Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase

Abstract

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here, we report that two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization.

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: Drosophila kinesin-13 proteins affect different parameters of microtubule dynamic instability in interphase S2 cells.
Figure 2: KLP10A and KLP59C localize to microtubule plus ends in interphase S2 cells.
Figure 3: KLP10A moves towards the cell cortex whereas KLP59C moves towards the cell interior.
Figure 4: KLP10A trails the +TIP protein EB1 on most microtubule plus ends.
Figure 5: EB1 is required for proper KLP10A localization at microtubule plus ends.
Figure 6: EB1 binds KLP10A in vitro.
Figure 7: Model for interphase kinesin-13 function.

Accession codes

Accessions

BINDPlus

References

  1. 1

    Howard, J. & Hyman, A.A. Dynamics and mechanics of the microtubule plus end. Nature 422, 753–758 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Mitchison, T. & Kirschner, M. Dynamic instability of microtubule growth. Nature 312, 237–242 (1984).

    CAS  Article  Google Scholar 

  3. 3

    Cassimeris, L. & Spittle, C. Regulation of microtubule-associated proteins. Int. Rev. Cytol. 210, 163–226 (2001).

    CAS  Article  Google Scholar 

  4. 4

    Belmont, L.D., Hyman, A.A., Sawin, K.E. & Mitchison, T.J. Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62, 579–589 (1990).

    CAS  Article  Google Scholar 

  5. 5

    Small, J.V. & Kaverina, I. Microtubules meet substrate adhesions to arrange cell polarity. Curr. Opin. Cell Biol. 15, 40–47 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Krylyshkina, O. et al. Nanometer targeting of microtubules to focal adhesions. J. Cell Biol. 161, 853–859 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Wittmann, T., Bokoch, G.M. & Waterman-Storer, C.M. Regulation of leading edge microtubule and actin dynamics downstream of Rac1. J. Cell Biol. 161, 845–851 (2003).

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

    Vale, R.D. & Fletterick, R.J. The design plan of kinesin motors. Annu. Rev. Cell Dev. Biol. 13, 745–777 (1997).

    CAS  Article  Google Scholar 

  10. 10

    Desai, A., Verma, S., Mitchison, T.J. & Walczak, C.E. Kin I kinesins are microtubule-destabilizing enzymes. Cell 96, 69–78 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Walczak, C.E., Mitchison, T.J. & Desai, A. XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. Cell 84, 37–47 (1996).

    CAS  Article  Google Scholar 

  12. 12

    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 

  13. 13

    Kline-Smith, S.L., Khodjakov, A., Hergert, P. & Walczak, C.E. Depletion of centromeric MCAK leads to chromosome congression and segregation defects due to improper kinetochore attachments. Mol. Biol. Cell 15, 1146–1159 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Kline-Smith, S.L. & Walczak, C.E. The microtubule-destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells. Mol. Biol. Cell 13, 2718–2731 (2002).

    CAS  Article  Google Scholar 

  15. 15

    Homma, N. et al. Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension. Cell 114, 229–239 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Holmfeldt, P., Stenmark, S. & Gullberg, M. Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis. EMBO J. 23, 627–637 (2004).

    CAS  Article  Google Scholar 

  17. 17

    Tournebize, R. et al. Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts. Nature Cell Biol. 2, 13–19 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Kinoshita, K., Arnal, I., Desai, A., Drechsel, D.N. & Hyman, A.A. Reconstitution of physiological microtubule dynamics using purified components. Science 294, 1340–1343 (2001).

    CAS  Article  Google Scholar 

  19. 19

    Gandhi, R. et al. The Drosophila kinesin-like protein KLP67A is essential for mitotic and male meiotic spindle assembly. Mol. Biol. Cell 15, 121–131 (2003).

    Article  Google Scholar 

  20. 20

    Miller, R.K. et al. The kinesin-related proteins, Kip2p and Kip3p, function differently in nuclear migration in yeast. Mol. Biol. Cell 9, 2051–2068 (1998).

    CAS  Article  Google Scholar 

  21. 21

    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 

  22. 22

    Tran, P.T., Walker, R.A. & Salmon, E.D. A metastable intermediate state of microtubule dynamic instability that differs significantly between plus and minus ends. J. Cell Biol. 138, 105–117 (1997).

    CAS  Article  Google Scholar 

  23. 23

    Galjart, N. & Perez, F. A plus-end raft to control microtubule dynamics and function. Curr. Opin. Cell Biol. 15, 48–53 (2003).

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  25. 25

    Tirnauer, J.S., O'Toole, E., Berrueta, L., Bierer, B.E. & Pellman, D. Yeast Bim1p promotes the G1-specific dynamics of microtubules. J. Cell Biol. 145, 993–1007 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Rogers, S.L., Wiedemann, U., Hacker, U., Turck, C. & Vale, R.D. Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner. Curr. Biol. 14, 1827–1833 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Su, L.K. et al. APC binds to the novel protein EB1. Cancer Res. 55, 2972–2977 (1995).

