Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay
Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay
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DOI：
10.1091/mbc.e03-07-0482
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发表时间：
2004-08-01
影响因子：
3.3
通讯作者：
Schebye, XM
中科院分区：
文献类型：
--
作者：
Tanudji, M;Shoemaker, J;Schebye, XM
着丝粒蛋白-E（CENP-E）是一种类似于驱动蛋白的运动蛋白，在前中期染色体的聚集过程中起重要作用。通过各种方法对CENP-E的功能干扰导致一致的表型，即，有丝分裂时染色体不对齐。以前的研究中一个尚未解决的问题是，细胞是否完成有丝分裂或维持有丝分裂停滞在存在未对齐的染色体。利用RNA干扰和视频显微镜，我们分析了缺乏CENP-E的HeLa（H2 B）-GFP细胞有丝分裂进程的动态过程。我们的研究结果表明，这些细胞启动后期后，由于存在未对齐的染色体延迟有丝分裂的进展。在某些分裂细胞中，染色体在分裂后期不排列，导致一些姐妹染色单体不分离，产生非整倍体子细胞。与非洲爪蟾提取物不同，HeLa细胞中CENP-E的丢失不会损害总检查点激活，因为细胞在有丝分裂中响应微管干扰剂而被阻止。然而，在着丝粒处缺乏CENP-E减少了有丝分裂期间BubR 1检查点蛋白的过度磷酸化，这可以解释在缺乏CENP-E的情况下细胞对一些未对齐染色体的敏感性丧失。我们还发现，用诺考达唑预处理使细胞对CENP-E的耗尽敏感，导致更多的未对齐染色体、更长的停滞和细胞死亡。
Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.