Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The Nup107-160 complex and γ-TuRC regulate microtubule polymerization at kinetochores

Nature Cell Biology volume 12, pages 164–169 (2010)Cite this article

Subjects

Abstract

The metazoan nuclear pore complex (NPC) disassembles during mitosis, and many of its constituents distribute onto spindles and kinetochores, including the Nup107-160 sub-complex1,2. We have found that Nup107-160 interacts with the γ-tubulin ring complex (γ-TuRC), an essential and conserved microtubule nucleator3,4, and recruits γ-TuRC to unattached kinetochores. The unattached kinetochores nucleate microtubules in a manner that is regulated by Ran GTPase5; such microtubules contribute to the formation of kinetochore fibres (k-fibres)6, microtubule bundles connecting kinetochores to spindle poles. Our data indicate that Nup107-160 and γ-TuRC act cooperatively to promote spindle assembly through microtubule nucleation at kinetochores: HeLa cells lacking Nup107-160 or γ-TuRC were profoundly deficient in kinetochore-associated microtubule nucleation. Moreover, co-precipitated Nup107-160– γ-TuRC complexes nucleated microtubule formation in assays using purified tubulin. Although Ran did not regulate microtubule nucleation by γ-TuRC alone, Nup107-160–γ-TuRC complexes required Ran–GTP for microtubule nucleation. Collectively, our observations show that Nup107-160 promotes spindle assembly through Ran–GTP-regulated nucleation of microtubules by γ-TuRC at kinetochores, and reveal a relationship between nucleoporins and the microtubule cytoskeleton.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Nup107-160 binds γ-TuRC.
Figure 2: γ-TuRC is recruited to kinetochores by Nup107-160.
Figure 3: Nup107-160 is critical for microtubule nucleation at kinetochores in HeLa cells.
Figure 4: Ran–GTP regulates microtubule nucleation by xNup107-160/γ-TuRC complexes.
Figure 5: Model for Nup107-160 and γ-TuRC regulation at kinetochores.

References

  1. Orjalo, A. V. et al. The Nup107–160 nucleoporin complex is required for correct bipolar spindle assembly. Mol. Biol. Cell 17, 3806–3818 (2006).

    Article  CAS  Google Scholar 

  2. Loiodice, I. et al. The entire Nup107–160 complex, including three new members, is targeted as one entity to kinetochores in mitosis. Mol. Biol. Cell 15, 3333–3344 (2004).

    Article  CAS  Google Scholar 

  3. Luders, J. & Stearns, T. Microtubule-organizing centres: a re-evaluation. Nature Rev. Mol. Cell Biol. 8, 161–167 (2007).

    Article  Google Scholar 

  4. Wiese, C. & Zheng, Y. Microtubule nucleation: γ-tubulin and beyond. J. Cell Sci. 119, 4143–4153 (2006).

    Article  CAS  Google Scholar 

  5. Torosantucci, L., De Luca, M., Guarguaglini, G., Lavia, P. & Degrassi, F. Localized RanGTP accumulation promotes microtubule nucleation at kinetochores in somatic mammalian cells. Mol. Biol. Cell 19, 1873–1882 (2008).

  6. O'Connell, C. B. & Khodjakov, A. L. Cooperative mechanisms of mitotic spindle formation. J. Cell Sci. 120, 1717–1722 (2007).

    Article  CAS  Google Scholar 

  7. Belgareh, N. et al. An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells. J. Cell Biol. 154, 1147–1160 (2001).

    Article  CAS  Google Scholar 

  8. Aitchison, J. D., Blobel, G. & Rout, M. P. Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport. J. Cell Biol. 131, 1659–1675 (1995).

    Article  CAS  Google Scholar 

  9. Zuccolo, M. et al. The human Nup107-160 nuclear pore subcomplex contributes to proper kinetochore functions. EMBO J. 26, 1853–1864 (2007).

    Article  CAS  Google Scholar 

  10. Wilde, A. & Zheng, Y. Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran. Science 284, 1359–1362 (1999).

    Article  CAS  Google Scholar 

  11. Heald, R. et al. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425 (1996).

    Article  CAS  Google Scholar 

  12. Earnshaw, W. C. & Rothfield, N. Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma 91, 313–321 (1985).

    Article  CAS  Google Scholar 

  13. Tulu, U. S., Fagerstrom, C., Ferenz, N. P. & Wadsworth, P. Molecular requirements for kinetochore-associated microtubule formation in mammalian cells. Curr. Biol. 16, 536–541 (2006).

