title: Analysis of nucleocytoplasmic transport in digitonin-permeabilized cells under different cellular conditions.
abstract: The regulation of nucleocytoplasmic transport is crucial not only for basic cellular activities but also for physiological adaptation to specific situation during the cell cycle, development, or stress. Although a wide variety of transport pathways have been identified in eukaryotic cells, the functional significance of their multiplicity remains unclear. The best-characterized nuclear transport receptors (NTRs) are the members of the importin β family (karyopherin, transportin) whose association with specific cargoes is regulated by the GTPase Ran. In this chapter, we first provide an overview of the various expression vectors used to purify recombinant NTRs. We then describe two sets of recent examples of using well-established digitonin-permeabilized cell-free transport systems in mammalian cells to mimic different cellular conditions in living cells: normal/heat-shock conditions and interphase/mitosis. In the former case, physiological regulation impacts different transport pathways in opposite ways. In the latter case, the importin β-Ran system is used at different cell-cycle stages but with the same biochemical principle to specify the nuclear localization and chromatin loading of a specific protein, respectively. This in vitro transport assay, when adapted to specific cellular conditions or particular substrates, should help to uncover specific transport pathways or transport factors function under different cellular conditions.
authors.affiliation: Department of Molecular Genetics, National Institute of Genetics, The Graduate University for Advanced Studies Sokendai, Mishima, Shizuoka, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Furuta
authors.affiliation: Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kose
authors.affiliation: Department of Molecular Biology, Faculty of Medicine, Georg-August-University of Göttingen, Göttingen, Germany.
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Kehlenbach
authors.affiliation: Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Imamoto
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D045744:Cell Line, Tumor; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000235:genetics; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D008938:Mitosis / Q000235:genetics; D022022:Nuclear Pore / Q000378:metabolism; D015398:Signal Transduction; D028901:alpha Karyopherins / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C098764:KIF22 protein, human; D028901:alpha Karyopherins; D028961:beta Karyopherins; D016547:Kinesins; D020931:ran GTP-Binding Protein; D004072:Digitonin
keywords: Digitonin; HeLa cells; Heat shock; Importins; In vitro assay; Karyopherins; Mitosis; Nuclear import; Nuclear transport factors