CYTOPLASMIC MYOSIN FUNCTION IN VITRO AND IN VIVO

我们的目标是确定哪些细胞运动需要肌球蛋白才能产生力量 生产并在分子水平上理解 肌球蛋白在体外和体内的功能。我们的方法一直是生产和 鉴定抗胞浆肌球蛋白的单抗为 棘阿米巴肌球蛋白功能的探讨。我们将分析作用力 在无细胞模型系统中用纯化的蛋白质生产肌球蛋白和 通过显微注射在活细胞中。棘阿米巴包含两类 形态截然不同的肌球蛋白。这两个肌球蛋白的作用 细胞运动中的作用尚不清楚。在体外，我们已经并将绘制 抗体-肌球蛋白复合体抗体结合部位的电子显微镜研究 并通过抗体与肌球蛋白多肽结合。我们已经并将分析 抗体与纯化的收缩蛋白相互作用确定 肌球蛋白分子中不同的结构域对肌球蛋白细丝的贡献 肌动蛋白的形成、结合和肌动蛋白激活的ATPase活性。我们有和 将抗体应用于无细胞收缩模型系统。至 到目前为止，我们已经鉴定了23种针对At的单抗 从肌球蛋白II的头部到其顶端至少有15个独特的位点分布 尾巴。2株抗体阻断聚合，12株阻断肌动球蛋白-II ATPase 阿米巴凝胶提取物的活性和14抑制收缩 细胞质。我们的数据显示，肌球蛋白尾端附近的区域是 聚合所需，肌球蛋白尾巴上的另一个结构域， 靠近肌球蛋白头部，对ATPase的活动和力量是必不可少的 制作。最后，我们已经并将从整体上探讨肌球蛋白的功能。 细胞。固定细胞的荧光抗体染色，电子显微镜 抗体染色的冰冻切片和标记的分布 将肌球蛋白显微注射到活细胞中将定位肌球蛋白。多数 值得注意的是，我们将把抗体微量注射到活细胞中 确定哪种运动(运动、胞质分裂等)需要肌球蛋白 功能。我们对微量注射的初步研究感到鼓舞 棘阿米巴，并通过我们的抗体微量注射研究肌球蛋白的功能 在海星卵中，这表明肌球蛋白是细胞质分裂所必需的，但 而不是染色体运动。通过分析抗体对生化的影响 体外收缩功能对肌球蛋白整体定位的影响 细胞，以及活体内的运动性，我们希望阐明分子 肌球蛋白在活细胞中的作用机制。

Our goal is to determine which cellular movements require myosin for force production and to understand, on the molecular level, the mechanisms of myosin function in vitro and in vivo. Our approach has been to produce and characterize monoclonal, site-specific antibodies to cytoplasmic myosin as probes of myosin function in Acanthamoeba. We will analyze force production by myosin with purified proteins, in cell free model systems and in living cells through microinjection. Acanthamoeba contains two classes of myosin that are morphologically distinct. The role these two myosins play in cell movement is not known. In vitro, we have and will map antibody binding sites by electron microscopy of antibody-myosin complexes and by antibody binding to myosin peptides. We have and will analyze the interaction of antibodies with purified contractile proteins to determine how distinct domains in the myosin molecule contribute to myosin filament formation, actin binding and actin-activated ATPase activity. We have and will apply the antibodies to cell free model systems of contractility. To date we have characterized 23 monoclonal antibodies directed against at least 15 unique sites distributed from the myosin-II head to the tip of its tail. Two antibodies block polymerization, 12 block actomyosin-II ATPase activity and 14 inhibit contraction of gelled extracts of amoeba cytoplasm. Our data shows that regions near the tip of the myosin tail are required for polymerization and that another domain on the myosin tail, close to the myosin head, is essential for ATPase activity and force production. Finally, we have and will probe myosin function in whole cells. Fluorescent antibody staining of fixed cells, electron microscopy of antibody stained frozen thin sections, and the distribution of labeled myosin microinjected into living cells will localize myosin. Most significantly, we will microinject the antibodies into living cells to determine which motilities (locomotion, cytokinesis, etc.) require myosin function. We are encouraged by preliminary microinjection studies on Acanthamoeba, and by our antibody-microinjection studies on myosin function in starfish eggs, which showed that myosin is necessary for cytokinesis but not chromosome movement. By analyzing antibody effects on biochemical function, on contractility in vitro, on the location of myosin in whole cells, and on motility in vivo, we expect to elucidate the molecular mechanism of myosin function in living cells.