Regulation of Protozoan Myosin-I Function

这项建议的目标是鉴定和表征调节膜结合的马达分子肌球蛋白-I与磷脂表面(如细胞膜)相互作用的蛋白质。肌球蛋白-I是已知的唯一使用脂质表面作为运动底物的运动分子，由于细胞运动伴随着细胞前沿的运动事件，肌球蛋白-I在细胞前沿聚集，因此肌球蛋白-I可能是驱动细胞运动的候选分子。为了寻找肌球蛋白-I的调节蛋白，我们将用三种不同的体外测定肌球蛋白-I活性的方法来筛选刺五加阿米巴提取物：(1)直接结合试验，以确定调节蛋白对肌球蛋白-I与膜结合的影响；(2)，ATPase活性，以评价调节蛋白对肌球蛋白-I酶活性的影响；以及(3)运动性试验，观察调节蛋白对肌球蛋白I移动肌动蛋白细丝能力的影响。在这些检测中鉴定出调控蛋白后，将使用标准的生化程序纯化特定的蛋白，并产生针对每种蛋白的多克隆抗体。这些抗体将用于上述检测，以测试它们对调节蛋白活性的影响，并通过免疫染色在细胞中定位调节蛋白。这些实验将提供关于肌球蛋白-I如何在细胞膜上组织以及肌球蛋白-I的活动如何被调节的重要信息。细胞运动是单细胞生物生命过程的基础，也是高等生物许多生理活动所必需的，如发育、神经元生长、血管生成、防御机制、伤口愈合和生物运动。细胞运动需要膜和细胞骨架系统的协调。由于其与脂质结合的特性，细胞骨架运动蛋白Myosin-I参与了许多膜介导的运动过程。然而，与其他马达蛋白(如肌球蛋白-II、动蛋白或动力蛋白)不同的是，到目前为止还不可能证明肌球蛋白-I是某些普遍的细胞过程所必需的。肌球蛋白-I的作用问题可能不是仅仅通过研究肌球蛋白-I就能轻易解决的，因为似乎有多个基因编码一系列功能重叠的肌球蛋白-I分子。这项研究的结果将有助于理解肌球蛋白-I在细胞运动中的作用。

The goal of this proposal is to identify and characterize proteins that reguate the interaction of the membrane-bound motor molecule, myosin-I, with phospholipid surfaces such as cell membranes. Myosin-I is the only motor molecule known to use a lipid surface as a substrate for motility, and because cellular movement is accompanied by motile events at the leading edge of the cell where myosin-I is concentrated, myosin-I is a likely candidate for driving cell movement. To search for proteins that regulate myosin-I, extracts from Acanthamoeba castellanii will be screened with three in vitro assays that measure different activities of myosin-I: (1), a direct binding assay, to determine the effect of regulatory proteins on myosin-I's association with membranes; (2), ATPase activity to evaluate the effect of regulatory proteins on myosin-I's enzyme activity; and (3), a motility assay to observe the effect of regulatory proteins on myosin-I's ability to move actin filaments. After regulatory proteins have been identified in these assays, specific proteins will be purified using standard biochemical procedures and polyclonalantibodies to each of these proteins will be produced. These antibodies will be used in the above assays to test their effect on regulatory protein activities and in cells to localize the regulatory proteins by immunostaining. These experiments will provide important information on how myosin-I is organized at the cell membrane and how myosin-I's activity is regulated. %%% Cellular motility is fundamental to the life processes of unicellular organisms and essential to many physiological activities of higher organisms, such as development, neuronal growth, angiogenesis, defense mechanisms, wound healing, and organismal locomotion. Cellular movement requires the coordination of membrane and cytoskeletal systems. As a result of its lipid- binding properties, the cytoskeletal motor protein myosin-I has been implicated in many membrane-mediated motile processes. However, unlike other motor proteins (e.g., myosin-II, kinesin, or dynein) it has not been possible thus far to show that mysoin-I is required for some generalized cellular process. The problem of what myosin-I does may not be easily solved solely by studying mysoin-I, since it appears likely that multiple genes encode a family of myosin-I molecules with overlapping functions. The results of this study will contribute to the understanding of myosin-I's role in cell motility.