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，识别肌球蛋白的成分和结构有助于产生力的超分子复合物细胞形状变化并研究其分子机制肌球蛋白的功能。我们关注非肌肉的功能果蝇中的（细胞质）肌球蛋白有两个原因。首先，一个各种有趣的动作提供了结构基础用于胚胎的空间分化并且具有特征其他物种发育过程中看到的各种运动整个系统发育。它们包括核分裂和迁移、极细胞形成和细胞化，复杂的原肠胚形成及之后的细胞迁移和形状变化胚胎、幼虫和蛹形态发生的阶段。其次，这些运动可以通过一系列强有力的方法来研究。我们使用经典的蛋白质生化和免疫学方法纯化和表征果蝇细胞的细胞质肌球蛋白在培养物中，然后表明在发育中的胚胎中，肌球蛋白是定位于与其在细胞化中的作用一致的模式和细胞片运动。果蝇的真正优势在于它遗传、分子生物学和现代分子的可及性基因操作，所以我们克隆了编码其 3 的基因多肽亚基。我们使用反向遗传方法来恢复重链突变并合作突变在监管轻链中。这项工作确定了肌球蛋白是细胞形状改变所必需的，无论是在细胞片中发育过程中的形态发生和胞质分裂。这实验提供了已知化学力学之间的第一个联系力产生蛋白质或运动，以及形态发生以及进一步表明细胞形状因胞质分裂和形态发生而变化之间有着密切的、机制上的联系。本提案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 处的拉链基因座和一个新的编码 35B、C处的肌球蛋白重链基因。我们的重点是遗传旨在确定哪些运动需要这些的策略肌球蛋白的正确执行和细胞生物学方法确定肌球蛋白靶向的分子机制对于膜下肌动蛋白皮层的离散域，它是如何活动受到调节以及它如何促进细胞形状变化在开发过程中。这些研究共同形成了一个全面的常规非肌肉肌球蛋白机制的研究功能及其在细胞运动中的作用。