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.

Poly(ADP-ribose) is required for spindle assembly and structure

Abstract

The mitotic spindle is typically thought of as an array of microtubules, microtubule-associated proteins and motors that self-organizes to align and segregate chromosomes1. The major spindle components consist of proteins and DNA, the primary structural elements of the spindle1. Other macromolecules including RNA and lipids also associate with spindles, but their spindle function, if any, is unknown. Poly(ADP-ribose) (PAR) is a large, branched, negatively charged polymeric macromolecule whose polymerization onto acceptor proteins is catalysed by a family of poly(ADP-ribose) polymerases (PARPs)2. Several PARPs localize to the spindle in vertebrate cells, suggesting that PARPs and/or PAR have a role in spindle function2. Here we show that PAR is enriched in the spindle and is required for spindle function—PAR hydrolysis or perturbation leads to rapid disruption of spindle structure, and hydrolysis during spindle assembly blocks the formation of bipolar spindles. PAR exhibits localization dynamics that differ from known spindle proteins and are consistent with a low rate of turnover in the spindle. Thus, PAR is a non-proteinaceous, non-chromosomal component of the spindle required for bipolar spindle assembly and function.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: PAR is enriched in the spindle.
Figure 2: PAR is required for bipolar spindle assembly and function.
Figure 3: PAR exchange between the spindle and cytoplasm is slow.

References

  1. Wittmann, T., Hyman, A. & Desai, A. The spindle: a dynamic assembly of microtubules and motors. Nature Cell Biol. 3, E28–E34 (2001)

    CAS  Article  Google Scholar 

  2. Smith, S. The world according to PARP. Trends Biochem. Sci. 26, 174–179 (2001)

    CAS  Article  Google Scholar 

  3. Tulin, A., Stewart, D. & Spradling, A. C. The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development. Genes Dev. 16, 2108–2119 (2002)

    CAS  Article  Google Scholar 

  4. Hatakeyama, K., Nemoto, Y., Ueda, K. & Hayaishi, O. Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose). J. Biol. Chem. 261, 14902–14911 (1986)

    CAS  PubMed  Google Scholar 

  5. Saxena, A., Saffery, R., Wong, L. H., Kalitsis, P. & Choo, K. H. Centromere proteins Cenpa, Cenpb, and Bub3 interact with poly(ADP-ribose) polymerase-1 protein and are poly(ADP-ribosyl)ated. J. Biol. Chem. 277, 26921–26926 (2002)

    CAS  Article  Google Scholar 

  6. Kickhoefer, V. A. et al. The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase. J. Cell Biol. 146, 917–928 (1999)

    CAS  Article  Google Scholar 

  7. Smith, S. & de Lange, T. Tankyrase promotes telomere elongation in human cells. Curr. Biol. 10, 1299–1302 (2000)

    CAS  Article  Google Scholar 

  8. Sbodio, J. I. & Chi, N. W. Identification of a tankyrase-binding motif shared by IRAP, TAB182, and human TRF1 but not mouse TRF1. NuMA contains this RXXPDG motif and is a novel tankyrase partner. J. Biol. Chem. 277, 31887–31892 (2002)

    CAS  Article  Google Scholar 

  9. Bakondi, E. et al. Detection of poly(ADP-ribose) polymerase activation in oxidatively stressed cells and tissues using biotinylated NAD substrate. J. Histochem. Cytochem. 50, 91–98 (2002)

    CAS  Article  Google Scholar 

  10. Earle, E. et al. Poly(ADP-ribose) polymerase at active centromeres and neocentromeres at metaphase. Hum. Mol. Genet. 9, 187–194 (2000)

    CAS  Article  Google Scholar 

  11. Smith, S. & de Lange, T. Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes. J. Cell Sci. 112, 3649–3656 (1999)

    CAS  PubMed  Google Scholar 

  12. Sawin, K. E. & Mitchison, T. J. Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol. 112, 925–940 (1991)

    CAS  Article  Google Scholar 

  13. Slama, J. T. et al. Specific inhibition of poly(ADP-ribose) glycohydrolase by adenosine diphosphate (hydroxymethyl)pyrrolidinediol. J. Med. Chem. 38, 389–393 (1995)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  15. Sawin, K. E., LeGuellec, K., Philippe, M. & Mitchison, T. J. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature 359, 540–543 (1992)

    ADS  CAS  Article  Google Scholar 

  16. Compton, D. A., Szilak, I. & Cleveland, D. W. Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis. J. Cell Biol. 116, 1395–1408 (1992)

