Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Nat Cell Biol. 2012 May 13;14(6):593-603. doi: 10.1038/ncb2489.
Cdt1, a protein critical for replication origin licensing in G1 phase, is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase owing to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle-assembly-checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kMT attachment.
Cdt1 是 G1 期复制起始许可所必需的蛋白质，在 S 期降解，但在 G2 期重新积累。我们现在证明，人类 Cdt1 具有可分离的必需有丝分裂功能。Cdt1 通过与 Ndc80 复合物的 Hec1 成分相互作用，在有丝分裂期间定位于动粒。由于动粒微管（kMT）连接异常不稳定和 Mad1 依赖性纺锤体组装检查点活性，G2 期特异性耗尽 Cdt1 会导致细胞在晚前期停滞。Cdt1 结合 Hec1 杆状结构域上的一个独特环，我们证明该环对于 kMT 连接也是必需的。环结构域的突变会阻止 Cdt1 向动粒的定位，并导致细胞停滞在前期。超分辨率荧光显微镜表明，Cdt1 与 Hec1 环结构域的结合促进了体内 Ndc80 复合物的微管依赖性构象变化。这些结果支持这样的结论，即 Cdt1 与 Hec1 的结合对于延长的 Ndc80 构型和稳定的 kMT 连接是必需的。
