ROLE OF ACTIN-BASED MOTORS IN ORGANELLE MOTILITY

一个新的小肌球蛋白基因abmA已被发现， 网骨藻 abmA基因编码一种蛋白质， 连接到带正电荷的尾部区域，该区域类似于 阿米巴肌球蛋白I膜结合域。 缺乏abmA的细胞 基因产物在单细胞水平上进行了检测， 结果显示，这些突变体显著降低了 细胞内颗粒运动。 本授权书中描述的实验 建议是针对进一步解剖abmA在细胞中的作用， 运动，特别是细胞器运输，和生化 表征abmA蛋白。 网骨藻是一种 对于此类研究，因为它允许研究者采用多方面的 方法，将分子遗传学实验与传统的 蛋白质生物化学和细胞生物学来剖析ABMS在 细胞器运输或其他形式的细胞运动。 第一步 将是abmA特异性抗体的产生和 应用免疫荧光技术对网骨藻中abmA蛋白的表达进行了研究。 突变体和 将野生型abmA基因导入abmA-细胞， 细胞内颗粒运动的评估。 这些突变将包括 调节磷酸化位点的改变（这将影响 分子的功能）或尾部区域的变化（其将 影响蛋白质的细胞内定位）。 二是 本研究拟建立一套从网骨藻中纯化abmA蛋白的方法 并测定abmA蛋白的肌球蛋白样性质。 细胞 将产生过表达野生型abmA蛋白的细胞系， 有助于abmA蛋白的纯化。 最后一系列 实验将解决abmA与细胞间的相互作用， 颗粒或其他膜状元素。 囊泡将从 网骨藻及其沿沿着定向丽藻的移位能力 将检查肌动蛋白电缆。 肌动蛋白为基础的马达的身份 附着在这些囊泡或膜元件上的分子量将由 免疫印迹实验和囊泡纯化的组合 电机 更多的实验将集中在如何识别ABM是 附着在囊泡或膜元件上，无论是通过直接 通过静电相互作用或如果存在 存在于膜中的abmA受体蛋白 以上所述的目的是明确地理解在体内的作用， abmA蛋白，但它也广泛适用于研究其他 参与细胞器运输的肌动蛋白基马达。

A novel small myosin gene, abmA, has been identified in Dictyostelium. The abmA gene encodes a protein that has a myosin head attached to a positively charged tail region that is similar to the Acanthamoeba myosin I membrane binding domain. Cells devoid of the abmA gene product were examined at the single cell level and preliminary results revealed that these mutants have significantly reduced rates of intracellular particle movement. The experiments described in this grant proposal are directed at further dissecting the role of abmA in cellular motility, specifically organelle transport, and biochemically characterizing the abmA protein. Dictyostelium is the organism of choice for such studies as it allows the investigator to employ a multi-faceted approach, combining molecular genetic experiments with traditional protein biochemistry and cell biology to dissect the role of abms in organelle transport or other forms of cellular motility. The first step will be the generation of abmA-specific antibodies and localization of the abmA protein in Dictyostelium using immunofluorescence. Mutant and wild-type abmA genes will be introduced into abmA- cells and the recovery of intracellular particle movement assessed. The mutations will include alteration of the regulatory phosphorylation site (which would affect the function of the molecule) or changes in the tail region (which would influence the intracellular localization of the protein). Secondly, a protocol will be developed to purify the abmA protein from Dictyostelium and the myosin-like properties of the abmA protein determined. A cell line that overexpresses the wild-type abmA protein will be generated to assist in the purification of the abmA protein. The final series of experiments will address the interaction of abmA with intercellular particles or other membranous elements. Vesicles will be isolated from Dictyostelium and their ability to be translocated along oriented Nitella actin cables will be examined. The identity of the actin-based motor attached to such vesicles or membranous elements will be determined by a combination of immunoblotting experiments and purification of the vesicle motor. Additional experiments will focus on how the identified abm is attached to the vesicle or membranous element, whether it is by direct association of the motor via electrostatic interaction or if there is an abmA receptor protein present in the membrane The approach described above is specifically directed at understanding the in vivo role of the abmA protein, but it is also broadly applicable to the study of other actin-based motors involved in organelle transport.