3D STRUCTURE OF MITOTIC MICROTUBULES

微管在真核细胞中参与了许多重要的活动。 它们构成了帮助确定整体细胞形状的脚手架 作为内部细胞器的排列。细胞内通信和 囊泡的运输依赖于微管网络。在除法中 细胞、微管之间循环的广义多功能 相间网络，提供对单元每一个角落的访问，以及 有丝分裂器官位于中心并专门完成这项任务的有丝分裂装置 分离染色体的方法。对于如此频繁使用的 对象，干扰微管功能的试剂在 心脏的许多细胞活动，往往具有深刻的生理意义 效果。其中一些药物在人类身上有治疗应用： 秋水仙碱，用于痛风性关节炎的抗炎特性； 长春新碱、长春新碱和紫杉醇，用于抗有丝分裂作用 抗多种肿瘤；抗真菌药灰黄霉素；以及 其他。除了这些药用用途外，影响微管的制剂还有 对人类健康同样重要，尽管不具有治疗作用 例如，它们被广泛用作杀虫剂和杀菌剂。基本信息 因此，关于微管的信息具有不同寻常的直接和 与美国国立卫生研究院的任务有直接联系。 正如从它们不同的活动中所预期的那样，微管与许多 其他细胞蛋白质。以某种方式理解这些相互作用 将允许合理地操纵基于微管的现象，这 肯定是一种强大的治疗和诊断能力，我们必须 非常详细地了解微管的结构。我们还必须知道 使用微管的超分子组件的结构 提供运动产生、形状确定和细胞器定位 单元格内的功能。幸运的是，值得注意的是，基本结构 微管的大小似乎是恒定的，而不考虑特定的 它所参与的装配。因此，如果我们能确定结构 对于微管的任何一种功能类别，我们都会了解到 很多关于所有微管的信息。 这份提案描述了我们成功地准备了原生细胞 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。我们报道了一些 冷冻电子检查这些微管的初步结果 显微镜，并提出一套实验，将产生一个高 分辨天然微管的三维结构。

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.