title: Cell-fusion method to visualize interphase nuclear pore formation.
abstract: 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.
authors.affiliation: 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.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Maeshima
authors.affiliation: Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan; Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Funakoshi
authors.affiliation: Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Imamoto
mesh_terms: D002459:Cell Fusion / Q000379:methods*; D045744:Cell Line, Tumor; D059585:G1 Phase Cell Cycle Checkpoints; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000502:physiology; D046912:Multiprotein Complexes / Q000096:biosynthesis / Q000378:metabolism*; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000096:biosynthesis / Q000378:metabolism*; D038761:Photobleaching; D011092:Polyethylene Glycols / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering; D013501:Surface-Active Agents / Q000494:pharmacology
chemical_list: D046912:Multiprotein Complexes; D028861:Nuclear Pore Complex Proteins; D034741:RNA, Small Interfering; D013501:Surface-Active Agents; D011092:Polyethylene Glycols
keywords: Cell cycle; Cell fusion; Heterokaryons; Interphase; NPC; Nuclear structure; Nucleocytoplasmic transport; Nups; PEG