MOLECULAR BASIS OF AXONAL TRANSPORT

本研究项目的总体目标是开发一个更完整的 分子间的相互作用决定了 细胞器在轴突中沿沿着运输。 我们提出的 研究特别关注驱动蛋白和动力蛋白两种 促进相反方向运动的可溶性、产生力的ATP酶 沿着微管的方向，与特定的 细胞器，被编程为朝向或远离细胞体移动。 在最近的实验中，纯化的细胞器的运动依赖于 存在含有许多蛋白质的轴浆胞质组分 除了驱动蛋白和动力蛋白。 这项建议的具体目的是 为了更精确地鉴定出 细胞器的运动。 我们将通过AMP-PNP诱导的微管 亲和力，然后是盐或核苷酸诱导的释放， 胞质蛋白质形成轴浆，足以促进细胞器 运动我们提出的实验将确定纯化的动力蛋白是否 轴浆中的驱动蛋白可以单独驱动细胞器运动， 如前所述，还需要可溶性“辅助因素”， 问题研究 我们还将探讨最近提出的可能性， 实验，动力蛋白和驱动蛋白都需要逆行 细胞器运动 我们将通过重组纯化的 组件和测试他们的能力，以促进定向运动的 此外，细胞器的电子显微镜研究将 定位驱动蛋白和动力蛋白，以确定它们是否结合在一起 在细胞器表面。 最后是nm尺度的运动分析， 先前表明驱动蛋白包被的珠沿着沿着单个 原丝，而动力蛋白包被的珠子在微管上“徘徊” 表面，将应用于细胞器的运动，以测试如果这些相同的 驱动蛋白和动力蛋白驱动运动的特征是明显的。 跟踪 nm水平的细胞器运动将提供分子水平的测定。 重组系统中特定蛋白质之间的相互作用。

The general aim of this research project is to develop a more complete understanding of the molecular interactions which determine how organelles are transported along microtubules in axons. Our proposed investigations are particularly concerned with how kinesin and dynein two soluble, force-generating ATPases that promote movement in opposite directions along microtubules, interact with specific populations of organelles, programmed to move either toward or away from the cell body. In recent experiments, the movement of purified organelles depended on the presence of an axoplasmic cytosol fraction containing many proteins in addition to kinesin and dynein. The specific aim of this proposal is to identify, more precisely, the particular cytosolic factors required for organelle movement. We will isolate, by AMP-PNP induced microtubule affinity, followed by salt or nucleotide induced release, a subset of cytosolic proteins form axoplasm, sufficient to promote organelle movement. Our proposed experiments will determine whether purified dynein and kinesin from axoplasm can alone drive organelle movement, or whether soluble "accessory factors" are also required, as suggested by previous studies. We will also explore the possibility, suggested by recent experiments, that both dynein and kinesin are required for retrograde organelle movement. We will do so in part by recombining the purified components and testing their ability to promote directed movement of organelles; in addition, electron microscopic studies of organelles will localize kinesin and dynein, to determine whether they are bound together on the organelle surface. Finally, nm-scale motion analysis, which previously indicated that kinesin-coated beads track along single protofilaments, while dynein-coated beads "wander" over the microtubule surface, will be applied to organelle movement to test if these same features of kinesin and dynein driven movement are apparent. Tracking of organelle movement at the nm level will provide an assay for molecular interactions between particular proteins in the reconstituted system.