Holt Sarah V, Vergnolle Mailys A S, Hussein Deema, Wozniak Marcin J, Allan Victoria J, Taylor Stephen S
Faculty of Life Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK.
J Cell Sci. 2005 Oct 15;118(Pt 20):4889-900. doi: 10.1242/jcs.02614.
Cenp-F is an unusual kinetochore protein in that it localizes to the nuclear matrix in interphase and the nuclear envelope at the G2/M transition; it is farnesylated and rapidly degraded after mitosis. We have recently shown that farnesylation of Cenp-F is required for G2/M progression, its localization to kinetochores, and its degradation. However, the role Cenp-F plays in mitosis has remained enigmatic. Here we show that, following repression of Cenp-F by RNA interference (RNAi), the processes of metaphase chromosome alignment, anaphase chromosome segregation and cytokinesis all fail. Although kinetochores attach to microtubules in Cenp-F-deficient cells, the oscillatory movements that normally occur following K-fibre formation are severely dampened. Consistently, inter-kinetochore distances are reduced. In addition, merotelic associations are observed, suggesting that whereas kinetochores can attach microtubules in the absence of Cenp-F, resolving inappropriate interactions is inhibited. Repression of Cenp-F does not appear to compromise the spindle checkpoint. Rather, the chromosome alignment defect induced by Cenp-F RNA interference is accompanied by a prolonged mitosis, indicating checkpoint activation. Indeed, the prolonged mitosis induced by Cenp-F RNAi is dependent on the spindle checkpoint kinase BubR1. Surprisingly, chromosomes in Cenp-F-deficient cells frequently show a premature loss of chromatid cohesion. Thus, in addition to regulating kinetochore-microtubule interactions, Cenp-F might be required to protect centromeric cohesion prior to anaphase commitment. Intriguingly, whereas most of the sister-less kinetochores cluster near the spindle poles, some align at the spindle equator, possibly through merotelic or lateral orientations.
Cenp-F是一种不同寻常的动粒蛋白，它在间期定位于核基质，在G2/M期转换时定位于核膜；它被法尼基化，并在有丝分裂后迅速降解。我们最近发现，Cenp-F的法尼基化对于G2/M期进程、其在动粒上的定位及其降解是必需的。然而，Cenp-F在有丝分裂中所起的作用仍然是个谜。在这里我们表明，通过RNA干扰（RNAi）抑制Cenp-F后，中期染色体排列、后期染色体分离和胞质分裂过程均失败。尽管在缺乏Cenp-F的细胞中动粒与微管相连，但在K纤维形成后通常发生的振荡运动受到严重抑制。一致的是，动粒间距离减小。此外，观察到了错向连接，这表明尽管在没有Cenp-F的情况下动粒可以连接微管，但解决不适当的相互作用受到抑制。抑制Cenp-F似乎不会损害纺锤体检查点。相反，由Cenp-F RNA干扰诱导的染色体排列缺陷伴随着有丝分裂延长，表明检查点被激活。事实上，由Cenp-F RNAi诱导的有丝分裂延长依赖于纺锤体检查点激酶BubR1。令人惊讶的是，缺乏Cenp-F的细胞中的染色体经常显示出染色单体凝聚力过早丧失。因此，除了调节动粒-微管相互作用外，Cenp-F可能还需要在后期开始前保护着丝粒凝聚力。有趣的是，虽然大多数无姐妹动粒聚集在纺锤体极附近，但有些则排列在纺锤体赤道上，可能是通过错向或侧向取向。
