title: Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport.
abstract: Karyopherin-dependent molecular transport through the nuclear pore complex is maintained by constant recycling pathways of karyopherins coupled with the Ran-dependent cargo catch-and-release mechanism. Although many studies have revealed the bidirectional dynamics of karyopherins, the entire kinetics of the steady-state dynamics of karyopherin and cargo is still not fully understood. In this study, we used fluorescence recovery after photobleaching and fluorescence loss in photobleaching on live cells to provide convincing in vivo proof that karyopherin-mediated nucleocytoplasmic transport of cargoes is bidirectional. Continuous photobleaching of the cytoplasm of live cells expressing NLS cargoes led to progressive decrease of nuclear fluorescence signals. In addition, experimentally obtained kinetic parameters of karyopherin complexes were used to establish a kinetic model to explain the entire cargo import and export transport cycles facilitated by importin β. The results strongly indicate that constant shuttling of karyopherins, either free or bound to cargo, ensures proper balancing of nucleocytoplasmic distribution of cargoes and establishes effective regulation of cargo dynamics by RanGTP.
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Ogheneochukome
authors.initials: O
authors.lastname: Lolodi
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Hiroya
authors.initials: H
authors.lastname: Yamazaki
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Otsuka
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kumeta
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan yoshimura@lif.kyoto-u.ac.jp.
authors.forename: Shige H
authors.initials: SH
authors.lastname: Yoshimura
mesh_terms: D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism*; D003198:Computer Simulation; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D019913:Nuclear Localization Signals / Q000378:metabolism*; D022022:Nuclear Pore / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism
chemical_list: D028884:Karyopherins; D019913:Nuclear Localization Signals; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein