MOLECULAR BASIS OF MICROTUBULE BASED VESICLE TRANSPORT

这项建议的总体目标是制定一个更基本的 了解分子间的相互作用， 在真核细胞中沿着沿着运输。 我们的研究会继续 把注意力集中在鱿鱼巨大轴突的轴浆上， 制备物高度富集泡状细胞器， 沿着沿着微管运输。 一个长期目标是阐明 以驱动蛋白和动力蛋白为目标的生化机制， 在微管上向相反方向运动， 细胞器的群体被编程为向正或负方向移动， 微管的负端。 针对这一问题，我们提出两个 鉴定与这些蛋白相互作用的囊泡膜蛋白的方法 马达蛋白 在一种方法中，生物素化的细胞器， 僵硬的微管，将洗涤剂提取，离开电机- 受体复合物附着在微管上。 运动受体复合体 将用ATP洗脱，并进一步纯化组分;生物素化 蛋白质将在凝胶后通过抗生物素蛋白荧光法进行分析。 电泳 在鉴定驱动蛋白的第二种方法中， 受体，驱动蛋白重链的C-末端尾部结构域， 被认为与细胞器相互作用，将被表达为谷胱甘肽S- 转移酶融合蛋白，与谷胱甘肽琼脂糖偶联，并用于 筛选高度纯化的囊泡的去污剂提取物， 与尾部结构域相互作用。 我们还提出了旨在 识别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试验， 光学镊子来筛选导致单个细胞器 改变它们的运动方向。 我们研究的第二个长期目标 是建立一个驱动蛋白和动力蛋白驱动运动的分子模型。 在 在这些研究中，光学镊子将用于操纵携带 单马达蛋白质到微管;这些珠子的运动将是 用图像处理技术以nm级分辨率跟踪， 目的是对运动性背后的分子力学事件进行成像。 这些 将用重组驱动蛋白运动域进行研究， 作为具有与珠子特异性偶联的位点的融合蛋白， 将驱动蛋白一级结构的元素与 机械化学转导 在合作研究中，我们将采用 用于跟踪珠运动的仪器具有显著更高的 时间分辨率高于视频。 这些研究的目的 是为了更详细地描述动力冲程期间的运动。

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.