ROLE OF ACTIN-BASED MOTORS IN ORGANELLE MOTILITY

基于肌动蛋白的马达在细胞器运动中的作用

• 批准号: 3305926
• 负责人: MARGARET A TITUS
• 金额: $ 12.02万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305926
• 关键词: Dictyostelium; actins; adenosine triphosphate; antibody specificity; autoradiography; binding proteins; cell motility; chimeric proteins; enzyme mechanism; immunofluorescence technique; immunoprecipitation; intracellular transport; laboratory rabbit; molecular genetics; myosins; organelles; polymerase chain reaction; posttranslational modifications; protein purification; protein structure function; site directed mutagenesis; western blottings

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.

一种新的小肌球蛋白基因 abmA 已在 盘基网柄菌。 abmA 基因编码一种具有肌球蛋白头的蛋白质 连接到带正电的尾部区域，类似于 棘阿米巴肌球蛋白 I 膜结合域。 缺乏 abmA 的细胞 在单细胞水平上检查基因产物并初步 结果表明，这些突变体的发生率显着降低 细胞内颗粒运动。 本补助金中描述的实验 该提案旨在进一步剖析 abmA 在细胞中的作用 运动性，特别是细胞器运输和生化 表征 abmA 蛋白。 盘基网柄菌是首选生物体 对于此类研究，因为它允许研究者采用多方面的方法 方法，将分子遗传学实验与传统实验相结合 蛋白质生物化学和细胞生物学剖析 abms 在 细胞器运输或其他形式的细胞运动。 第一步 将产生 abmA 特异性抗体并定位 使用免疫荧光法检测盘基网柄菌中的 abmA 蛋白。 突变体和 野生型abmA基因将被引入abmA-细胞并恢复 评估细胞内颗粒运动。 突变将包括 调节性磷酸化位点的改变（这会影响 分子的功能）或尾部区域的变化（这将 影响蛋白质的细胞内定位）。 其次，一个 将开发从盘基网柄菌中纯化 abmA 蛋白的方案 并测定了 abmA 蛋白的肌球蛋白样特性。 一个细胞 将生成过度表达野生型 abmA 蛋白的细胞系 协助 abmA 蛋白的纯化。 最后的系列 实验将解决 abmA 与细胞间质的相互作用 颗粒或其他膜状元素。 囊泡将被分离 盘基网柄菌及其沿着定向 Nitella 易位的能力 将检查肌动蛋白电缆。 基于肌动蛋白的马达的身份 附着在此类囊泡或膜元件上的物质将由 免疫印迹实验和囊泡纯化的结合 发动机。 额外的实验将集中于如何识别出反导弹 附着在囊泡或膜元件上，无论是直接 通过静电相互作用或如果存在电机关联 膜中存在的 abmA 受体蛋白 描述的方法 上述内容专门针对了解体内作用 abmA 蛋白，但它也广泛适用于其他 参与细胞器运输的肌动蛋白马达。