Analysis of a RanGTP-regulated gradient in mitotic somatic cells (original) (raw)

Nature volume 440, pages 697–701 (2006)Cite this article

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Weis, K. Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112, 441–451 (2003)
    Article CAS Google Scholar
  2. Pemberton, L. F. & Paschal, B. M. Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6, 187–198 (2005)
    Article CAS 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)
    Article CAS 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)
    Article CAS 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)
    Article CAS Google Scholar
  7. Arnaoutov, A. & Dasso, M. The Ran GTPase regulates kinetochore function. Dev. Cell 5, 99–111 (2003)
    Article CAS 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)
    Article CAS 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)
    Article CAS 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)
    Article CAS 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)
    Article ADS CAS Google Scholar
  12. Suhling, K., French, P. M. & Phillips, D. Time-resolved fluorescence microscopy. Photochem. Photobiol. Sci. 4, 13–22 (2005)
    Article CAS Google Scholar
  13. Becker, W. et al. Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc. Res. Tech. 63, 58–66 (2004)
    Article CAS 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)
    Article CAS 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)
    Article CAS 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)
    Article CAS 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)
    Article CAS Google Scholar

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, California, 94720-3200, Berkeley, USA
    Petr Kaláb, Arnd Pralle, Ehud Y. Isacoff, Rebecca Heald & Karsten Weis

Authors

  1. Petr Kaláb
    You can also search for this author inPubMed Google Scholar
  2. Arnd Pralle
    You can also search for this author inPubMed Google Scholar
  3. Ehud Y. Isacoff
    You can also search for this author inPubMed Google Scholar
  4. Rebecca Heald
    You can also search for this author inPubMed Google Scholar
  5. Karsten Weis
    You can also search for this author inPubMed Google Scholar

Corresponding authors

Correspondence toRebecca Heald or Karsten Weis.

Ethics declarations

Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Figures 1–9, Supplementary Table 1, Supplementary Methods and additional references. (DOC 730 kb)

Rights and permissions

About this article

Cite this article

Kaláb, P., Pralle, A., Isacoff, E. et al. Analysis of a RanGTP-regulated gradient in mitotic somatic cells.Nature 440, 697–701 (2006). https://doi.org/10.1038/nature04589

Download citation

This article is cited by