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.

  • Article
  • Published:

Examining how the spatial organization of chromatin signals influences metaphase spindle assembly

Nature Cell Biology volume 8, pages 924–932 (2006)Cite this article

Abstract

During cell division, the proper assembly of a microtubule-based bipolar spindle depends on signals from chromatin. However, it is unknown how the spatial organization of chromatin signals affects spindle morphology. Here, we use paramagnetic chromatin beads, and magnetic fields for their alignment in cell-free extracts, to examine the spatial components of signals that regulate spindle assembly. We find that for linear chromatin-bead arrays that vary by eightfold in length, metaphase spindle size and shape are constant. Our findings indicate that, although chromatin provides cues for microtubule formation, metaphase spindle organization, which is controlled by microtubule-based motors, is robust to changes in the shape of chromatin signals.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Formation of linear chromatin-bead structures in Xenopus egg extracts.
Figure 2: Microtubule organization of spindles assembled on aligned chromatin-bead structures.
Figure 3: Time course of spindle assembly on linear chromatin structures.
Figure 4: Spindle organization is not sensitive to complex chromatin-bead geometries.
Figure 5: Dynamic microtubules explore the region around the entire chromatin-bead structure.
Figure 6: Dynein–dynactin are required for the establishment and maintenance of spindle width on linear chromatin-bead structures.
Figure 7: Kinesin-5 inhibition results in radial microtubule arrays associated with chromatin structures.

Similar content being viewed by others

References

  1. Nedelec, F., Surrey, T. & Karsenti, E. Self-organisation and forces in the microtubule cytoskeleton. Curr. Opin. Cell Biol. 15, 118–124 (2003).

    Article  CAS  Google Scholar 

  2. Gadde, S. & Heald, R. Mechanisms and molecules of the mitotic spindle. Curr. Biol. 14, R797–R805 (2004).

    Article  CAS  Google Scholar 

  3. Compton, D. A. Spindle assembly in animal cells. Annu. Rev. Biochem. 69, 95–114 (2000).

    Article  CAS  Google Scholar 

  4. Wadsworth, P. & Khodjakov, A. E pluribus unum: towards a universal mechanism for spindle assembly. Trends Cell Biol. 14, 413–419 (2004).

    Article  CAS  Google Scholar 

  5. 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 

  6. Khodjakov, A., Cole, R. W., Oakley, B. R. & Rieder, C. L. Centrosome-independent mitotic spindle formation in vertebrates. Curr. Biol. 10, 59–67 (2000).

    Article  CAS  Google Scholar 

  7. Hinchcliffe, E. H., Miller, F. J., Cham, M., Khodjakov, A. & Sluder, G. Requirement of a centrosomal activity for cell cycle progression through G1 into S phase. Science 291, 1547–1550 (2001).

    Article  CAS  Google Scholar 

  8. Bonaccorsi, S., Giansanti, M. G. & Gatti, M. Spindle self-organization and cytokinesis during male meiosis in asterless mutants of Drosophila melanogaster. J. Cell Biol. 142, 751–761 (1998).

    Article  CAS  Google Scholar 

  9. Megraw, T. L., Kao, L. R. & Kaufman, T. C. Zygotic development without functional mitotic centrosomes. Curr. Biol. 11, 116–120 (2001).

    Article  CAS  Google Scholar 

  10. Karsenti, E. & Vernos, I. The mitotic spindle: a self-made machine. Science 294, 543–547 (2001).

    Article  CAS  Google Scholar 

  11. Ohba, T., Nakamura, M., Nishitani, H. & Nishimoto, T. Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran. Science 284, 1356–1358 (1999).

    Article  CAS  Google Scholar 

  12. Andersen, S. S. et al. Mitotic chromatin regulates phosphorylation of Stathmin/Op18. Nature 389, 640–643 (1997).

    Article  CAS  Google Scholar 

  13. Niethammer, P., Bastiaens, P. & Karsenti, E. Stathmin-tubulin interaction gradients in motile and mitotic cells. Science 303, 1862–1866 (2004).

