ROLE OF ACTIN-BASED MOTORS IN ORGANELLE MOTILITY

一种新的小肌球蛋白基因abma已在 网柄网柄菌属。Abma基因编码一种带有肌球蛋白头部的蛋白质。 连接到一个带正电的尾部区域，该尾部区域类似于 棘阿米巴肌球蛋白I膜结合区。缺乏ABMA的细胞 基因产物在单细胞水平上进行了检测，并初步 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 建议旨在进一步剖析abma在细胞内的作用。 运动性，特别是细胞器运输，以及生物化学 确定了abma蛋白的特征。网柄网柄菌是首选的有机体 对于这样的研究，因为它允许调查员使用多方面的 分子遗传学实验与传统方法相结合 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 用免疫荧光法检测网柄苔藓中的Abma蛋白。突变型和 野生型ABMA基因将导入ABMA细胞并恢复 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(这将 影响蛋白质在细胞内的定位)。其次，a 将开发一种从网柄苔藓中提纯Abma蛋白的方案 并测定了ABMA蛋白的肌球蛋白样属性。一间牢房 过表达野生型Abma蛋白的菌株将产生 协助纯化ABMA蛋白。系列的最后一集 实验将解决abma与细胞间的相互作用。 颗粒或其他膜元素。囊泡将从 网生网柄菌及其沿定向网状菌的移位能力 将检查肌动蛋白电缆。肌动蛋白为基础的马达的身份 附着到这样的囊泡或膜元素上将由 免疫印迹实验与囊泡纯化的结合 马达。其他实验将集中在已识别的abm是如何进行的。 附着于囊泡或膜元素，无论它是通过直接 通过静电相互作用或如果存在 ABMA受体蛋白存在于膜上，该方法描述了 以上是专门针对了解在体内的作用 ABMA蛋白，但它也广泛适用于其他 基于肌动蛋白的马达参与细胞器的运输。

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.