Letter | Published:

Analysis of a RanGTP-regulated gradient in mitotic somatic cells

Nature volume 440, pages 697701 (30 March 2006) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

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

  2. 2.

    & Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6, 187–198 (2005)

  3. 3.

    , & The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly. Nature Cell Biol. 4, E177–E184 (2002)

  4. 4.

    & Importin-β: conducting a much larger cellular symphony. Mol. Cell 16, 319–330 (2004)

  5. 5.

    , , , & GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol. Cell 5, 1013–1024 (2000)

  6. 6.

    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)

  7. 7.

    & The Ran GTPase regulates kinetochore function. Dev. Cell 5, 99–111 (2003)

  8. 8.

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

  9. 9.

    , & 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)

  10. 10.

    , , & An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnol. 22, 445–449 (2004)

  11. 11.

    , & Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts. Science 295, 2452–2456 (2002)

  12. 12.

    , & Time-resolved fluorescence microscopy. Photochem. Photobiol. Sci. 4, 13–22 (2005)

  13. 13.

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

  14. 14.

    , & Dynamic elastic behaviour of alpha-satellite DNA domains visualized in situ in living human cells. J. Cell Biol. 135, 545–557 (1996)

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

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

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

Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA

    • Petr Kaláb
    • , Arnd Pralle
    • , Ehud Y. Isacoff
    • , Rebecca Heald
    •  & Karsten Weis

Authors

  1. Search for Petr Kaláb in:

  2. Search for Arnd Pralle in:

  3. Search for Ehud Y. Isacoff in:

  4. Search for Rebecca Heald in:

  5. Search for Karsten Weis in:

Competing interests

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

Corresponding authors

Correspondence to Rebecca Heald or Karsten Weis.

Supplementary information

Word documents

  1. 1.

    Supplementary Notes

    This file contains Supplementary Figures 1–9, Supplementary Table 1, Supplementary Methods and additional references.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature04589

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.