HVEM TOMOGRAPHY OF YEAST SPINDLE POLE BODIES & EARLY MITOTIC SPINDLES

纺锤体是微管的主要组织结构 芽殖酵母的中心，是功能等同于 高等真核生物的中心体。 我们用的是速冻的 冷冻替代细胞结合HVEM断层扫描， SPB的精细结构和早期纺锤体事件 阵 SPB由不同的层组成， 物理耦合;它被锚定在核膜 具有钩状结构。 单个微管（MT）及其 核质内的相互作用在5-10 nm的三维成像 分辨率，明显优于传统的 电镜 MT负端被加帽并被拴系到 SPB内部斑块，而大多数核MT具有明显的 在它们的正端张开。 未出芽的酵母细胞含有一个 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 其中值长度为约150 nm。 MT增长， 重复但不分离的SPB具有约130 nm的中值长度， 在桥上交错。 当形成双极主轴时， 中位MT长度增加至约300 nm，然后急剧降低至约100 nm。 后期细胞中约30 nm。 3D模型证实没有 常规中期和后期A发生。 这些研究 补充和扩展了已知的三维结构， 酵母有丝分裂纺锤体通过进一步了解 完整细胞中SPB的组织（见研究亮点#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).