Role of Myosin and Actin Filaments in Fast Axonal Transport

肌球蛋白和肌动蛋白丝在快速轴突运输中的作用

• 批准号: 9974709
• 负责人: George Langford
• 金额: $ 14.1万
• 依托单位: Dartmouth College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9974709&HistoricalAwards=false
• 关键词: Role; Myosin; Actin; Filaments; Fast

Fast axonal transport, the anterograde and retrograde movement of vesicles and mitochondria in the neuronal axon, is a fundamental process in nerve cells. It is now well established that the transport of vesicles requires cytoskeletal filaments (both microtubules and actin microfilaments) and molecular motors (kinesin, dynein, and myosin). It is also well established that vesicles are targeted to either the axon or dendrites to maintain cell polarity, and that anterograde and retrograde transport are coordinated to maintain the proper distribution of organelles throughout the cell.The long-term objectives of this project are to determine the role of myosin and actin filaments in fast axonal transport. In particular, the role of myosin V in vesicle transport will be determined. Recently published data from Dr. Langford's laboratory has shown that myosin V transports ER vesicles in the squid giant axon. The specific aim of this project is to test the hypothesis that the membrane receptor for myosin V is a member of the protein 4.1 family of proteins and that the DIL motif of myosin V is involved in its binding to vesicles. Several approaches to determine the membrane proteins to which myosin binds will be used: (1) Affinity column chromatography will be performed with a synthetic peptide used to produce the myosin-V function-blocking antibody. Extracts of axoplasm and squid brain homogenates will be passed over the column and specific interacting proteins recovered. (2) Cross-linking reagents will be used to couple myosin V to its binding partners in the vesicle membrane and co-immunoprecipitation will be used to isolate the myosincomplex. (3) Dominant-negative constructs will be expressed to determine whether the DIL motif represents the membrane binding sequence. The full-length clone of squid myosin-V has been obtained by RT-PCR. The myosin-V gene will be fused to GFP and the fusion construct ligated into an expression vector for myosin V expression in cultured cells. The three experimental plans of this specific aim will be conducted in parallel.

轴突快速运输是神经细胞中的一个基本过程，即轴突中囊泡和线粒体的顺行和逆行运动。 现在已经确定，囊泡的运输需要细胞骨架丝（微管和肌动蛋白微丝）和分子马达（驱动蛋白、动力蛋白和肌球蛋白）。 它也是公认的，囊泡的轴突或树突为目标，以保持细胞极性，顺行和逆行运输协调，以保持整个cell.The长期目标的细胞器的正确分布是确定快速轴突运输中的肌球蛋白和肌动蛋白丝的作用。特别是，肌球蛋白V在囊泡运输中的作用将被确定。最近发表的数据从博士兰福德的实验室表明，肌球蛋白V运输ER囊泡在鱿鱼巨大的轴突。该项目的具体目的是检验肌球蛋白V的膜受体是蛋白质4.1家族的成员，并且肌球蛋白V的DIL基序参与其与囊泡的结合的假设。将使用几种方法来确定肌球蛋白结合的膜蛋白：（1）将使用用于产生肌球蛋白-V功能阻断抗体的合成肽进行亲和柱层析。轴浆和鱿鱼脑匀浆的提取物将通过柱，并回收特定的相互作用蛋白。(2)将使用交联试剂将肌球蛋白V偶联到囊泡膜中的结合伴侣，并使用免疫共沉淀分离肌球蛋白复合物。(3)将表达显性阴性构建体以确定DIL基序是否代表膜结合序列。通过RT-PCR获得了鱿鱼肌球蛋白-V的全长克隆。将肌球蛋白-V基因与GFP融合，并将融合构建体连接到表达载体中，用于在培养的细胞中表达肌球蛋白V。 这一具体目标的三个实验计划将同时进行。