Rath Uttama, Wang Dong, Ding Yun, Xu Ying-Zhi, Qi Hongying, Blacketer Melissa J, Girton Jack, Johansen Jørgen, Johansen Kristen M
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
J Cell Biochem. 2004 Nov 15;93(5):1033-47. doi: 10.1002/jcb.20243.
We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.
我们利用酵母双杂交相互作用分析鉴定出了染色质结合蛋白(Chromator),这是一种含有新型染色质结构域的蛋白质,它能直接与假定的纺锤体基质蛋白骨架蛋白(Skeletor)相互作用。免疫细胞化学表明,染色质结合蛋白和骨架蛋白在整个细胞周期中都有广泛的共定位。在间期,染色质结合蛋白定位于染色体的间带染色质区域,其模式与骨架蛋白的模式重叠。然而,在有丝分裂期间,染色质结合蛋白和骨架蛋白都从染色体上脱离,并在类似纺锤体的结构中排列在一起。在S2细胞中进行的缺失构建体分析表明,没有染色质结构域的染色质结合蛋白的COOH末端对于细胞核定位和纺锤体定位都足够了。对染色质结合蛋白基因座中的P元素突变分析表明,染色质结合蛋白是一种必需蛋白。此外,在S2细胞中通过RNA干扰耗尽染色质结合蛋白会导致异常的微管纺锤体形态和染色体分离缺陷。这些发现表明,染色质结合蛋白是一种核蛋白,在有丝分裂期间对正常的纺锤体动力学起作用。