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，是指神经元轴突内囊泡和线粒体的逆行和顺行运动。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（运动蛋白、动力蛋白和肌球蛋白）。此外，囊泡以轴突或树突为目标来维持细胞极性，并且顺行和逆行运输是协调的，以维持细胞器在细胞中的适当分布。该项目的长期目标是确定肌凝蛋白和肌动蛋白丝在快速轴突运输中的作用。特别是，肌凝蛋白V在囊泡运输中的作用将被确定。Langford博士实验室最近公布的数据显示，肌凝蛋白V在鱿鱼巨大的轴突中运输内质网囊泡。本项目的具体目的是验证肌凝蛋白V的膜受体是蛋白4.1蛋白家族成员的假设，以及肌凝蛋白V的DIL基元参与其与囊泡的结合。确定肌球蛋白结合的膜蛋白的几种方法将被使用：(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进行。轴质和鱿鱼脑匀浆的提取物将通过柱和特定的相互作用蛋白回收。(2)使用交联试剂将肌球蛋白V与囊泡膜上的结合伙伴偶联，使用共免疫沉淀法分离肌球蛋白复合物。(3)通过表达显性阴性结构体来确定DIL基序是否代表膜结合序列。利用RT-PCR技术获得了鱿鱼肌球蛋白- v的全长克隆。将myosin-V基因融合到GFP中，并将融合构建体连接到myosin-V表达载体中，在培养细胞中表达。这一具体目标的三个实验计划将并行进行。

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.