Vesicle Microtubule Interaction and Fast Axonal Transport

9316540斯洛博达 在这个建议中概述的实验将扩展以前的研究微管为基础的细胞内颗粒运输。 这项工作的重点是两个高分子量微管相关蛋白的特性，这是参与运输的膜结合囊泡在鱿鱼巨轴突，称为MAP H1和MAP H2。 MAP H2是鱿鱼细胞质动力蛋白的类似物。 MAP H1是一种囊泡表面蛋白，也可以与微管结合。 MAP H1结合ATP，并且MAP H1的抗体阻断挤压鱿鱼轴浆中的囊泡运输。 MAP H1多肽具有大于400，000的分子量;迄今为止已克隆了约25%的多肽，并由此推导出其氨基酸序列。 在这个更新项目中提出的实验旨在扩大我们对MAP H1及其在微管运动中的作用的认识。 将完成MAP H1多肽的克隆和测序。 该分子的功能结构域将在细菌细胞中表达，纯化，并分析它们与微管、囊泡表面蛋白和神经细胞细胞骨架的其他元素相互作用的能力。 在用组织培养细胞进行的转染实验中，MAP H1的过度表达或MAP H1的表达不足（使用反义构建体）的影响将通过运动性测定和免疫荧光显微术来评估。 最后，将进行免疫沉淀和特异性放射性标记实验，以确定这些多肽的轴质和/或囊泡表面与MAP H1能够相互作用。 真核细胞是一种高度组织化的结构，这种高度的空间组织对细胞功能至关重要。 在神经细胞的特殊情况下，其功能是细胞间的通讯，这种通讯部分是通过从邻近细胞的轴突尖端释放分泌物质而发生的，轴突是一个细长的细胞过程。 从轴突尖端释放的分泌物质在轴突突的另一端的神经细胞主体中制造。 因此，这些物质沿着轴突的长度运输的机制对正常的神经功能至关重要。 这种机制涉及含有分泌物质的膜封闭囊泡沿沿着轴突微管的运动，并不是神经组织所特有的，因为微管介导的运动是一种几乎普遍存在的定向细胞内颗粒运动的机制。 然而，轴突，特别是鱿鱼巨轴突，是一个特别有用的模型，用于研究微管介导的运输。 在这一领域的最新进展导致发现的马达蛋白参与这种运输。 然而，单靠发动机显然不是故事的全部。 该项目的结果将导致更全面地了解神经元中囊泡运输的机制，并将提供有关可运输囊泡表面上的组分的重要信息，这些组分允许囊泡与基于微管的运输装置特异性相互作用。 这些知识不仅对理解系统的生物学具有重要意义，而且对非生物应用中微管介导的运输现象的下游开发具有潜在的重要意义，例如先进材料和纳米纤维。 ***

9316540 Sloboda The experiments outlined in this proposal will extend previous studies on microtubule based intracellular particle transport. The work focuses on the characterization of two high molecular weight microtubule associated proteins which are involved in the transport of membrane bound vesicles in squid giant axon, termed MAP H1 and MAP H2. MAP H2 is the squid analog of cytoplasmic dynein. MAP H1 is a vesicle surface protein that can also bind to microtubules. MAP H1 binds ATP, and antibodies to MAP H1 block vesicle transport in extruded squid axoplasm. The MAP H1 polypeptide has a molecular weight greater than 400,000; approximately 25% of the polypeptide has thus far been cloned and its amino acid sequence thereby deduced. The experiments proposed in this renewal project are designed to expand our knowledge of MAP H1 and its role in microtubule based motility. The cloning and sequencing of the MAP H1 polypeptide will be completed. Functional domains of the molecule will be expressed in bacterial cells, purified, and their characteristics analyzed with respect to their ability to interact with microtubules, vesicle surface proteins, and other elements of the cytoskeleton of nerve cells. In transfection experiments with tissue culture cells, the effect of over expression of MAP H1 or of under expression (using antisense constructs) of MAP H1 will be assessed via motility assays and immunofluorescence microscopy. Finally, immunoprecipitation and specific radiolabelling experiments will be performed to identify those polypeptides of axoplasm and/or of the vesicle surface with which MAP H1 is capable of interacting. %%% The eukaryotic cell is a highly organized structure, and this high degree of spatial organization is critical to cellular function. In the special case of nerve cells, whose function is intercellular communication, this communication occurs in part by the release of secretory materials from the tip of the axon, a long slen der cellular process, in the vicinity of a neighboring cell. The secretory materials released from the axonal tip are manufactured in the main body of the nerve cell, at the other end of the axonal process. The mechanism whereby these materials are transported down the length of the axon is thus critical to proper nerve function. This mechanism, which involves the movement of membrane-enclosed vesicles containing the secretory materials along axonal microtubules, is not really unique to nerve tissue, since microtubule-mediated motility is a nearly ubiquitous mechanism for directed intracellular particle movements. However, the axon, particularly the squid giant axon, is an especially useful model for studying microtubule mediated transport. Recent advances in this field have led to the discovery of motor proteins which are involved in this transport. However, the motors alone are clearly not the whole story. The results of this project will lead to a more complete understanding of the mechanism of vesicle transport in neurons, and will provide important information about the components on the surfaces of the transportable vesicles that allow the vesicles to specifically interact with the microtubule based transport apparatus. This knowledge will not only be of significance to understanding the biology of the system, but is potentially important for downstream exploitation of microtubule-mediated transport phenomena in non-biological applications, such as advanced materials and nanofabrication. ***