    CAS  Article  Google Scholar 

  17. Dionne, M. A., Howard, L. & Compton, D. A. NuMA is a component of an insoluble matrix at mitotic spindle poles. Cell Motil. Cytoskel. 42, 189–203 (1999)

    CAS  Article  Google Scholar 

  18. Olmsted, J. B., Stemple, D. L., Saxton, W. M., Neighbors, B. W. & McIntosh, J. R. Cell cycle-dependent changes in the dynamics of MAP2 and MAP4 in cultured cells. J. Cell Biol. 109, 211–223 (1989)

    CAS  Article  Google Scholar 

  19. Kapoor, T. M. & Mitchison, T. J. Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix. J. Cell Biol. 154, 1125–1133 (2001)

    CAS  Article  Google Scholar 

  20. Saxton, W. M. et al. Tubulin dynamics in cultured mammalian cells. J. Cell Biol. 99, 2175–2186 (1984)

    CAS  Article  Google Scholar 

  21. Wittmann, T., Wilm, M., Karsenti, E. & Vernos, I. TPX2, A novel Xenopus MAP involved in spindle pole organization. J. Cell Biol. 149, 1405–1418 (2000)

    CAS  Article  Google Scholar 

  22. Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C. & Mandel, P. Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure. Proc. Natl Acad. Sci. USA 79, 3423–3427 (1982)

    ADS  CAS  Article  Google Scholar 

  23. Aoufouchi, S. & Shall, S. Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation. Biochem. J. 325, 543–551 (1997)

    CAS  Article  Google Scholar 

  24. Chi, N. W. & Lodish, H. F. Tankyrase is a golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles. J. Biol. Chem. 275, 38437–38444 (2000)

    CAS  Article  Google Scholar 

  25. Rouleau, M., Aubin, R. A. & Poirier, G. G. Poly(ADP-ribosyl)ated chromatin domains: access granted. J. Cell Sci. 117, 815–825 (2004)

    CAS  Article  Google Scholar 

  26. Pickett-Heaps, J. D., Forer, A. & Spurck, T. Traction fibre: toward a “tensegral” model of the spindle. Cell Motil. Cytoskel. 37, 1–6 (1997)

    CAS  Article  Google Scholar 

  27. Troll, W., Garte, S. & Frenkel, K. Suppression of tumor promotion by inhibitors of poly(ADP)ribose formation. Basic Life Sci. 52, 225–232 (1990)

    CAS  PubMed  Google Scholar 

  28. Tirnauer, J. S., Salmon, E. D. & Mitchison, T. J. Microtubule plus-end dynamics in Xenopus egg extract spindles. Mol. Biol. Cell 15, 1776–1784 (2004)

    CAS  Article  Google Scholar 

  29. Lin, W., Ame, J. C., Aboul-Ela, N., Jacobson, E. L. & Jacobson, M. K. Isolation and characterization of the cDNA encoding bovine poly(ADP-ribose) glycohydrolase. J. Biol. Chem. 272, 11895–11901 (1997)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A. Pollington, B. Ward, J. Tirnauer and B. Brieher for critical reading of the manuscript; A. Straight for CENPE antibodies; A. Groen and D. Miyamoto for TPX2, NuMA and Eg5 antibodies; Z. Perlman for assistance with data analysis; D. L. Coyle for technical support; and the Nikon Imaging Facility at Harvard Medical School for use of microscopes. This work was supported by NIH grants to T.M.J. and M.K.J.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Paul Chang.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure S1

PAR Localization to the kinetochore. Xenopus extract spindles were incubated with Alexa 488-labeled PAR antibody, and X-rhodamine-labeled CENPE antibody (CENP) and, visualized using spinning disk confocal microscopy every 30 s for 20 min. PAR co-localized with CENPE at every time point. In Merge, PAR is green and CENPE, red. (JPG 36 kb)

Supplementary Movie M1

Real time spinning disk confocal microscopy of Xenopus egg extract spindles treated with 100 µg/ml PARG. Images were obtained every 30s for 15 min. (MP4 156 kb)

Supplementary Movie M2

Real time spinning disk confocal microscopy of Xenopus egg extract spindles treated with 500 µg/ml anti PAR antibody. Images were obtained every 30s for 15 min. (MP4 186 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chang, P., Jacobson, M. & Mitchison, T. Poly(ADP-ribose) is required for spindle assembly and structure. Nature 432, 645–649 (2004). https://doi.org/10.1038/nature03061

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature03061

Further reading

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.

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