title: Imaging the assembly, structure, and function of the nuclear pore inside cells.
abstract: 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.
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Otsuka
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Szymborska
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Ellenberg
mesh_terms: D000818:Animals; D001692:Biological Transport; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008853:Microscopy / Q000379:methods*; D008938:Mitosis; D046912:Multiprotein Complexes / Q000032:analysis*; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000032:analysis*; D051381:Rats; D059807:S Phase Cell Cycle Checkpoints
chemical_list: D046912:Multiprotein Complexes; D028861:Nuclear Pore Complex Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Live-cell imaging; Nuclear envelope; Nuclear transport; Nucleoporin; Particle averaging; Photoswitching; STORM; Super-resolution microscopy