MYOSIN I FUNCTION IN VIVO

肌球蛋白 I 在体内发挥作用

• 批准号: 2022495
• 负责人: MARGARET A TITUS
• 金额: $ 22.17万
• 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 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022495
• 关键词: Dictyostelium; actins; binding proteins; cell membrane; chemical binding; intermolecular interaction; membrane structure; microorganism genetics; mutant; myosins; protein structure function

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.

一个新颖的小肌球蛋白基因ABMA已在 dictyostelium。 ABMA基因编码具有肌球蛋白头的蛋白质 附着在带正电荷的尾部区域上，与 Acanthamoeba肌球蛋白I膜结合域。 缺乏ABMA的细胞 在单细胞水平和初步检查中检查了基因产物 结果表明，这些突变体的速率显着降低 细胞内粒子运动。 这笔赠款中描述的实验 提案针对进一步剖析ABMA在细胞中的作用 运动性，特别是细胞器的运输和生化 表征ABMA蛋白。 dictyostelium是选择的生物 对于研究，它允许研究人员采用多方面的 方法，将分子遗传实验与传统的遗传实验相结合 蛋白质生物化学和细胞生物学剖析ABM的作用 细胞器转运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定位 使用免疫荧光在dictyostelium中的ABMA蛋白。 突变和 野生型ABMA基因将被引入ABMA细胞，并恢复 评估的细胞内粒子运动。 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（这将 影响蛋白质的细胞内定位）。 其次，a 将开发协议以纯化dictyostelium的ABMA蛋白 以及确定ABMA蛋白的肌球蛋白样性能。 一个单元 过表达过表达野生型ABMA蛋白的行将产生 有助于纯化ABMA蛋白。 最后一系列 实验将解决ABMA与细胞间的相互作用 颗粒或其他膜元素。 囊泡将与 dictyostelium及其沿定向的Nitella易位的能力 将检查肌动蛋白电缆。 基于肌动蛋白的电机的身份 附着在此类囊泡或膜元件上的 免疫印迹实验和囊泡纯化的组合 发动机。 其他实验将集中于确定的ABM的方式 连接到囊泡或膜元件，无论是直接 电动机通过静电相互作用或是否存在 膜中存在的ABMA受体蛋白描述的方法 以上专门针对理解体内的角色 ABMA蛋白质，但它也广泛适用于其他 基于肌动蛋白的电动机参与细胞器运输。