The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis.
The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis.
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NDC80复合蛋白NUF2和HEC1在有丝分裂中对动型微管的附着做出了不同的贡献。
DOI：
10.1091/mbc.e10-08-0671
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发表时间：
2011-03-15
影响因子：
3.3
通讯作者：
Deluca JG
中科院分区：
文献类型：
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作者：
Sundin LJ;Guimaraes GJ;Deluca JG
已知NDC 80复合物在有丝分裂过程中在着丝粒-微管附着中起作用。我们分析了三个独立的结构基序内的复杂的有丝分裂的作用，发现Nuf 2 CH结构域，Hec 1 CH结构域，和Hec 1尾结构域每个在kinetochore-microtubule接口作出不同的贡献。成功的有丝分裂需要着丝粒稳定地附着在纺锤体微管的正端。产生这些附着的核心是NDC 80复合物，由四种蛋白质Spc 24，Spc 25，Nuf 2和Hec 1/Ndc 80组成。结构研究表明，Hec 1和Nuf 2 N末端的部分折叠成钙调蛋白同源（CH）结构域，已知其介导某些蛋白质中的微管结合。Hec 1还包含一个基本的，带正电荷的氨基酸延伸，位于其CH结构域之前，称为“尾巴”。在这里，使用基因沉默和拯救的方法在HeLa细胞中，我们表明，CH结构域的Hec 1，CH结构域的Nuf 2，和Hec 1尾各自有助于以不同的方式kinetochore-microtubule附件。最严重的缺陷，在着丝粒微管附件中观察到获救的细胞与Hec 1 CH结构域突变体，其次是获救的Hec 1尾结构域突变体。然而，用Nuf 2 CH结构域突变体拯救的细胞产生稳定的动粒-微管附着，但未能产生野生型动粒间张力，并且未能及时进入后期。这些数据表明，CH和尾部结构域的Hec 1产生的着丝粒和微管在细胞中的重要联系，而Nuf 2 CH结构域没有。
The NDC80 complex is known to function in kinetochore-microtubule attachment during mitosis. We analyzed the mitotic roles of three separate structural motifs within the complex and found that the Nuf2 CH domain, the Hec1 CH domain, and the Hec1 tail domain each make distinct contributions at the kinetochore-microtubule interface. Successful mitosis requires that kinetochores stably attach to the plus ends of spindle microtubules. Central to generating these attachments is the NDC80 complex, made of the four proteins Spc24, Spc25, Nuf2, and Hec1/Ndc80. Structural studies have revealed that portions of both Hec1 and Nuf2 N termini fold into calponin homology (CH) domains, which are known to mediate microtubule binding in certain proteins. Hec1 also contains a basic, positively charged stretch of amino acids that precedes its CH domain, referred to as the “tail.” Here, using a gene silence and rescue approach in HeLa cells, we show that the CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contributes to kinetochore–microtubule attachment in distinct ways. The most severe defects in kinetochore–microtubule attachment were observed in cells rescued with a Hec1 CH domain mutant, followed by those rescued with a Hec1 tail domain mutant. Cells rescued with Nuf2 CH domain mutants, however, generated stable kinetochore–microtubule attachments but failed to generate wild-type interkinetochore tension and failed to enter anaphase in a timely manner. These data suggest that the CH and tail domains of Hec1 generate essential contacts between kinetochores and microtubules in cells, whereas the Nuf2 CH domain does not.