Vesicle Microtubule Interaction and Fast Axonal Transport

囊泡微管相互作用和快速轴突运输

• 批准号: 9316540
• 负责人: Roger Sloboda
• 金额: $ 30万
• 依托单位: Dartmouth College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1997-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9316540&HistoricalAwards=false
• 关键词: Vesicle; Microtubule; Interaction; Fast; Axonal

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. ***

本提案中概述的实验将扩展先前基于微管的细胞内颗粒运输的研究。本文研究了鱿鱼巨轴突中参与膜结合囊泡运输的两个高分子量微管相关蛋白MAP H1和MAP H2的特性。maph2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。maph1结合ATP， maph1抗体阻断膨化鱿鱼轴质中的囊泡运输。MAP H1多肽分子量大于40万；到目前为止，大约25%的多肽已被克隆，并由此推断出其氨基酸序列。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。完成MAP H1多肽的克隆和测序。该分子的功能域将在细菌细胞中表达、纯化，并分析其与微管、囊泡表面蛋白和神经细胞骨架的其他元素相互作用的能力。在组织培养细胞转染实验中，MAP H1过表达或过表达（使用反义结构）的影响将通过运动测定和免疫荧光显微镜来评估。最后，将进行免疫沉淀和特异性放射性标记实验，以确定哪些轴质和/或囊泡表面的多肽能够与MAP H1相互作用。真核细胞是一个高度组织化的结构，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，其功能是细胞间通讯，这种通讯部分是通过在邻近细胞附近的轴突尖端释放分泌物质而发生的，这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向下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着轴突微管的运动，并不是神经组织所独有的，因为微管介导的运动几乎是细胞内颗粒定向运动的普遍机制。然而，轴突，特别是鱿鱼巨轴突，是研究微管介导转运的一个特别有用的模型。这一领域的最新进展导致了参与这种运输的运动蛋白的发现。然而，马达本身显然不是故事的全部。该项目的结果将导致对神经元中囊泡运输机制的更完整理解，并将提供有关可运输囊泡表面成分的重要信息，这些成分允许囊泡特异性地与基于微管的运输装置相互作用。这些知识不仅对理解该系统的生物学意义重大，而且对于在非生物应用中开发微管介导的运输现象具有潜在的重要意义，例如先进材料和纳米制造。* * *