Role of Actin Filaments in Fast Axonal Transport

这个项目将研究神经细胞中细胞器相关肌球蛋白的特定功能。在过去的几年中，通过分子和遗传技术鉴定的肌球蛋白新马达的数量呈爆炸式增长，但这些新马达的功能作用尚未确定。这个项目测试了这样的假设，即微管依赖的马达驱动细胞器长距离运动，而动作蛋白依赖的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马达，肌动蛋白依赖和微管依赖的细胞器运动形式之间的相互关系，以及参与细胞器运动的马达被调节的机制。鱿鱼巨大的轴突和视叶（脑）将是这些研究中使用的主要生物材料。鱿鱼轴质是少数可以直接研究肌球蛋白依赖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来研究(a)细胞器相关肌球蛋白，(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。挤压的轴质对运动研究特别有用，但对于肌球蛋白运动的生化纯化来说，材料的量太少了。然而，鱿鱼脑是肌球蛋白纯化的另一种材料来源，因此，在轴质中发现的马达随后将从鱿鱼视叶中分离出来。本研究项目的主要目标是利用鱿鱼巨轴突中的管泡细胞器（tubulovesicular organelles, TVOs）来研究与肌球蛋白相关的细胞器特性和调控。tvo的净化将通过产生轴浆鬼来实现；轴质高度提取的残余纯化后的tvo将在肌动蛋白纤维上重建运动，并通过定量运动分析确定运动参数。这些区室作为钙储存的功能将用钙指示剂来测定。此外，针对已知光滑内质网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小管泡细胞器结合的因素。肌动蛋白依赖马达在TVOs表面的位置将使用免疫荧光显微镜确定。这项技术将有可能确定马达是均匀分布还是局限于TVOs的囊泡域。将确定马达的性质，例如马达是否是外周蛋白，以及是否有可溶性因子参与其附着在膜上。用常规生化方法或免疫沉淀法从鱿鱼脑中纯化肌球蛋白。纯化后的马达将使用体外运动测定进行生化和功能表征。一种针对鱿鱼脑肌球蛋白V的抗体已经产生，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，并用于抑制挤压轴质中的运动，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，细胞运动和细胞器的组织和运输。从这些研究中获得的新知识在生物技术和分子纳米结构的研究中得到了应用。＊＊＊

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. ***