Lab for Cell Function Dynamics, BSI, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan.
Development. 2013 Nov;140(22):4624-32. doi: 10.1242/dev.099226. Epub 2013 Oct 23.
The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in 'licensing' of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APC(Cdh1) and SCF(Skp2), which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APC(Cdh1) and SCF(Skp2). By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APC(Cdh1) and SCF(Skp2) in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.
大多数哺乳动物体细胞维持二倍体基因组。然而，一些哺乳动物细胞类型会经历多次基因组复制（内复制），这是正常发育和分化的一部分。例如，胎盘中的滋养层巨细胞（TGC）通过内复制（绕过有丝分裂）成为多倍体，骨髓中的巨核细胞（MKC）通过内有丝分裂（失败的有丝分裂）成为多倍体。在正常的有丝分裂细胞周期中，geminin 和 Cdt1 参与复制原点的“许可”，这确保了复制在细胞周期中仅发生一次。它们的蛋白积累直接受两种 E3 泛素连接酶活性 APC（Cdh1）和 SCF（Skp2）的调节，这两种活性在细胞周期中相互反相波动。尽管在进行内复制的果蝇细胞中已经记录了蛋白的降解介导的振荡积累，但控制正常细胞周期转变的泛素振荡器是否也在哺乳动物内复制过程中发挥作用尚不清楚。在这项研究中，我们使用表达 Fucci 荧光细胞周期探针的转基因小鼠，该探针报告 APC（Cdh1）和 SCF（Skp2）的活性。通过进行长期、高时间分辨率的 Fucci 成像，我们能够在我们设计的培养孔中生长的分化的 TGC 和 MKC 中可视化 APC（Cdh1）和 SCF（Skp2）的相互激活。我们发现 TGC 和 MKC 都跳过胞质分裂，但方式不同，并且 MKC 中泛素振荡器的相互激活随多倍体水平而变化。我们还获得了整个固定的小鼠胎盘内高度多倍体 TGC 的三维重建。因此，Fucci 技术能够揭示分化过程中内复制细胞周期的时空调节。
