CYTOPLASMIC MYOSIN FUNCTION IN VITRO AND IN VIVO

我们的目标是研究需要 肌球蛋白功能，确定肌球蛋白的组件和架构 有助于产生能量的超分子络合物 细胞形态改变及其分子机制的研究 肌球蛋白起作用。我们关注的是非肌肉的功能 果蝇体内的(细胞质)肌球蛋白有两个原因。首先，一个 各种有趣的运动提供了结构基础 对于胚胎的空间分化是有特征的 在其他物种的发展过程中看到的各种运动 在整个系统发展史中。它们包括核部门和 迁移，极细胞形成和细胞化，复合体 原肠胚及以后的细胞迁移和形态变化 胚胎、幼虫和蛹的形态发生阶段。第二，这些 运动可以通过一系列强有力的方法来研究。 我们使用了经典的蛋白质生化和免疫学方法 果蝇细胞胞浆肌球蛋白的纯化与鉴定 在培养中，然后表明在发育中的胚胎中，肌球蛋白是 以与其在细胞化中的作用一致的模式定位 和单元格的移动。果蝇的真正优势是它 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恢复了 重链的突变，并共同研究了一种突变 在监管轻链中。这项工作确立了 肌球蛋白是细胞形状改变所必需的，两者都存在于细胞片中 发育过程中的形态发生和胞质分裂。这个 实验提供了已知的化学机械之间的第一个联系 产生蛋白质或运动的力量，以及形态发生和进一步 提示为了胞质分裂和形态发生，细胞形状发生了变化 在机制上是密切相关的。本建议书要求 继续对非肌肉组织进行多学科分析的资金 6OE9位拉链基因编码的肌球蛋白重链和一个新的 肌球蛋白重链基因35B，C，我们的重点将放在遗传上 旨在确定哪些运动需要这些 肌球蛋白的正确执行和细胞生物学方法 以确定肌球蛋白作为靶点的分子机制 膜下肌动蛋白皮质的离散区域，它是如何 活动受到调节，以及它如何影响细胞形状的变化 在开发过程中。这些研究结合在一起形成了一个全面的 常规非肌肉肌球蛋白作用机制的研究 功能及其在细胞运动中的作用。

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.