Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
J Cell Biol. 2010 Jun 14;189(6):937-43. doi: 10.1083/jcb.200912022.
To define the molecular architecture of the kinetochore in vertebrate cells, we measured the copy number of eight kinetochore proteins that link kinetochore microtubules (MTs [kMTs]) to centromeric DNA. We used a fluorescence ratio method and chicken DT40 cell lines in which endogenous loci encoding the analyzed proteins were deleted and complemented using integrated green fluorescent protein fusion transgenes. For a mean of 4.3 kMTs at metaphase, the protein copy number per kMT is between seven and nine for members of the MT-binding KNL-1/Mis12 complex/Ndc80 complex network. It was between six and nine for four members of the constitutive centromere-associated network: centromere protein C (CENP-C), CENP-H, CENP-I, and CENP-T. The similarity in copy number per kMT for all of these proteins suggests that each MT end is linked to DNA by six to nine fibrous unit attachment modules in vertebrate cells, a conclusion that indicates architectural conservation between multiple MT-binding vertebrate and single MT-binding budding yeast kinetochores.
为了定义脊椎动物细胞中的动粒分子结构，我们测量了连接动粒微管（kMTs）和着丝粒 DNA 的八种动粒蛋白的拷贝数。我们使用荧光比率法和鸡 DT40 细胞系，其中内源编码分析蛋白的基因被删除，并使用整合的绿色荧光蛋白融合转基因进行互补。对于中期平均 4.3 个 kMT，MT 结合 KNL-1/Mis12 复合物/Ndc80 复合物网络成员的每个 kMT 的蛋白拷贝数在 7 到 9 之间。在组成性着丝粒相关网络的四个成员中，有四个成员的蛋白拷贝数在 6 到 9 之间：着丝粒蛋白 C（CENP-C）、CENP-H、CENP-I 和 CENP-T。所有这些蛋白质的 kMT 拷贝数相似，这表明每个 MT 末端通过六个到九个纤维单位附着模块与 DNA 相连，这一结论表明脊椎动物和单 MT 结合芽殖酵母动粒之间的多个 MT 结合结构具有结构保守性。
