West R R, Vaisberg E V, Ding R, Nurse P, McIntosh J R
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA.
Mol Biol Cell. 1998 Oct;9(10):2839-55. doi: 10.1091/mbc.9.10.2839.
The "cut" mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11(+) suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11(+) encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.
粟酒裂殖酵母的“cut”突变体在纺锤体形成和/或染色体分离方面存在缺陷，但它们能完成细胞周期，最终导致致死。对cut11(+)温度敏感等位基因的分析表明，该基因对于功能性双极纺锤体的形成是必需的。用微管蛋白和DNA荧光探针揭示了纺锤体结构的缺陷。通过连续切片和电子显微镜对突变纺锤体进行三维重建显示，纺锤极体（SPB）要么无法完成正常复制，要么在核质中自由漂浮。用绿色荧光蛋白标记的Cut11p的定位显示，在整个细胞周期中核膜呈点状染色，从前期早期到中期后期SPB呈染色。这种SPB定位与SPB插入核膜的细胞周期时间相关。免疫电子显微镜证实了Cut11p定位于有丝分裂SPB和核孔复合体。克隆和测序表明，cut11(+)编码一种具有七个推定跨膜结构域的新蛋白，与酿酒酵母基因NDC1具有同源性。这些数据表明，Cut11p作为核膜中的锚定物与核孔复合体和有丝分裂SPB相关联；这一作用对有丝分裂至关重要。
