MOLECULAR BASIS OF MICROTUBULE-BASED VESICLE TRANSPORT

这项提议的总体目标是发展一种更根本的 对指导细胞器的分子相互作用的理解 真核细胞中的微管交通。我们的研究将继续下去 把重点放在从鱿鱼巨型轴突中挤出的轴浆，因为这 为泡状细胞器高度浓缩的制剂是 沿着微管运输。一个长期目标是澄清 针对运动蛋白和动力蛋白这两种运动蛋白的生化机制 对于微管上相反方向的运动，到特定的 细胞器的数量被编程为向加号或加号移动 微管的减端。为了解决这个问题，我们提出了两个 鉴定与这些蛋白相互作用的囊泡膜蛋白的方法 马达蛋白质。在一种方法中，生物素化细胞器通过 到微管的硬度，将被洗涤剂提取，留下马达- 附着在微管上的受体复合体。运动-受体复合体 将用三磷酸腺苷洗脱，并进一步纯化；生物素化 蛋白质将在凝胶后用亲和素化学发光法进行分析。 电泳法。在识别Kinesin的第二种方法中 受体，运动蛋白重链的C末端尾部结构域，它是 据信与细胞器相互作用，将被表达为谷胱甘肽S- 转移酶融合蛋白，偶联到谷胱甘肽琼脂糖凝胶上，用于 筛选高纯度囊泡的洗涤剂提取物中的蛋白质 与尾域交互。我们还提出了一些实验，旨在 确定调节细胞器运输方向的机制。 在这些研究中，我们将使用体外细胞器运动分析和 光学镊子用于筛选导致单个细胞器 改变他们的移动方向。我们研究的第二个长期目标 就是开发一个由动蛋白和动力蛋白驱动的运动的分子模型。在……里面 在这些研究中，光学镊子将被用来操纵携带珠子 单个马达蛋白到微管上；这些珠子的运动将是 使用图像处理技术以纳米级分辨率跟踪，使用 目的是对运动性背后的分子力学事件进行成像。这些 研究将用重组运动蛋白运动域进行，表达 作为与珠子特异偶联的融合蛋白，在一项努力中 将激动素一级结构的元素与 机械力化学转导。在协作研究中，我们将采用 用于跟踪珠子运动的仪器具有显著更高的 比视频更高的时间分辨率。这些研究的目的是 就是越来越详细地描述力量划水过程中的运动。

The general aim of this proposal is to develop a more fundamental understanding of the molecular interactions which serve to direct organelle traffic along microtubules in eukaryotic cells. Our studies will continue to focus on axoplasm extruded from the squid giant axon because this preparation is highly enriched for vesicular organelles that are transported along microtubules. One long-term aim is to elucidate the biochemical mechanisms that target kinesin and dynein, two motor proteins for movement in opposite directions on microtubules, to specific populations of organelles programmed to move toward either the plus- or minus-ends of microtubules. In pursuit of this question, we propose two approaches to identify vesicular membrane proteins that interact with these motor proteins. In one approach, biotinylated organelles, attached by rigor to microtubules, will be detergent extracted, leaving the motor- receptor complex attached to the microtubule. The motor-receptor complex will be eluted with ATP and the components further purified; biotinylated proteins will be analyzed by an avidin-chemiluminescent procedure after gel electrophoresis. In a second approach toward identifying a kinesin receptor, the C-terminal tail domain of the kinesin heavy chain, which is believed to interact with organelles, will be expressed as a glutathione S- transferase fusion protein, coupled to glutathione sepharose, and used to screen detergent extracts of highly purified vesicles for proteins that interact with the tail domain. We also propose experiments aimed at identifying mechanisms that regulate the direction of organelle transport. In these studies, we will use an in vitro organelle motility assay and optical tweezers to screen for factors that cause individual organelles to change their direction of movement. A second long-term aim of our research is to develop a molecular model for kinesin and dynein-driven movement. In these studies, optical tweezers will be used to manipulate beads carrying single motor proteins onto microtubules; the motion of these beads will be tracked at nm-scale resolution with an image processing technique, with the aim of imaging the molecular mechanical events underlying motility. These studies will be executed with recombinant kinesin motor domains, expressed as fusion proteins with sites for specific coupling to beads, in an effort to relate elements of the primary structure of kinesin to the process of mechanochemical transduction. In collaborative studies, we will employ instrumentation for tracking the motion of beads with significantly higher temporal resolution than is possible with video. The aim of these studies is to characterize motion during the power stroke in increasing detail.