3D STRUCTURE OF MITOTIC MICROTUBULES

微管在真核细胞中参与许多重要的活动。 它们形成的支架也决定了细胞的整体形状 内部细胞器的排列。细胞内通讯和 囊泡的运输依赖于微管网络。在分裂 细胞、微管之间的循环广义多功能 相间网络，提供对单元的每个角落的访问，以及 一种位于中心并专门用于 分离染色体。正如人们所预料的那样， 因此，干扰微管功能的药物会攻击微管， 心脏的许多细胞活动，往往有深刻的生理 方面的影响.这些药物中的一些在人类中具有治疗应用： 秋水仙碱，用于痛风性关节炎的抗炎特性; 长春新碱、长春碱和紫杉醇，用于抗有丝分裂作用 灰黄霉素，一种抗真菌剂; 他人除了这些医药用途外，影响微管的试剂是 对人类健康同样重要，但非治疗性 方法，如它们作为杀虫剂和杀菌剂的广泛使用。基本 因此，关于微管的信息具有异常直接和 与国家卫生研究院的使命有直接联系。 正如从它们的多样性活动中所预期的那样，微管与许多 其他细胞蛋白质。为了理解这些相互作用， 将允许合理操纵基于微管的现象， 肯定是一种有效的治疗和诊断能力，我们必须 对微管的结构有很详细的了解。我们还必须知道 使用微管的超分子组装结构， 提供运动产生、形状决定和细胞器定位 细胞内的功能。幸运的是，值得注意的是， 的微管似乎是恒定的，无论特定的 它所参与的集会。因此如果我们能确定 任何一种微管的功能类别，我们将了解到 很多关于微管的知识。 该建议描述了我们成功制备天然细胞 适用于低温电子结构测定的微管 未固定、未染色、冷冻水合样品的显微镜检查。我们报告一些 冷冻电子显微镜检查这些微管的初步结果 显微镜，并提出了一套实验，将产生高 原生微管的分辨率3D结构。

Microtubules are engaged in many important activities in eukaryotic cells. They form the scaffolding that helps determine overall cell shape, as well as the arrangement of interior organelles. Intracellular communication and the traffic of vesicles depend upon the microtubule network. In dividing cells, the microtubules cycle between the generalized multifunctional interphase network that provides access to every corner of the cell, and a mitotic apparatus that is centrally located and specialized for the task of separating chromosomes. As would be expected for such a heavily used object, agents that interfere with microtubule function strike at the heart of many cellular activities and often have profound physiological effects. Some of these agents have therapeutic applications in humans: colchicine, used for its anti-inflammatory properties in gouty arthritis; vincristine, vinblastine, and taxol, used for their anti-mitotic effects against a variety of neoplasms; griseofulvin, an anti-fungal agent; and others. Besides these medicinal uses, agents that affect microtubules are significant for human health in equally important, though non-therapeutic ways, such as their widespread use as pesticides and fungicides. Basic information about microtubules therefore has an unusually direct and immediate connection to the mission of the NIH. As expected from their diverse activities, microtubules interact with many other cellular proteins. To understand these interactions in a way that will allow rational manipulation of microtubule-based phenomena, which would certainly be a potent therapeutic and diagnostic capability, we must know the structure of microtubules in great detail. We must also know the structure of the supramolecular assemblies that use microtubules to provide the motion-producing, shape-determining, and organelle-positioning functions within cells. Fortunately, and remarkably, the basic structure of the microtubule seems to be constant regardless of the particular assembly in which it is involved. Thus if we can determine the structure of any one of the functional classes of microtubule, we will have learned a lot about all microtubules. This proposal describes our successful preparation of native cellular microtubules suitable for structure determination by cryo-electron microscopy of unfixed, unstained, frozen hydrated samples. We report some preliminary results from examining these microtubules by cryo-electron microscopy, and propose a set of experiments that will yield a high resolution 3D structure of the native microtubule.