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.

Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres

Abstract

The motion of a chromosome during mitosis is mediated by a bundle of microtubules, termed a kinetochore fibre (K-fibre), which connects the kinetochore of the chromosome to a spindle pole. Once formed, mature K-fibres maintain a steady state length because the continuous addition of microtubule subunits onto microtubule plus ends at the kinetochore is balanced by their removal at their minus ends within the pole. This condition is known as 'microtubule poleward flux'1. Chromosome motion and changes in position are then driven by changes in K-fibre length, which in turn are controlled by changes in the rates at which microtubule subunits are added at the kinetochore and/or removed from the pole2. A key to understanding the role of flux in mitosis is to identify the molecular factors that drive it. Here we use Drosophila melanogaster S2 cells expressing α-tubulin tagged with green fluorescent protein, RNA interference, laser microsurgery and photobleaching to show that the kinetochore protein MAST/Orbit — the single CLASP orthologue in Drosophila — is an essential component for microtubule subunit incorporation into fluxing K-fibres.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: CLASP is required for maintaining spindle and kinetochore fibre length in Drosophila S2T cells.
Figure 2: CLASP is required for microtubule poleward flux.
Figure 3: CLASP is required for subunit incorporation into K-fibre microtubules at their kinetochore-attached plus ends.
Figure 4: Schematic depicting the role of CLASP at kinetochores.

References

  1. 1

    Mitchison, T. J. Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J. Cell Biol. 109, 637–652 (1989).

    CAS  Article  Google Scholar 

  2. 2

    Rogers, G. C. et al. Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. Nature 427, 364–370 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Carvalho, P., Tirnauer, J. S. & Pellman, D. Surfing on microtubule ends. Trends Cell Biol. 13, 229–237 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Lemos, C. L. et al. Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization. EMBO J. 19, 3668–3682 (2000).

    CAS  Article  Google Scholar 

  5. 5

    Maiato, H. et al. Human CLASP1 is an outer kinetochore component that regulates spindle microtubule dyanmics. Cell 113, 891–904 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Maiato, H. et al. MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity. J. Cell Biol. 157, 749–760 (2002).

    CAS  Article  Google Scholar 

  7. 7

    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).

    CAS  Article  Google Scholar 

  8. 8

    Maiato, H., Rieder, C. L. & Khodjakov, A. Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during mitosis in animals. J. Cell Biol. DOI: 10.1083/jcb.200407090.

  9. 9

    Ganem, N. J. & Compton, D. A. The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK. J. Cell Biol. 166, 473–478 (2004).

    CAS  Article  Google Scholar 

  10. 10

    Lawrence, C. J. et al. A standardized kinesin nomenclature. J. Cell Biol. 167, 19–22 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Yin, H., You, L., Pasqualone, D., Kopski, K. M. & Huffaker, T. C. Stu1p is physically associated with β-tubulin and is required for structural integrity of the mitotic spindle. Mol. Biol. Cell 13, 1881–1892 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Sharp, D. J. MAST sails through mitosis. Curr. Biol. 12, R585–R587 (2002).

    CAS  Article  Google Scholar 

  13. 13

    Desai, A. et al. KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans. Genes Dev. 17, 2421–2435 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Hoffman, D. B., Pearson, C. G., Yen, T. J., Howell, B. J. & Salmon, E. D. Microtubule dependent changes in the assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at kinetochores. Mol. Biol. Cell 12, 1995–2009 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Tirnauer, J. S., Canman, J. C., Salmon, E. D. & Mitchison, T. J. EB1 targets to kinetochores with attached, polymerizing microtubules. Mol. Biol. Cell 13, 4308–4316 (2002).

    CAS  Article  Google Scholar 

  16. 16

    Rogers, S. L., Rogers, G. C., Sharp, D. J. & Vale, R. D. Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol. 158, 873–884 (2002).

    CAS  Article  Google Scholar 

  17. 17

    Waters, J. C., Mitchison, T. J., Rieder, C. L. & Salmon, E. D. The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work. Mol. Biol. Cell 7, 1547–1558 (1996).

    CAS  Article  Google Scholar 

  18. 18

    Czaban, B. B., Forer, A. & Bajer, A. S. Ultraviolet microbeam irradiation of chromosomal spindle fibres in Haemanthus katherinae endosperm. I. Behaviour of the irradiated region. J. Cell Sci. 105, 571–578 (1993).

    PubMed  Google Scholar 

  19. 19

    Forer, A., Spurck, T. & Pickett-Heaps, J. Ultaviolet microbeam irradiations of spindle fibers in crane-fly spermatocytes and newt epithelial cells: resolution of previously conflicting observations. Protoplasma 197, 230–240 (1997).

    Article  Google Scholar 

  20. 20

    Gordon, G. W. The Control of Mitotic Motility as Influenced by Ultraviolet Microbeam Irradiation of Kinetochore Fibers. Thesis, Univ. Pennsylvania (1980).

    Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

    Chen, W. & Zhang, D. Kinetochore fibre dynamics outside the contex of the spindle during anaphase. Nature Cell Biol. 6, 227–231 (2004).

    Article  Google Scholar 

  23. 23

    LaFountain, J. R. Jr., Cohan, C. S., Siegel, A. J. & LaFountain, D. J. Direct visualization of microtubule flux during metaphase and anaphase in crane-fly spermatocytes. Mol. Biol. Cell 15, 5724–5732 (2004).

    CAS  Article  Google Scholar 

  24. 24

    Mitchison, T. J. & Salmon, E. D. Poleward kinetochore fiber movement occurs during both metaphase and anaphase-A in newt lung cell mitosis. J. Cell Biol. 119, 569–582 (1992).

    CAS  Article  Google Scholar 

  25. 25

    Brust-Mascher, I. & Scholey, J. M. Microtubule flux and sliding in mitotic spindles of Drosophila embryos. Mol. Biol. Cell 13, 3967–3975 (2002).

    CAS  Article  Google Scholar 

  26. 26

    Maddox, P., Desai, A., Oegema, K., Mitchison, T. J. & Salmon, E. D. Poleward microtubule flux is a major component of spindle dynamics and anaphase A in mitotic Drosophila embryos. Curr. Biol. 12, 1670–1674 (2002).

    CAS  Article  Google Scholar 

  27. 27

    Skibbens, R. V., Skeen, V. P. & Salmon, E. D. Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a push-pull mechanism. J. Cell Biol. 122, 859–875 (1993).

    CAS  Article  Google Scholar 

  28. 28

    Khodjakov, A. & Rieder, C. L. Kinetochores moving away from their associated pole do not exert a significant pushing force on the chromosome. J. Cell Biol. 135, 315–327 (1996).

    CAS  Article  Google Scholar 

  29. 29

    Maiato, H., Sunkel, C. E. & Earnshaw, W. C. Dissecting mitosis by RNAi in Drosophila tissue culture cells. Biol. Proced. 5, 153–161 (2003).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank G. Goshima, S. Rogers and R. Vale (University of California, San Francisco, CA) for the S2T cells and EB1 antibodies, and C. Sunkel (University of Porto, Portugal) for communicating results before publication. This work was supported by National Institutes of Health grants GMS 40198 (to C.L.R.), GMS 59363 (to A.K.) and a postdoctoral research fellowship from Fundação para a Ciência e a Tecnologia of Portugal (SFRH/BPD/1159/2002 to H.M.).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Conly L. Rieder.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Maiato, H., Khodjakov, A. & Rieder, C. Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres. Nat Cell Biol 7, 42–47 (2005). https://doi.org/10.1038/ncb1207

Download citation

Further reading

Search

Quick links