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Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control

Abstract

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide–disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

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Figure 1: Kip3-mediated microtubule-sliding activity.
Figure 2: Kip3-mediated sliding of dynamic microtubules.
Figure 3: Kip3-CC is a sliding-proficient but depolymerizing-deficient Kip3 variant.
Figure 4: Kip3-CC retains comparable motility to Kip3.
Figure 5: Counterbalancing effects between the sliding and destabilizing activity of Kip3 in the control of spindle length.
Figure 6: The sliding activity of Kip3 promotes spindle assembly.
Figure 7: A slide–disassemble model to explain the complex effects of Kip3 on regulating spindle length and stability.

References

  1. Severin, F., Habermann, B., Huffaker, T. & Hyman, T. Stu2 promotes mitotic spindle elongation in anaphase. J. Cell Biol. 153, 435–442 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Goshima, G., Saitoh, S. & Yanagida, M. Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation. Genes Dev. 13, 1664–1677 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Goshima, G. & Scholey, J. M. Control of mitotic spindle length. Annu. Rev. Cell Dev. Biol. 26, 21–57 (2010).

    CAS  Article  PubMed  Google Scholar 

  4. Meunier, S. & Vernos, I. Microtubule assembly during mitosis—from distinct origins to distinct functions? J. Cell Sci. 125, 2805–2814 (2012).

    CAS  Article  PubMed  Google Scholar 

  5. Gatlin, J. C. & Bloom, K. Microtubule motors in eukaryotic spindle assembly and maintenance. Semin. Cell Dev. Biol. 21, 248–254 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Winey, M. & Bloom, K. Mitotic spindle form and function. Genetics 190, 1197–1224 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Tanenbaum, M. E., Macurek, L., Galjart, N. & Medema, R. H. Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly. EMBO J. 27, 3235–3245 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Saunders, W., Lengyel, V. & Hoyt, M. A. Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors. Mol. Biol. Cell 8, 1025–1033 (1997).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W. & Scholey, J. M. Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos. Nat. Cell Biol. 1, 51–54 (1999).

    CAS  Article  PubMed  Google Scholar 

  10. Gupta, M. L. Jr, Carvalho, P., Roof, D. M. & Pellman, D. Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle. Nat. Cell Biol. 8, 913–923 (2006).

    CAS  Article  PubMed  Google Scholar 

  11. Varga, V. et al. Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner. Nat. Cell Biol. 8, 957–962 (2006).

    CAS  Article  PubMed  Google Scholar 

  12. Mayr, M. I. et al. The human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression. Curr. Biol. 17, 488–498 (2007).

    CAS  Article  PubMed  Google Scholar 

  13. Du, Y., English, C. A. & Ohi, R. The kinesin-8 Kif18A dampens microtubule plus-end dynamics. Curr. Biol. 20, 374–380 (2010).

    CAS  Article  PubMed  Google Scholar 

  14. Gardner, M. K., Zanic, M., Gell, C., Bormuth, V. & Howard, J. Depolymerizing kinesins Kip3 and MCAK shape cellular microtubule architecture by differential control of catastrophe. Cell 147, 1092–1103 (2011).

    CAS  Article  PubMed  Google Scholar 

  15. Su, X., Ohi, R. & Pellman, D. Move in for the kill: motile microtubule regulators. Trends Cell Biol. 22, 567–575 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Goshima, G., Wollman, R., Stuurman, N., Scholey, J. M. & Vale, R. D. Length control of the metaphase spindle. Curr. Biol. 15, 1979–1988 (2005).

    CAS  Article  PubMed  Google Scholar 

  17. West, R. R., Malmstrom, T. & McIntosh, J. R. Kinesins klp5(+) and klp6(+) are required for normal chromosome movement in mitosis. J. Cell Sci. 115, 931–940 (2002).

    CAS  PubMed  Google Scholar 

  18. Garcia, M. A., Koonrugsa, N. & Toda, T. Two kinesin-like Kin I family proteins in fission yeast regulate the establishment of metaphase and the onset of anaphase A. Curr. Biol. 12, 610–621 (2002).

    CAS  Article  PubMed  Google Scholar 

  19. Stumpff, J., von Dassow, G., Wagenbach, M., Asbury, C. & Wordeman, L. The kinesin-8 motor Kif18A suppresses kinetochore movements to control mitotic chromosome alignment. Dev. Cell 14, 252–262 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Wargacki, M. M., Tay, J. C., Muller, E. G., Asbury, C. L. & Davis, T. N. Kip3, the yeast kinesin-8, is required for clustering of kinetochores at metaphase. Cell Cycle 9, 2581–2588 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Su, X. et al. Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8. Mol. Cell 43, 751–763 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Woodruff, J. B., Drubin, D. G. & Barnes, G. Mitotic spindle disassembly occurs via distinct subprocesses driven by the anaphase-promoting complex, Aurora B kinase, and kinesin-8. J. Cell Biol. 191, 795–808 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Varga, V., Leduc, C., Bormuth, V., Diez, S. & Howard, J. Kinesin-8 motors act cooperatively to mediate length-dependent microtubule depolymerization. Cell 138, 1174–1183 (2009).

    CAS  Article  PubMed  Google Scholar 

  24. Fink, G. et al. The mitotic kinesin-14 Ncd drives directional microtubule–microtubule sliding. Nat. Cell Biol. 11, 717–723 (2009).

    CAS  Article  PubMed  Google Scholar 

  25. Braun, M., Drummond, D. R., Cross, R. A. & McAinsh, A. D. The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP-dependent sorting mechanism. Nat. Cell Biol. 11, 724–730 (2009).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  27. Van den Wildenberg, S. M. et al. The homotetrameric kinesin-5 KLP61F preferentially crosslinks microtubules into antiparallel orientations. Curr. Biol. 18, 1860–1864 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Portran, D., Gaillard, J., Vantard, M. & