Maeshima Kazuhiro, Funakoshi Tomoko, Imamoto Naoko
Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan; Biological Macromolecules Laboratory, Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka, Japan; Department of Genetics, School of Life Science, Graduate University for Advanced Studies (Sokendai), Mishima, Shizuoka, Japan.
Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan; Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, Japan.
Methods Cell Biol. 2014;122:239-54. doi: 10.1016/B978-0-12-417160-2.00011-4.
In eukaryotic cells, the nucleus is a complex and sophisticated organelle that organizes genomic DNA to support essential cellular functions. The nuclear surface contains many nuclear pore complexes (NPCs), channels for macromolecular transport between the cytoplasm and nucleus. It is well known that the number of NPCs almost doubles during interphase in cycling cells. However, the mechanism of NPC formation is poorly understood, presumably because a practical system for analysis does not exist. The most difficult obstacle in the visualization of interphase NPC formation is that NPCs already exist after nuclear envelope formation, and these existing NPCs interfere with the observation of nascent NPCs. To overcome this obstacle, we developed a novel system using the cell-fusion technique (heterokaryon method), previously also used to analyze the shuttling of macromolecules between the cytoplasm and the nucleus, to visualize the newly synthesized interphase NPCs. In addition, we used a photobleaching approach that validated the cell-fusion method. We recently used these methods to demonstrate the role of cyclin-dependent protein kinases and of Pom121 in interphase NPC formation in cycling human cells. Here, we describe the details of the cell-fusion approach and compare the system with other NPC formation visualization methods.
在真核细胞中，细胞核是一个复杂而精密的细胞器，它组织基因组DNA以支持细胞的基本功能。核表面含有许多核孔复合体（NPC），这些是细胞质与细胞核之间大分子运输的通道。众所周知，在循环细胞的间期，NPC的数量几乎会翻倍。然而，NPC形成的机制却鲜为人知，大概是因为不存在用于分析的实用系统。观察间期NPC形成时最困难的障碍是，在核膜形成后NPC就已经存在了，而这些现有的NPC会干扰对新生NPC的观察。为了克服这一障碍，我们开发了一种利用细胞融合技术（异核体法）的新系统，该技术之前也用于分析大分子在细胞质与细胞核之间的穿梭，以此来观察新合成的间期NPC。此外，我们使用了一种光漂白方法来验证细胞融合法。最近，我们利用这些方法证明了细胞周期蛋白依赖性蛋白激酶和Pom121在循环人类细胞间期NPC形成中的作用。在这里，我们描述细胞融合方法的细节，并将该系统与其他NPC形成可视化方法进行比较。
