Yoder Tennessee J, McElwain Mark A, Francis Susan E, Bagley Joy, Muller Eric G D, Pak Brian, O'Toole Eileen T, Winey Mark, Davis Trisha N
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Mol Biol Cell. 2005 Jan;16(1):141-52. doi: 10.1091/mbc.e04-08-0703. Epub 2004 Nov 3.
The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.
纺锤体极体（SPB）是酿酒酵母中的微管组织中心。SPB的一项重要任务是确保双极纺锤体的组装，这需要在微管和染色体上的力达到适当平衡。SPB组件Spc110p将纺锤体微管的末端连接到SPB的核心。我们之前报道了分离出一个突变等位基因spc110 - 226，它会导致纺锤体形成30分钟后纺锤体断裂和SPB解体。通过用绿色荧光蛋白（GFP）- Tub1p或Spc97p - GFP对突变细胞进行活细胞成像，我们发现spc110 - 226突变细胞在纺锤体组装方面存在早期缺陷。短纺锤体形成但无法发育到1.5微米阶段并经常崩溃。突变细胞中的动粒排列不正常。在70%的细胞中，没有发生稳定的双定向，所有动粒仅与一个SPB相关联。通过电子显微镜断层扫描和荧光显微镜对SPB残余物的检查表明，Spc110 - 226p/钙调蛋白复合物从SPB的中央板上剥离，并在核质中聚合并形成一个成核结构。中央板组件Spc42p和Spc29p留在核膜中。通过荧光共振能量转移分析检测到，这种分层可能是由于Spc42p和Spc110 - 226p之间相互作用受到干扰所致。我们认为，染色体双定向施加在SPB上的力将Spc110 - 226p从SPB中拉出；姐妹染色单体的凝聚所施加的力的消除使碎片化减少了四倍。细胞质微管施加的力的消除对碎片化没有影响。我们的结果为动粒和细胞质微管对双极纺锤体形成过程中所涉及的力的相对贡献提供了见解。
