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Analysis of a RanGTP-regulated gradient in mitotic somatic cells


The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-β family1,2. The Ran–importin-β system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation1,3,4. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran–importin-β system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-β by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran–importin-β–cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports6,7,8, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

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Figure 1: Characterization of the Rango–importin-β interaction in vitro.
Figure 2: Detection of the Ran-regulated mitotic Rango gradient in HeLa cells by FLIM.
Figure 3: Comparison of Rango gradient in mitotic HeLa cells and meiotic X. laevis egg extracts.
Figure 4: Mitotic spindle phenotypes induced by Ran system perturbations in somatic cells.


  1. Weis, K. Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112, 441–451 (2003)

    CAS  Article  Google Scholar 

  2. Pemberton, L. F. & Paschal, B. M. Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6, 187–198 (2005)

    CAS  Article  Google Scholar 

  3. Hetzer, M., Gruss, O. J. & Mattaj, I. W. The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly. Nature Cell Biol. 4, E177–E184 (2002)

    CAS  Article  Google Scholar 

  4. Harel, A. & Forbes, D. J. Importin-β: conducting a much larger cellular symphony. Mol. Cell 16, 319–330 (2004)

    CAS  PubMed  Google Scholar 

  5. Hetzer, M., Bilbao-Cortes, D., Walther, T. C., Gruss, O. J. & Mattaj, I. W. GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol. Cell 5, 1013–1024 (2000)

    CAS  Article  Google Scholar 

  6. Ciciarello, M. et al. Importin-β is transported to spindle poles during mitosis and regulates Ran-dependent spindle assembly factors in mammalian cells. J. Cell Sci. 117, 6511–6522 (2004)

    CAS  Article  Google Scholar 

  7. Arnaoutov, A. & Dasso, M. The Ran GTPase regulates kinetochore function. Dev. Cell 5, 99–111 (2003)

    CAS  Article  Google Scholar 

  8. Arnaoutov, A. et al. Crm1 is a mitotic effector of Ran-GTP in somatic cells. Nature Cell Biol. 7, 626–632 (2005)

    CAS  Article  Google Scholar 

  9. Huber, J., Dickmanns, A. & Luhrmann, R. The importin-β binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro. J. Cell Biol. 156, 467–479 (2002)

    CAS  Article  Google Scholar 

  10. Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnol. 22, 445–449 (2004)

    CAS  Article  Google Scholar 

  11. Kalab, P., Weis, K. & Heald, R. Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts. Science 295, 2452–2456 (2002)

    ADS  CAS  Article  Google Scholar 

  12. Suhling, K., French, P. M. & Phillips, D. Time-resolved fluorescence microscopy. Photochem. Photobiol. Sci. 4, 13–22 (2005)

    CAS  Article  Google Scholar 

  13. Becker, W. et al. Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc. Res. Tech. 63, 58–66 (2004)

    CAS  Article  Google Scholar 

  14. Shelby, R. D., Hahn, K. M. & Sullivan, K. F. Dynamic elastic behaviour of alpha-satellite DNA domains visualized in situ in living human cells. J. Cell Biol. 135, 545–557 (1996)

    CAS  Article  Google Scholar 

  15. Gorlich, D., Seewald, M. J. & Ribbeck, K. Characterization of Ran-driven cargo transport and the RanGTPase system by kinetic measurements and computer simulation. EMBO J. 22, 1088–1100 (2003)

    Article  Google Scholar 

  16. Riddick, G. & Macara, I. G. A systems analysis of importin-α-β mediated nuclear protein import. J. Cell Biol. 168, 1027–1038 (2005)

    CAS  Article  Google Scholar 

  17. Nachury, M. V. et al. Importin-β is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104, 95–106 (2001)

    CAS  Article  Google Scholar 

  18. Wollman, R. et al. Efficient chromosome capture requires a bias in the 'search-and-capture' process during mitotic-spindle assembly. Curr. Biol. 15, 828–832 (2005)

    CAS  Article  Google Scholar 

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The authors wish to thank T. Nishimoto, M. Dasso, J. Fang, M. A. Rizzo, D. W. Piston and F. Melchior for providing reagents, and C. Weirich for performing fluorescence polarization assays. We are grateful to A. Arnaoutov for discussion and sharing unpublished results, C. Weirich, M. Blower, A. Madrid and H. Aaron for critical reading of the manuscript, and members of the Heald and Weis laboratories for discussions. The research described in this article was supported in part by Philip Morris USA Inc. and Philip Morris International (R.H.), and by grants from the National Institute of Health (E.Y.I., R.H. and K.W.). Author Contributions P.K. and A.P. contributed equally to this project.

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Correspondence to Rebecca Heald or Karsten Weis.

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Kaláb, P., Pralle, A., Isacoff, E. et al. Analysis of a RanGTP-regulated gradient in mitotic somatic cells. Nature 440, 697–701 (2006).

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