HVEM TOMOGRAPHY OF YEAST SPINDLE POLE BODIES & EARLY MITOTIC SPINDLES

我们使用冷冻固定材料和双轴HVEM断层扫描 为了研究纺锤体的精细结构， 纺锤体的形成。 酵母是 高压冷冻，冷冻替代和包埋在Lowicryl中 HM20。 酵母细胞在适当阶段的三维重建 使用双轴HVEM断层摄影术获得细胞周期。 部分 在细胞周期的不同阶段， 重建：1个来自G1中的细胞，2个显示G1中SPB的细胞， 早期复制，3个细胞具有复制但未分离的SPB， 2个细胞由约1.2 μ m的纺锤体分隔。 初步 来自这些细胞的数据显示，在SPB复制期间， 比女儿大得多， 微管 此外，许多微管显示出向外的 在其正端张开。 HVEM提供的更高分辨率 固定良好的材料的断层扫描应该可以让我们研究3D SPB的结构和有丝分裂形成的结构 使用标准串行部分无法实现的主轴 重建方法 这些研究应该提供洞察如何 动粒微管的排列方式与有丝分裂纺锤体的排列方式相同， 形成并提供用于野生型和 突变株

We are using cryofixed material and dual tilt-axis HVEM tomography to study the fine structure of the spindle pole body (SPB) and early spindle formation in the yeast, Saccharomyces cerevisiae. Yeast are high pressure frozen, freeze substituted and embedded in Lowicryl HM20. 3-D reconstructions of yeast cells at the appropriate stage of the cell cycle are obtained using dual-axis HVEM tomography. Portions of eight cells at different stages of the cell cycle have been reconstructed: 1 from a cell in G1, 2 cells that show the SPB in early duplication, 3 cells that have duplicated but unseparated SPBs, and 2 cells that SPBs separated by a ~ 1.2um spindle. Preliminary data from these cells show that during SPB duplication, the mother SPB is significantly larger than the daughter and initiates more microtubules. In addition, many of the microtubules show an outward flare at their plus ends. The increased resolution afforded by HVEM tomography of well-fixed material should allow us to study the 3D structure of both the SPB and the structure of forming mitotic spindles that would not be possible using standard serial section reconstruction methods. These studies should provide insight into how kinetochore microtubules are organized as the mitotic spindle is forming and provide a benchmark for comparison between wild-type and mutant strains.