CYTOPLASMIC MYOSIN FUNCTION IN VIVO AND IN VITRO

我们假设，在果蝇中可用的经典遗传学和分子遗传学方法为研究非肌肉肌球蛋白II的分子基础提供了一个独特的机会。非肌肉肌球蛋白-II是一种高度保守的运动蛋白，参与胞质分裂、细胞表面受体的分布、有丝分裂后细胞形态发生的改变、细胞运动和伤口愈合。此前，我们已经证明在果蝇中，编码非肌肉肌球蛋白II重链的单个基因zipper(Zipper)和编码非肌肉肌球蛋白调节轻链的单个基因意大利面南瓜(Sqh)。这些基因编码的多肽与它们的哺乳动物同源基因非常相似(调节轻链蛋白有81%相同，93%相似)。因此，苍蝇蛋白与它们的哺乳动物同源物比来自其他无脊椎动物模型系统的肌球蛋白-II更相似，这些无脊椎动物模型系统服从于经典的遗传学或分子遗传学方法。我们使用了各种方法来确定哪些细胞形状的改变需要肌球蛋白II的功能。在这里，我们计划通过使用遗传、分子、细胞生物学和蛋白质生化方法来研究肌球蛋白在活细胞中的功能，以确定肌球蛋白功能所需的蛋白质，然后表征它们的功能。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相互作用的基因，以影响胞质分裂和形态发生。2)我们将研究新的肌球蛋白等位基因和可能的相互作用基因。3)我们将鉴定新的肌球蛋白等位基因和可能的相互作用基因。最后，我们将使用多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的功能。我们将评估a)表征肌球蛋白突变的分子缺陷；b)相互作用的基因产物的分子同一性；以及c)表征实际上调节肌球蛋白执行其功能的能力的相互作用基因座的等位基因的分子缺陷之间的联系。我们的策略将是研究运动的变化，肌动蛋白细胞骨架的结构和肌球蛋白在适当突变动物中的定位。我们将描述不同肌球蛋白突变纯合子、交互作用子突变纯合子、肌球蛋白/交互作用子双重纯合子的这些表型，如果有启发的话，还有其他各种可能的组合。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。

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.