    Article  CAS  Google Scholar 

  14. Zheng, Y., Wong, M. L., Alberts, B. & Mitchison, T. Nucleation of microtubule assembly by a γ-tubulin-containing ring complex. Nature 378, 578–583 (1995).

    Article  CAS  Google Scholar 

  15. Stewart, M. Molecular mechanism of the nuclear protein import cycle. Nature Rev. Mol. Cell Biol. 8, 195–208 (2007).

    Article  CAS  Google Scholar 

  16. Klebe, C., Bischoff, F. R., Ponstingl, H. & Wittinghofer, A. Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1. Biochemistry 34, 639–647 (1995).

    Article  CAS  Google Scholar 

  17. Harel, A. et al. Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores. Mol. Cell 11, 853–864 (2003).

    Article  CAS  Google Scholar 

  18. Walther, T. C. et al. The conserved Nup107-160 complex is critical for nuclear pore complex assembly. Cell 113, 195–206 (2003).

    Article  CAS  Google Scholar 

  19. Kutay, U. & Hetzer, M. W. Reorganization of the nuclear envelope during open mitosis. Curr. Opin. Cell Biol. 20, 669–677 (2008).

    Article  CAS  Google Scholar 

  20. Joseph, J., Liu, S. T., Jablonski, S. A., Yen, T. J. & Dasso, M. The RanGAP1–RanBP2 complex is essential for microtubule–kinetochore interactions in vivo. Curr. Biol. 14, 611–617 (2004).

    Article  CAS  Google Scholar 

  21. Maiato, H., Rieder, C. L. & Khodjakov, A. Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis. J. Cell Biol. 167, 831–840 (2004).

    Article  CAS  Google Scholar 

  22. Goshima, G. et al. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316, 417–421 (2007).

    Article  CAS  Google Scholar 

  23. 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 

  24. Resendes, K. K., Rasala, B. A. & Forbes, D. J. Centrin 2 localizes to the vertebrate nuclear pore and plays a role in mRNA and protein export. Mol. Cell Biol. 28, 1755–1769 (2008).

    Article  CAS  Google Scholar 

  25. Rebollo, E., Llamazares, S., Reina, J. & Gonzalez, C. Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes. PLoS Biol. 2, E8 (2004).

    Article  Google Scholar 

  26. Seltzer, V. et al. Arabidopsis GCP2 and GCP3 are part of a soluble γ-tubulin complex and have nuclear envelope targeting domains. Plant J. 52, 322–331 (2007).

    Article  CAS  Google Scholar 

  27. Knop, M., Pereira, G. & Schiebel, E. Microtubule organization by the budding yeast spindle pole body. Biol. Cell 91, 291–304 (1999).

    Article  CAS  Google Scholar 

  28. Goshima, G., Mayer, M., Zhang, N., Stuurman, N. & Vale, R. D. Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle. J. Cell Biol. 181, 421–429 (2008).

    Article  CAS  Google Scholar 

  29. Desai, A., Murray, A., Mitchison, T. J. & Walczak, C. E. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61, 385–412 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

R.K.M., A.A. and M.D. were supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development Intramural funds (Z01 HD008816 and Z01 HD008740). B.M.A.F. and P.C. were supported by National Institutes of Health R01 GM07159 and Texas HEB (Higher Education Board) 010019-0022-2006.

Author information

Authors and Affiliations

  1. Laboratory of Gene Regulation and Development, NICHD/NIH, Bethesda, 20892, MD, USA

    Ram Kumar Mishra, Alexei Arnaoutov & Mary Dasso

  2. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, 75390, Texas, USA

    Papia Chakraborty & Beatriz M.A. Fontoura

Authors
  1. Ram Kumar Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Papia Chakraborty
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexei Arnaoutov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Beatriz M.A. Fontoura
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mary Dasso
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to experimental design. R.K.M., A.A. and P.C. performed the experiments and analysed the data; M.D. wrote the manuscript.

Corresponding author

Correspondence to Mary Dasso.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 541 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mishra, R., Chakraborty, P., Arnaoutov, A. et al. The Nup107-160 complex and γ-TuRC regulate microtubule polymerization at kinetochores. Nat Cell Biol 12, 164–169 (2010). https://doi.org/10.1038/ncb2016

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2016

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing