Wang Limin, Zhu Guixin, Yang Dong, Li Qianyi, Li Yongzhe, Xu Xingzhi, He Dacheng, Zeng Changqing
Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing China.
Cell Motil Cytoskeleton. 2008 Jul;65(7):581-93. doi: 10.1002/cm.20286.
In an attempt to discover novel proteins functioning in both interphase nucleus and mitotic spindle as NuMA does, we carried out cDNA library screening with pooled autoimmune antibodies. Among positive clones we found a recently identified transcription regulatory protein (CDCA4) with the distinctive nuclear-mitotic apparatus distribution. CDCA4 localizes at metaphase spindle poles and the midzone in later stages. Additionally, an intensive CDCA4 accumulation parallel to spindle was observed in half of metaphase cells but not in later stages, implying a transient form of CDCA4 binding to midzone from anaphase. Mitotic arrest dissolved CDCA4 from centrosomes but during the spindle recovery, CDCA4 invariably colocalized with the microtubule nucleation foci as a component of microtubule organization center. RNA interference of CDCA4 resulted in significant increase of multinuclei and multipolar spindles, suggesting impaired function in chromosome segregation or cytokinesis. However, the spindle checkpoint and the centrosome cycle appeared not to be affected by such interference. Furthermore, CDCA4 depletion resulted in accelerated cell proliferation, perhaps due to the disruption of CDCA4 nuclear function as a transcription suppressor. Interphase CDCA4 is localized in nucleoli by immunofluorescence, although GFP-CDCA4 expressed in the nucleoplasm. An N-terminal KRKC domain appears to be the nuclear localization signal as identified by sequence alignment and the expression of truncated mutants. Taken together, our results suggested that as a novel nuclearmitotic apparatus protein, CDCA4 is involved in spindle organization from prometaphase. When anaphase begins, CDCA4 may play a different role as a midzone factor involved in chromosome segregation or cytokinesis.
为了像核有丝分裂器蛋白（NuMA）那样发现同时在间期细胞核和有丝分裂纺锤体中发挥作用的新蛋白质，我们用混合的自身免疫抗体进行了cDNA文库筛选。在阳性克隆中，我们发现了一种最近鉴定出的转录调节蛋白（CDCA4），其具有独特的核有丝分裂器分布。CDCA4定位于中期纺锤体极以及后期的中间区。此外，在一半的中期细胞中观察到与纺锤体平行的强烈CDCA4积累，但在后期则没有，这意味着从后期开始CDCA4以一种瞬时形式与中间区结合。有丝分裂阻滞使CDCA4从中心体解离，但在纺锤体恢复过程中，CDCA4始终与微管成核位点共定位，作为微管组织中心的一个组成部分。对CDCA4进行RNA干扰导致多核和多极纺锤体显著增加，表明其在染色体分离或胞质分裂中的功能受损。然而，纺锤体检查点和中心体周期似乎不受这种干扰的影响。此外，CDCA4缺失导致细胞增殖加速，这可能是由于作为转录抑制因子的CDCA4核功能被破坏所致。通过免疫荧光观察到间期CDCA4定位于核仁，尽管绿色荧光蛋白（GFP）标记的CDCA4在核质中表达。通过序列比对和截短突变体的表达确定，N端的KRKC结构域似乎是核定位信号。综上所述，我们的结果表明，作为一种新的核有丝分裂器蛋白，CDCA4在前期参与纺锤体组织。当后期开始时，CDCA4可能作为参与染色体分离或胞质分裂的中间区因子发挥不同的作用。
