Role of Actin Filaments in Fast Axonal Transport

小行星9506279 本计画将探讨细胞器相关肌球蛋白在神经细胞中的特殊功能。 在过去的几年里，通过分子和遗传技术鉴定的新肌球蛋白马达的数量爆炸性增长，但这些新马达的功能作用尚未确定。本研究旨在验证微管依赖性马达驱动细胞器长距离运动的假设，而肌动蛋白依赖性马达则驱动轴突沿着局部运动。该研究项目的广泛目标是确定参与快速轴突运输的肌动蛋白依赖性马达、肌动蛋白依赖性和微管依赖性形式的细胞器运动之间的相互关系以及参与细胞器运动的马达的调节机制。鱿鱼巨大的轴突和视叶（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的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，其中研究（a）细胞器相关肌球蛋白，（B）这些电机的调节和（c）微管和肌动蛋白丝上的膜运输之间的关系。 挤压轴浆是特别有用的运动研究，但材料的量太小的生化纯化肌球蛋白马达。鱿鱼脑，然而，代表了另一种来源的纯化肌球蛋白的材料，因此，电机轴浆中确定将随后从鱿鱼视叶分离。该研究项目的主要目标是使用一种已鉴定的细胞器，鱿鱼巨轴突中的管状泡状细胞器（TVOs），研究细胞器相关肌球蛋白的性质和调控。TVO的纯化将通过产生轴浆鬼来实现;轴浆的高度提取的残余物。纯化的TVO的运动性将在肌动蛋白纤维上重建，并且通过定量运动分析确定运动参数。这些隔室作为钙储存的功能将用钙指示剂染料测定。此外，将使用已知平滑ER蛋白的抗体来确定分离的隔室的同一性和纯度。另一个具体的目标是确定因素的影响绑定电机的tubulovesicular细胞器。将使用免疫荧光显微镜确定TVO表面上肌动蛋白依赖性马达的位置。这种技术将使它有可能建立是否电机是均匀分布或本地化的囊泡域的TVO。将确定马达的性质，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。肌球蛋白将通过常规的生化方法从鱿鱼脑中纯化或通过免疫沉淀从轴浆中纯化。纯化的马达将使用体外运动试验进行生化和功能表征。鱿鱼脑肌球蛋白V的抗体已经产生用于定位和功能研究。 其他细胞器相关的非常规肌球蛋白的抗体将被产生和用于抑制运动的挤压轴浆，从而验证这些电机在细胞器的运动中的作用。 本提案中描述的工作将提供分子马达的新知识，并将帮助我们理解快速轴突运输以及我们理解许多其他基本细胞过程，包括分泌，细胞分裂，细胞运动和细胞器的组织和运输。这些研究的新知识在生物技术和分子纳米结构研究中有应用。 ***

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. ***