Role of Myosin and Actin Filaments in Fast Axonal Transport

快速轴突运输是神经细胞的一个基本过程，即神经元轴突内小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细丝(微管和肌动蛋白微丝)和分子马达(动蛋白、动力蛋白和肌球蛋白)。囊泡被定位于轴突或树突以维持细胞的极性，顺行和逆行运输被协调以维持细胞器在整个细胞中的适当分布。该项目的长期目标是确定肌球蛋白和肌动蛋白细丝在快速轴突运输中的作用。特别是，肌球蛋白V在囊泡运输中的作用将被确定。朗福德博士的实验室最近公布的数据显示，肌球蛋白V运输鱿鱼巨型轴突中的内质网小泡。这个项目的具体目的是检验一种假设，即肌球蛋白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基序参与了它与小泡的结合。将使用几种方法来确定肌球蛋白结合的膜蛋白：(1)亲和柱层析将与用于产生肌球蛋白V功能阻断抗体的合成肽一起进行。轴浆和鱿鱼脑匀浆的提取物将通过柱子，回收特定的相互作用蛋白。(2)用交联剂将肌球蛋白V偶联到囊泡膜上的结合伙伴上，并用免疫共沉淀法分离肌球蛋白复合体。(3)表达显性-负性结构以确定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基因与绿色荧光蛋白融合，构建成肌球蛋白V在培养细胞中表达的表达载体。这一具体目标的三个试验计划将并行进行。

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.