Regulation of Protozoan Myosin-I Function

本提案的目标是确定和描述 调节膜结合马达相互作用的蛋白质 分子，肌球蛋白-I，具有磷脂表面，如细胞 膜。 肌球蛋白-I是已知的唯一一种使用 脂质表面作为运动的底物，并且因为细胞 运动是伴随着运动的事件在前沿的 肌球蛋白-I是一个可能的候选者 来驱动细胞运动。 为了寻找能够调节 肌球蛋白-I，卡氏阿米巴提取物， 用三种体外试验测量不同的活性， 肌球蛋白-I：（1），直接结合试验，以确定 肌球蛋白-I与膜结合的调节蛋白;（2）， ATP酶活性，以评估调节蛋白对细胞的影响。 肌球蛋白-I的酶活性;和（3），运动测定，以观察 调节蛋白对肌球蛋白-I运动能力的影响 肌动蛋白丝 在调节蛋白被发现后， 在这些测定中，将使用标准的 生化程序和多克隆抗体 蛋白质将被生产出来。 这些抗体将用于 上述测定以测试它们对调节蛋白活性的影响 并在细胞中通过免疫染色定位调节蛋白。 这些实验将提供重要的信息， 肌球蛋白-I是在细胞膜上组织的， 活动受到管制。 %%% 细胞运动是生命过程的基础， 单细胞生物和许多生理必需的 高等生物的活动，例如发育、神经元 生长、血管生成、防御机制、伤口愈合，以及 有机体运动 细胞运动需要协调 细胞膜和细胞骨架系统。 由于它的脂质- 结合特性，细胞骨架运动蛋白肌球蛋白-I具有 与许多膜介导的运动过程有关。 然而，与其他马达蛋白（例如，肌球蛋白-II、驱动蛋白或 到目前为止，还不可能证明mysoin-I是 是一些广义细胞过程所必需的。 的问题 肌球蛋白-I的作用仅仅通过研究 mysoin-I，因为它似乎很可能是多个基因编码的一个 具有重叠功能的肌球蛋白-I分子家族。 的 这项研究的结果将有助于了解 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.