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

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.

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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.

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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 

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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.

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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.

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Correspondence to Mary Dasso.

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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

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