Role of Actin Filaments in Fast Axonal Transport

肌动蛋白丝在快速轴突运输中的作用

• 批准号: 9506279
• 负责人: George Langford
• 金额: $ 43万
• 依托单位: Dartmouth College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-15 至 2000-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506279&HistoricalAwards=false
• 关键词: Role; Actin; Filaments; Fast; Axonal

9506279 Langford This project will investigate the specific functions of organelle-associated myosins in nerve cells. The number of new myosin motors identified by molecular and genetic techniques has grown explosively in the past few years but the functional roles of these new motors have not been determined. This project tests the hypothesis that microtubule-dependent motors drive movement of organelles over long distances while actin-dependent motors drive movement to local sites along the axon. The broad objectives of the research project are to determine the actin-dependent motors involved in fast axonal transport, the interrelationship between the actin-dependent and microtubule-dependent forms of organelle motility and the mechanism by which the motors involved in organelle motility are regulated. The squid giant axon and optic lobes (brain) will be the primary biological materials used in these studies. Squid axoplasm is one of a small number of in vitro systems where myosin-dependent organelle motility can be studied directly. Therefore, it represents a unique system in which to study (a) organelle associated myosins, (b) the regulation of these motors and (c) the relationship between membrane trafficking on microtubules and actin filaments. Extruded axoplasm is particularly useful for motility studies but the amount of material is too small for biochemical purification of myosin motors. Squid brain, however, represents an alternate source of material for purification of myosins, therefore, motors identified in axoplasm will be subsequently isolated from squid optic lobes. The primary goal of the research project is to use an identified organelle, the tubulovesicular organelles (TVOs) in the squid giant axon, to study the properties and regulation of organelle-associated myosins. Purification of TVOs will be achieved by generating axoplasmic ghosts; the highly extracted remnant of axoplasm. Motility of purified TVOs will be reconstituted on actin f ilaments and the parameters of movement determined by quantitative motion analysis. The function of these compartments as Ca stores will be determined with Ca-indicator dyes. In addition, antibodies to known smooth ER proteins will be used to establish the identity and purity of the isolated compartments. Another specific goal is to identify factors that influence binding of motors to the tubulovesicular organelles. The location of the actin-dependent motors on the surfaces of TVOs will be determined using immunofluorescence microscopy. This technique will make it possible to establish whether the motors are uniformly distributed or localized to the vesicular domains of TVOs. Properties of the motor, such as whether the motor is a peripheral protein and whether soluble factors are involved in its attachment to the membrane will be determined. Myosins will be purified from squid brain by conventional biochemical methods or from axoplasm by immunoprecipitation. Purified motors will be biochemically and functionally characterized using in vitro motility assays. An antibody to squid brain myosin V has been generated for use in localization and functional studies. Antibodies to other organelle-associated unconventional myosins will be generated and used to inhibit motility in extruded axoplasm thereby verifying the role of these motors in the movement of organelles. %%% The work described in this proposal will provide new knowledge on molecular motors and will aid our understanding of fast axonal transport as well as our understanding of many other fundamental cellular processes including secretion, cell division, cell motility and the organization and transport of organelles. The new knowledge from these studies has applications in biotechnology and research on molecular nanostructures. ***

9506279 Langford 该项目将研究神经细胞中细胞器相关肌球蛋白的特定功能。 在过去几年中，通过分子和遗传技术鉴定的新型肌球蛋白马达的数量呈爆炸性增长，但这些新马达的功能作用尚未确定。该项目测试了这样的假设：微管依赖性马达驱动细胞器长距离运动，而肌动蛋白依赖性马达驱动沿轴突局部位置的运动。该研究项目的主要目标是确定参与快速轴突运输的肌动蛋白依赖性马达、细胞器运动的肌动蛋白依赖性和微管依赖性形式之间的相互关系以及参与细胞器运动的马达的调节机制。鱿鱼巨型轴突和视叶（大脑）将是这些研究中使用的主要生物材料。鱿鱼轴浆是少数可以直接研究肌球蛋白依赖性细胞器运动的体外系统之一。因此，它代表了一个独特的系统，可以在其中研究（a）细胞器相关的肌球蛋白，（b）这些马达的调节以及（c）微管和肌动蛋白丝上的膜运输之间的关系。 挤出的轴浆对于运动研究特别有用，但材料的量太小，无法用于肌球蛋白马达的生化纯化。然而，鱿鱼脑代表了用于纯化肌球蛋白的替代材料来源，因此，轴浆中鉴定出的马达随后将从鱿鱼视叶中分离出来。该研究项目的主要目标是利用鱿鱼巨轴突中已确定的细胞器（TVO）来研究细胞器相关肌球蛋白的特性和调节。 TVO的纯化将通过产生轴浆鬼来实现；高度提取的轴浆残余物。纯化的 TVO 的运动性将在肌动蛋白丝上重建，运动参数由定量运动分析确定。这些隔室作为 Ca 储存的功能将由 Ca 指示剂染料确定。此外，已知平滑 ER 蛋白的抗体将用于确定分离区室的身份和纯度。另一个具体目标是确定影响马达与管泡细胞器结合的因素。 TVO 表面上肌动蛋白依赖性马达的位置将使用免疫荧光显微镜确定。这项技术将能够确定马达是否均匀分布或集中于 TVO 的囊泡域。将确定马达的特性，例如马达是否是外周蛋白以及可溶性因子是否参与其与膜的附着。通过常规生化方法从鱿鱼脑中纯化肌球蛋白，或通过免疫沉淀从轴浆中纯化肌球蛋白。将使用体外运动测定对纯化的电机进行生化和功能表征。一种针对鱿鱼脑肌球蛋白 V 的抗体已产生，可用于定位和功能研究。 将产生针对其他细胞器相关的非常规肌球蛋白的抗体，并用于抑制挤出的轴浆的运动，从而验证这些马达在细胞器运动中的作用。 %%% 本提案中描述的工作将提供关于分子马达的新知识，并将有助于我们理解快速轴突运输以及我们对许多其他基本细胞过程的理解，包括分泌、细胞分裂、细胞运动以及细胞器的组织和运输。这些研究的新知识可应用于生物技术和分子纳米结构的研究。 ***