Otsuka Shotaro, Szymborska Anna, Ellenberg Jan
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Methods Cell Biol. 2014;122:219-38. doi: 10.1016/B978-0-12-417160-2.00010-2.
The nuclear pore complex (NPC) mediates selective transport across the nuclear envelope (NE) and plays crucial roles in several additional cellular functions. In higher eukaryotes, the NPC and the NE disassemble and reassemble during cell division and live-cell imaging has been a powerful tool to analyze these dynamic processes. Here, we present a method for the kinetic analysis of postmitotic NPC assembly and reestablishment of transport competence in intact cells by multicolor 4D imaging and photoswitching. By applying the methods we have established previously using normal rat kidney to HeLa cells, we demonstrate the conservation of NPC assembly in different mammalian cells. We recently showed that the molecular organization of the NPC can be studied by combining stochastic super-resolution microscopy with single-particle averaging and present this method here in detail.
核孔复合体（NPC）介导物质跨核膜（NE）的选择性运输，并在其他几种细胞功能中发挥关键作用。在高等真核生物中，NPC和NE在细胞分裂过程中会解体并重新组装，而活细胞成像已成为分析这些动态过程的有力工具。在此，我们介绍一种通过多色4D成像和光开关对完整细胞有丝分裂后NPC组装及运输能力重建进行动力学分析的方法。通过将我们之前在正常大鼠肾细胞上建立的方法应用于HeLa细胞，我们证明了NPC组装在不同哺乳动物细胞中的保守性。我们最近表明，可以通过将随机超分辨率显微镜与单颗粒平均相结合来研究NPC的分子组织，并在此详细介绍此方法。
