Dynamic behavior of Nuf2-Hec1 complex that localizes to the centrosome and centromere and is essential for mitotic progression in vertebrate cells
Dynamic behavior of Nuf2-Hec1 complex that localizes to the centrosome and centromere and is essential for mitotic progression in vertebrate cells
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DOI：
10.1242/jcs.00645
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发表时间：
2003-08-15
影响因子：
4
通讯作者：
Fukagawa, T
中科院分区：
文献类型：
--
作者：
Hori, T;Haraguchi, T;Fukagawa, T
Nuf 2和Hec 1是进化上保守的着丝粒蛋白。为了阐明这些蛋白在脊椎动物细胞中的功能，我们在鸡DT 40细胞中对其进行了表征。我们产生了Nuf 2和Hec 1的GFP融合构建体，以详细研究这些蛋白在细胞周期中的定位。我们发现，Nuf 2是与Hec 1在整个细胞周期，这个复杂的是本地化的中心体在G1和S期，然后移动通过核膜的着丝粒在G2期。在有丝分裂过程中，这种复合物定位于着丝粒。我们还创造了Nuf 2和Hec 1的条件性功能丧失突变体。在这两种突变体中，细胞周期停滞在前中期，这表明Nuf 2-Hec 1复合物是必不可少的有丝分裂进程。内着丝粒蛋白CENP-A，-C，和-H和检查点蛋白BubR 1被定位到染色体的突变细胞在早中期被捕，但Mad 2本地化被废除。此外，光漂白实验表明，Nuf 2-Hec 1复合物是稳定的与着丝粒和该复合物与中心体的相互作用是动态的。
Nuf2 and Hec1 are evolutionarily conserved centromere proteins. To clarify the functions of these proteins in vertebrate cells, we characterized them in chicken DT40 cells. We generated GFP fusion constructs of Nuf2 and Hec1 to examine in detail the localization of these proteins during the cell cycle. We found that Nuf2 is associated with Hec1 throughout the cell cycle and that this complex is localized to the centrosomes during G1 and S phases and then moves through the nuclear membrane to the centromere in G2 phase. During mitosis, this complex is localized to the centromere.We also created conditional loss-of-function mutants of Nuf2 and Hec1. In both mutants, the cell cycle arrested at prometaphase, suggesting that the Nuf2-Hec1 complex is essential for mitotic progression. The inner centromere proteins CENP-A, -C, and -H and checkpoint protein BubR1 were localized to chromosomes in the mutant cells arrested at prometaphase, but Mad2 localization was abolished. Furthermore, photobleaching experiments revealed that the Nuf2-Hec1 complex is stably associated with the centromere and that interaction of this complex with the centrosome is dynamic.