    Article  CAS  Google Scholar 

  14. Tsai, M. Y. et al. A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly. Nature Cell Biol. 5, 242–248 (2003).

    Article  CAS  Google Scholar 

  15. Sampath, S. C. et al. The chromosomal passenger complex is required for chromatin-induced microtubule stabilization and spindle assembly. Cell 118, 187–202 (2004).

    Article  CAS  Google Scholar 

  16. Kalab, P., Pralle, A., Isacoff, E. Y., Heald, R. & Weis, K. Analysis of a RanGTP-regulated gradient in mitotic somatic cells. Nature 440, 697–701 (2006).

    Article  CAS  Google Scholar 

  17. 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  CAS  Google Scholar 

  18. Caudron, M., Bunt, G., Bastiaens, P. & Karsenti, E. Spatial coordination of spindle assembly by chromosome-mediated signaling gradients. Science 309, 1373–1376 (2005).

    Article  CAS  Google Scholar 

  19. Gierer, A. & Meinhardt, H. A theory of biological pattern formation. Kybernetik 12, 30–39 (1972).

    Article  CAS  Google Scholar 

  20. Carazo-Salas, R. E. et al. Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature 400, 178–181 (1999).

    Article  CAS  Google Scholar 

  21. Goshima, G. & Vale, R. D. The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. J. Cell Biol. 162, 1003–1016 (2003).

    Article  CAS  Google Scholar 

  22. Wilde, A. et al. Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities. Nature Cell Biol. 3, 221–227 (2001).

    Article  CAS  Google Scholar 

  23. Ems-McClung, S. C., Zheng, Y. & Walczak, C. E. Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal. Mol. Biol. Cell 15, 46–57 (2004).

    Article  CAS  Google Scholar 

  24. 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 

  25. Walczak, C. E., Vernos, I., Mitchison, T. J., Karsenti, E. & Heald, R. A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity. Curr. Biol. 8, 903–913 (1998).

    Article  CAS  Google Scholar 

  26. Gaetz, J. & Kapoor, T. M. Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles. J. Cell Biol. 166, 465–471 (2004).

    Article  CAS  Google Scholar 

  27. Mitchison, T. J. et al. Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles. Mol. Biol. Cell 16, 3064–3076 (2005).

    Article  CAS  Google Scholar 

  28. Houchmandzadeh, B. & Dimitrov, S. Elasticity measurements show the existence of thin rigid cores inside mitotic chromosomes. J. Cell Biol. 145, 215–223 (1999).

    Article  CAS  Google Scholar 

  29. Houchmandzadeh, B., Marko, J. F., Chatenay, D. & Libchaber, A. Elasticity and structure of eukaryote chromosomes studied by micromanipulation and micropipette aspiration. J. Cell Biol. 139, 1–12 (1997).

    Article  CAS  Google Scholar 

  30. Marshall, W. F., Marko, J. F., Agard, D. A. & Sedat, J. W. Chromosome elasticity and mitotic polar ejection force measured in living Drosophila embryos by four-dimensional microscopy-based motion analysis. Curr. Biol. 11, 569–578 (2001).

    Article  CAS  Google Scholar 

  31. Nicklas, R. B. Chromosome velocity during mitosis as a function of chromosome size and position. J. Cell Biol. 25 (Suppl), 119–135 (1965).

    Article  Google Scholar 

  32. Carazo-Salas, R. E. & Karsenti, E. Long-range communication between chromatin and microtubules in Xenopus egg extracts. Curr. Biol. 13, 1728–1733 (2003).

    Article  CAS  Google Scholar 

  33. Dogterom, M., Felix, M. A., Guet, C. C. & Leibler, S. Influence of M-phase chromatin on the anisotropy of microtubule asters. J. Cell Biol. 133, 125–140 (1996).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  Google Scholar 

  36. Heald, R., Tournebize, R., Habermann, A., Karsenti, E. & Hyman, A. Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization. J. Cell Biol. 138, 615–628 (1997).

