HVEM TOMOGRAPHY OF YEAST SPINDLE POLE BODIES & EARLY MITOTIC SPINDLES

纺锤体是微管的主要组织形式 发芽酵母菌的中心，在功能上相当于 高等真核生物中的中心体。我们用的是速冻， 冷冻替代细胞联合高能电子显微镜断层扫描研究 SPB的精细结构与早期纺锤体事件 队形。SPB由不同的层组成，这些层 物理耦合；它固定在核膜上 具有钩状结构。单个微管(MT)及其功能 核质内的相互作用在5-10 nm的3-D成像 分辨率，明显好于传统的 电子显微镜。MT的减端被封口并系在 SPB内部斑块，而大多数核MT有明显的 它们的正端都在发亮。未萌发的酵母细胞含有单一的 SPB保留16个MT，足以连接到预期的16个MT中的每一个 染色体。它们的平均长度为~150 nm。MTS发展自 复制但未分离的SPB的中位数长度为~130 nm 在桥上交错摆动。当形成两极心轴时 MT长度中值增加到~300 nm，然后急剧下降到 细胞分裂后期为30 nm左右。3-D模型证实没有 常规中期和后期A发生。这些研究 补充和扩展了关于三维结构的已知内容 通过进一步了解酵母有丝分裂纺锤体 完整细胞中SPB的组织(见研究重点3)。

The spindle pole body (SPB) is the major microtubule organizing center of budding yeasts and is the functional equivalent of the centrosome in higher eukaryotes. We are using fast-frozen, freeze-substituted cells in conjunction with HVEM tomography to study the fine structure of the SPB and the events of early spindle formation. The SPB is organized from distinct layers that are physically coupled; it is anchored in the nuclear envelope membrane with hook-like structures. Individual microtubules (MTs) and their interactions within the nucleoplasm are imaged at 5-10 nm 3-D resolution, significantly better than that achieved by conventional electron microscopy. MT minus ends are capped and are tethered to the SPB inner plaque, whereas the majority of nuclear MTs have a distinct flaring at their plus ends. Unbudded yeast cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ~150 nm. MTs growing from duplicated but not separated SPBs have a median length of ~130 nm and interdigitate over the bridge. As a bipolar spindle is formed the median MT length increases to ~300nm, then decreases dramatically to ~30 nm in cells in late anaphase. 3-D models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the 3-D structure of the yeast mitotic spindle by furthering our understanding of the organization of the SPB in intact cells (See Research Highlight #3).