CYTOPLASMIC MYOSIN FUNCTION IN VIVO AND IN VITRO

体内和体外细胞质肌球蛋白功能

• 批准号: 6199006
• 负责人: DANIEL PETER KIEHART
• 金额: $ 30.32万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6199006
• 关键词: Drosophilidae; arthropod genetics; binding proteins; embryogenesis; gene mutation; genetic screening; molecular cloning; myosins; phosphorylation; protein protein interaction; protein sequence; protein structure function

We hypothesize that the classical genetic and molecular genetic approaches available in Drosophila provide a unique opportunity to investigate the molecular basis of nonmuscle myosin-II. Nonmuscle myosin-II is a highly conserved motor protein that contributes to cytokinesis, the distribution of cell surface receptors, post mitotic cell shape changes for morphogenesis, cellular locomotion and wound healing. Previously, we showed that in fly a single gene, zipper (zip) encodes the nonmuscle myosin-II heavy chain and a single gene, spaghetti squash (sqh) encodes the nonmuscle myosin regulatory light chain. These genes encode polypeptides that are very similar to their mammalian orthologs (the regulatory light chain protein is 81 percent identical and 93 percent similar). Thus, the fly proteins are considerably more similar to their mammalian orthologs than are myosin-IIs from other invertebrate model systems that are amenable to classical genetic or molecular genetic approaches. We have used a variety of methods to establish which cell shape changes require myosin-II function. Here we plan to investigate how myosin functions in living cells by using genetic, molecular, cell biological and protein biochemical approaches to identify proteins that are required for myosin function and then to characterize HOW they function. Our Specific Aims are as follows. 1) We will continue to use simple and powerful F1 screens to recover genes whose products interact with myosin-II to effect cytokinesis and morphogenesis. 2) We will characterize GENETICALLY new myosin alleles and putative interacting loci. 3) We will characterize MOLECULARLY new myosin alleles and putative interacting loci. Finally, 4) we will use a variety of CELL BIOLOGICAL approaches to investigate how these interacting proteins facilitate myosin function. We will evaluate links between a) the molecular defects that characterize myosin mutations; b) the molecular identity of interacting gene products and c) the molecular defects that characterize those alleles of interacting loci that actually modulate the ability of the myosin to perform its functions. Our strategy will be to investigate alterations in movements, the structure of the actin cytoskeleton and the localization of myosin in appropriate mutant animals. We will characterize these phenotypes of various myosin mutation homozygotes, interactor mutation homozygotes, myosin/interactor double homozygotes and if instructive, various other potential combinations. Our goal is to ascertain the basis of myosin function at the cellular and molecular level.

我们假设，经典的遗传学和分子遗传学方法在果蝇提供了一个独特的机会，调查非肌肉肌球蛋白II的分子基础。非肌肉肌球蛋白-II是一种高度保守的运动蛋白，其有助于胞质分裂、细胞表面受体的分布、有丝分裂后细胞形态发生的变化、细胞运动和伤口愈合。 以前，我们发现，在飞一个单一的基因，拉链（zip）编码的非肌肉肌球蛋白-II重链和一个单一的基因，意大利南瓜（sqh）编码的非肌肉肌球蛋白调节轻链。 这些基因编码的多肽与它们的哺乳动物直系同源物非常相似（调节轻链蛋白有81%的相同性和93%的相似性）。 因此，苍蝇蛋白是相当多的类似于他们的哺乳动物直系同源物比肌球蛋白II类从其他无脊椎动物模型系统，是服从经典的遗传或分子遗传学的方法。我们已经使用了多种方法来确定哪些细胞形状的变化需要肌球蛋白II的功能。 在这里，我们计划研究肌球蛋白如何在活细胞中的功能，通过使用遗传，分子，细胞生物学和蛋白质生化方法来识别肌球蛋白功能所需的蛋白质，然后描述它们如何发挥作用。 我们的具体目标如下。1)我们将继续使用简单而强大的F1筛选来恢复其产物与肌球蛋白-II相互作用以影响胞质分裂和形态发生的基因。 2)我们将从遗传学上描述新的肌球蛋白等位基因和假定的相互作用位点。 3)我们将在分子水平上描述新的肌球蛋白等位基因和假定的相互作用位点。 最后，4）我们将使用各种细胞生物学方法来研究这些相互作用的蛋白质如何促进肌球蛋白功能。 我们将评估a）肌球蛋白突变特征的分子缺陷; B）相互作用基因产物的分子特性; c）实际调节肌球蛋白执行其功能的能力的相互作用位点的等位基因特征的分子缺陷之间的联系。 我们的策略将是调查运动的改变，肌动蛋白细胞骨架的结构和肌球蛋白在适当的突变动物的本地化。 我们将描述这些表型的各种肌球蛋白突变纯合子，相互作用突变纯合子，肌球蛋白/相互作用双纯合子，如果有指导意义，各种其他潜在的组合。 我们的目标是在细胞和分子水平上确定肌球蛋白功能的基础。