    Article  CAS  Google Scholar 

  37. Rusan, N. M., Tulu, U. S., Fagerstrom, C. & Wadsworth, P. Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport. J. Cell Biol. 158, 997–1003 (2002).

    Article  CAS  Google Scholar 

  38. Khodjakov, A., Copenagle, L., Gordon, M. B., Compton, D. A. & Kapoor, T. M. Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis. J. Cell Biol. 160, 671–683 (2003).

    Article  CAS  Google Scholar 

  39. Echeverri, C. J., Paschal, B. M., Vaughan, K. T. & Vallee, R. B. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell Biol. 132, 617–633 (1996).

    Article  CAS  Google Scholar 

  40. Quintyne, N. J. et al. Dynactin is required for microtubule anchoring at centrosomes. J. Cell Biol. 147, 321–334 (1999).

    Article  CAS  Google Scholar 

  41. Kashina, A. S., Rogers, G. C. & Scholey, J. M. The bimC family of kinesins: essential bipolar mitotic motors driving centrosome separation. Biochim. Biophys. Acta 1357, 257–271 (1997).

    Article  CAS  Google Scholar 

  42. Kapoor, T. M., Mayer, T. U., Coughlin, M. L. & Mitchison, T. J. Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5. J. Cell Biol. 150, 975–988 (2000).

    Article  CAS  Google Scholar 

  43. Kapoor, T. M. et al. Chromosomes can congress to the metaphase plate before biorientation. Science 311, 388–391 (2006).

    Article  CAS  Google Scholar 

  44. Karki, S. & Holzbaur, E. L. Cytoplasmic dynein and dynactin in cell division and intracellular transport. Curr. Opin. Cell Biol. 11, 45–53 (1999).

    Article  CAS  Google Scholar 

  45. Svoboda, K. & Block, S. M. Force and velocity measured for single kinesin molecules. Cell 77, 773–784 (1994).

    Article  CAS  Google Scholar 

  46. Mallik, R., Carter, B. C., Lex, S. A., King, S. J. & Gross, S. P. Cytoplasmic dynein functions as a gear in response to load. Nature 427, 649–652 (2004).

    Article  CAS  Google Scholar 

  47. Valentine, M. T., Fordyce, P. M., Krzysiak, T. C., Gilbert, S. P. & Block, S. M. Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro. Nature Cell Biol. 8, 470–476 (2006).

    Article  CAS  Google Scholar 

  48. Kapitein, L. C. et al. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature 435, 114–118 (2005).

    Article  CAS  Google Scholar 

  49. Wang, X. et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422, 897–901 (2003).

    Article  CAS  Google Scholar 

  50. Durand, D. Vertebrate evolution: doubling and shuffling with a full deck. Trends Genet. 19, 2–5 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Tirnauer for the EB1 expression plasmid; T. Schroer for the p150-CC1 expression plasmid; and L. Postow for helpful discussions. This work was supported by a National Institutes of Health grant to T.M.K. (GM 65933). J.G. is a Howard Hughes Medical Institute Predoctoral fellow. Z.G. acknowledges CNRS for support.

Author information

Author notes

  1. Zoher Gueroui

    Present address: GMCM CNRS UMR6626, Universite Rennes 1, France

  2. Jedidiah Gaetz and Zoher Gueroui: These authors contributed equally to this work.

Authors and Affiliations

  1. The Rockefeller University, 1230 York Avenue, New York, 10021, NY, USA

    Jedidiah Gaetz, Zoher Gueroui, Albert Libchaber & Tarun M. Kapoor

Authors
  1. Jedidiah Gaetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zoher Gueroui
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Albert Libchaber
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tarun M. Kapoor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Albert Libchaber or Tarun M. Kapoor.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures S1, S2, S3 and Methods (PDF 397 kb)

Supplementary Information

Supplementary Movie 1 (MOV 1869 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gaetz, J., Gueroui, Z., Libchaber, A. et al. Examining how the spatial organization of chromatin signals influences metaphase spindle assembly. Nat Cell Biol 8, 924–932 (2006). https://doi.org/10.1038/ncb1455

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1455

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