    CAS  PubMed  Google Scholar 

  28. 28

    Moores, C.A. et al. A mechanism for microtubule depolymerization by KinI kinesins. Mol. Cell 9, 903–909 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Newton, C.N., Wagenbach, M., Ovechkina, Y., Wordeman, L. & Wilson, L. MCAK, a Kin I kinesin, increases the catastrophe frequency of steady-state HeLa cell microtubules in an ATP-dependent manner in vitro. FEBS Lett. 572, 80–84 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Ovechkina, Y., Wagenbach, M. & Wordeman, L. K-loop insertion restores microtubule depolymerizing activity of a “neckless” MCAK mutant. J. Cell Biol. 159, 557–562 (2002).

    CAS  Article  Google Scholar 

  31. 31

    Tirnauer, J.S., Grego, S., Salmon, E.D. & Mitchison, T.J. EB1-microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules. Mol. Biol. Cell 13, 3614–3626 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Browning, H., Hackney, D.D. & Nurse, P. Targeted movement of cell end factors in fission yeast. Nature Cell Biol. 5, 812–818 (2003).

    CAS  Article  Google Scholar 

  33. 33

    Carvalho, P., Gupta, M.L. Jr, Hoyt, M.A. & Pellman, D. Cell cycle control of kinesin-mediated transport of Bik1 (CLIP-170) regulates microtubule stability and dynein activation. Dev. Cell 6, 815–829 (2004).

    CAS  Article  Google Scholar 

  34. 34

    Busch, K.E., Hayles, J., Nurse, P. & Brunner, D. Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules. Dev. Cell 6, 831–843 (2004).

    CAS  Article  Google Scholar 

  35. 35

    Hunter, A.W. et al. The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATP-hydrolyzing complex at microtubule ends. Mol. Cell 11, 445–457 (2003).

    CAS  Article  Google Scholar 

  36. 36

    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).

    CAS  Article  Google Scholar 

  37. 37

    Andrews, P.D. et al. Aurora B regulates MCAK at the mitotic centromere. Dev. Cell 6, 253–268 (2004).

    CAS  Article  Google Scholar 

  38. 38

    Lan, W. et al. Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity. Curr. Biol. 14, 273–286 (2004).

    CAS  Article  Google Scholar 

  39. 39

    Ohi, R., Coughlin, M.L., Lane, W.S. & Mitchison, T.J. An inner centromere protein that stimulates the microtubule depolymerizing activity of a KinI kinesin. Dev. Cell 5, 309–321 (2003).

    CAS  Article  Google Scholar 

  40. 40

    Ohi, R., Sapra, T., Howard, J. & Mitchison, T.J. Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation. Mol. Biol. Cell 15, 2895–2906 (2004).

    CAS  Article  Google Scholar 

  41. 41

    Kaverina, I., Rottner, K. & Small, J.V. Targeting, capture, and stabilization of microtubules at early focal adhesions. J. Cell Biol. 142, 181–190 (1998).

    CAS  Article  Google Scholar 

  42. 42

    Wittmann, T., Bokoch, G.M. & Waterman-Storer, C.M. Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1. J. Biol. Chem. 279, 6196–6203 (2004).

    CAS  Article  Google Scholar 

  43. 43

    Komarova, Y.A., Akhmanova, A.S., Kojima, S., Galjart, N. & Borisy, G.G. Cytoplasmic linker proteins promote microtubule rescue in vivo. J. Cell Biol. 159, 589–599 (2002).

    CAS  Article  Google Scholar 

  44. 44

    Lantz, V.A. & Miller, K.G. A class VI unconventional myosin is associated with a homologue of a microtubule-binding protein, cytoplasmic linker protein-170, in neurons and at the posterior pole of Drosophila embryos. J. Cell Biol. 140, 897–910 (1998).

    CAS  Article  Google Scholar 

  45. 45

    Miki, H., Setou, M., Kaneshiro, K. & Hirokawa, N. All kinesin superfamily protein, KIF, genes in mouse and human. Proc. Natl Acad. Sci. USA 98, 7004–7011 (2001).

    CAS  Article  Google Scholar 

  46. 46

    Clemens, J.C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl Acad. Sci. USA 97, 6499–6503 (2000).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank P. Sampaio and C. Sunkel (Porto, Portugal) for the anti-Mast antibody, E. Ghersi and L. D'adamio (AECOM) for advice on in vitro binding assays, M. Cammer of the AECOM analytical imaging facility for advice on image acquisition and analysis, and H. Sosa (AECOM) for helpful comments on the manuscript. We also thank R. Tsien (UCSD) for providing the mRFP construct, and K. Slep (UCSF) for the APC and some EB1 constructs. This work was supported by grants from the NIH to D.J.S. and R.D.V. V.M. is a Fulbright Fellow and D.J.S. is a Scholar of the Leukemia and Lymphoma Society.

Author information

Affiliations

Authors

Corresponding author

Correspondence to David J. Sharp.

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

Mennella, V., Rogers, G., Rogers, S. et al. Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase. Nat Cell Biol 7, 235–245 (2005). https://doi.org/10.1038/ncb1222

Download citation

Further reading

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