High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the Yeast Saccharomyces cerevisiae

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5–10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded 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 that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ∼300 nm and then decreases to ∼30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.

纺锤体极体（spindle pole body, SPB）是出芽酵母的核心微管组织中心，其功能等价于高等真核细胞中的中心体。本研究采用快速冷冻-冷冻替代细胞结合高压电子断层成像技术，对SPB的精细结构与纺锤体早期形成过程展开系统性探究。研究中以5–10 nm的分辨率实现了单个结构的三维成像，其成像质量显著优于连续切片电子显微镜技术所能达到的水平。

SPB由独特但相互偶联的层状结构组成，其中两层呈现有序的二维排布。SPB的中央斑块通过钩状结构锚定至核被膜。核微管（microtubules, MTs）的负端被封端，并锚定至SPB的内层斑块；而绝大多数微管的正端呈现出明显的膨大结构。

仅携带单个SPB的未出芽细胞中存在16条微管，其数量足以与预期的16条染色体一一结合，这些微管的中位长度约为150 nm。从已复制但未分离的SPB上生长出的微管，中位长度约为130 nm，并在连接两个SPB的桥状结构处相互交错。随着双极纺锤体形成，微管的中位长度增至约300 nm；而在后期晚期，微管的中位长度会降至约30 nm。三维模型证实，该过程不存在典型的有丝分裂中期，且存在后期A（anaphase A）。

本研究补充并拓展了当前关于酵母有丝分裂纺锤体三维结构的认知，进一步加深了我们对完整细胞内SPB组织方式的理解。