Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores
Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores
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DOI：
10.1016/j.cub.2003.10.056
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发表时间：
2003-12-02
期刊：
影响因子：
9.2
通讯作者：
Salmon, ED
中科院分区：
文献类型：
--
作者：
DeLuca, JG;Howell, BJ;Salmon, ED
Ndc 80/Nuf 2复合物的成员已在几个系统中显示在有丝分裂中稳定的着丝粒-微管附着和染色体排列的形成中是重要的[1-9]。在HeLa细胞中，我们已经证明，通过RNA干扰（RNAi）耗尽Nuf 2导致具有活性纺锤体检查点的强烈前中期阻滞，这与没有或很少有稳定动粒微管的动粒处的低但可检测的Mad 2相关[5]。另一项在HeLa细胞中进行的RNAi研究报道，Hec 1（人类Ndc 80同系物）是Mad 1和Mad 2与动粒结合所必需的，而动粒结合的Mad 2在产生和维持纺锤体组装检查点方面不起作用[6]。在这里，我们表明，无论是Nuf 2或Hec 1在HeLa细胞中的RNAi耗竭的结果在着丝粒和细胞质中的蛋白质减少。Mad 1和Mad 2在后期前期/早期前中期集中在动粒，但在前中期阻滞的过程中被耗尽5倍或更多，这是Mad 2依赖的。Mad 1和Mad 2的还原在纺锤体解聚时是可逆的。我们的观察结果支持一个模型，其中Nuf 2和Hec 1的功能，以防止微管依赖性剥离Mad 1和Mad 2的着丝粒尚未形成稳定的着丝粒微管附件。
Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis [1-9]. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules [5]. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint [6]. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.