MOLECULAR BASIS OF AXONAL TRANSPORT

这项研究项目的总体目标是开发一个更完整的 对分子相互作用的理解，分子相互作用决定了 细胞器沿轴突内的微管运输。我们的建议 调查特别关注激动素和动力蛋白两种 可溶性的、产生力量的ATPase，促进相反方向的运动 沿着微管的方向，与特定的人群相互作用 细胞器，被编程为朝向细胞体或远离细胞体。 在最近的实验中，纯化的细胞器的运动依赖于 含有多种蛋白质的轴浆组分的存在 除了激动素和动力蛋白。这项建议的具体目的是 为了更准确地确定所需的特定胞浆因子 用于细胞器的运动。我们将通过AMP-PNP诱导的微管分离 亲和力，然后是盐或核苷酸诱导的释放，是 胞浆蛋白形成轴浆，足以促进细胞器 有动静。我们提议的实验将确定纯化的动力蛋白 来自轴浆的激动素可以单独驱动细胞器的运动，或者 也需要可溶的“辅助因素”，正如前面所建议的 学习。我们还将探索最近提出的可能性 实验表明，逆行既需要动力蛋白，也需要动力蛋白 细胞器运动。我们将通过重新组合提纯的 组件并测试它们促进定向移动的能力 细胞器；此外，细胞器的电子显微镜研究将 本地化Kinesin和dynein，以确定它们是否绑定在一起 在细胞器表面。最后，进行了纳米尺度的运动分析 先前指出，动蛋白包裹的珠子沿着单一的轨迹 原丝，而包裹着动力蛋白的珠子则在微管上“游荡” 表面，将其应用于细胞器的运动以测试这些是否相同 动蛋白和动力蛋白驱动的运动特征明显。跟踪 细胞器在纳米水平上的运动将为分子水平的研究提供实验依据。 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.