Institute of Molecular Biology and Pathology, National Research Council of Italy, Rome, Italy.
PLoS One. 2011 Jan 28;6(1):e16307. doi: 10.1371/journal.pone.0016307.
Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.
METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.
CONCLUSIONS/SIGNIFICANCE: Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.
高度表达的癌症蛋白 1（Hec1）是 Ndc80 复合物的组成部分，Ndc80 复合物是动粒的一个组成部分，在稳定的动粒-微管附着、染色体排列和有丝分裂中的纺锤体检查点激活中具有基本作用。在几种癌细胞中发现 HEC1 RNA 上调，提示 HEC1 失调在癌症中的作用。有鉴于此，我们在人细胞的诱导表达系统中研究了实验驱动的 Hec1 表达对有丝分裂和染色体分离的影响。
方法/主要发现：在可诱导表达 C 端标记的 Hec1 或未标记的 Hec1 的 HeLa 克隆中，Hec1 的过表达从未获得过，这表明 Hec1 细胞水平受到严格控制。相反，带有 EGFP 标签融合到 Hec1 N 端的嵌合蛋白在细胞中积累，并破坏有丝分裂分裂。EGFP-Hec1 细胞在源自中心粒分裂的多极纺锤体中经历了改变的染色体分离。我们发现，EGFP-Hec1 组装了一种不能挽救 Hec1 耗竭的有丝分裂表型的突变 Ndc80 复合物。带有 EGFP-Hec1 的动粒形成持续的侧向微管-动粒相互作用，招募了微管解聚酶 MCAK 和微管稳定蛋白 HURP 在 K 纤维上。在这些条件下，正端动力蛋白 CENP-E 优先保留在动粒上。RNAi 介导的 CENP-E 耗竭进一步证明了 CENP-E 功能对于 EGFP-Hec1 表达细胞中的多极纺锤体形成是必需的。
结论/意义：我们的研究表明，Hec1 N 端尾巴的修饰可以改变动粒-微管附着的稳定性，并独立于蛋白质的细胞内水平影响 Ndc80 复合物的功能。N 端修饰的 Hec1 通过 CENP-E 介导的正端定向动粒拉力促进纺锤体极片段化，破坏了调节纺锤体两极性的动粒和中心体相关力之间的精细平衡。总的来说，我们的研究结果支持这样一种模型，即中心体的完整性受调节动粒-微管附着稳定性的途径影响。
