutations in the heavy chain and have collaborated on a mutation in the regulatory light chain. The work establishes that the myosin is required for cell shape change, both in cell sheet morphogenesis during development and for cytokinesis. The experiments provide the first link between a known chemomechanical force producing protein, or motor, and morphogenesis and further suggests that cell shape changes for cytokinesis and morphogenesis are intimately and mechanistically related. This proposal requests funds to continue a multidisciplinary analysis of the nonmuscle myosin heavy chain encoded by the zipper locus at 6OE9 and a new myosin heavy chain gene at 35B,C. Our emphasis will be on genetic strategies designed to establish which movements that require these myosins for their proper execution and cell biological approaches to ascertaining the molecular mechanism by which myosin is targeted to a discrete domain of the submembraneous actin cortex, how its activity is regulated and how it contributes to cell shape change during development. Together these studies forge a comprehensive investigation of the mechanism of conventional nonmuscle myosin function and its role in cellular movements.

我们的目标是检查需要的生物过程肌球蛋白功能，确定组件和体系结构超分子复合物有助于产生强制细胞形状改变并研究分子机制肌球蛋白功能。我们专注于非肌肉的功能果蝇中（细胞质）肌球蛋白有两个原因。首先，a 各种有趣的运动提供了结构性基础对于胚胎的空间分化，是特征在其他物种开发过程中看到的各种运动整个系统发育。它们包括核部门和迁移，极细胞的形成和细胞化，复合物细胞迁移和胃结构的形状变化，以后胚胎，幼虫和脓疱形态发生的阶段。第二，这些运动可以通过一系列强大的方法进行研究。我们使用经典蛋白质生化和免疫学方法从果蝇细胞中纯化和表征细胞质肌球蛋白在文化中，然后表明在发展胚胎时表明肌球蛋白是以与其在细胞化中作用一致的模式定位和细胞表的运动。果蝇的真正优势是遗传，分子生物学和现代分子的可及性基因操纵，因此我们克隆了编码其3的基因多肽亚基。我们使用反向遗传方法恢复重链中的突变，并进行了突变在监管轻链中。工作确定了肌球蛋白是细胞形状变化所必需的，两者都在细胞片中发育和细胞因子过程中的形态发生。这实验提供了已知的化学力学之间的第一个联系产生蛋白质或运动和形态发生的力以及进一步表明细胞形状变化的细胞因子和形态发生密切相关。该建议要求资金继续对非肌肉进行多学科分析肌球蛋白重链由6oe9的拉链基因座编码和新的肌球蛋白重链基因在35b，c。我们的重点将放在遗传上旨在确定需要这些运动的策略肌球蛋白的适当执行和细胞生物学方法确定肌球蛋白靶向的分子机制到肿瘤肌动蛋白皮质的离散域，其如何调节活动及其如何促进细胞形状变化在开发过程中。这些研究共同创造了一个全面的调查常规非肌肉肌球蛋白的机制功能及其在细胞运动中的作用。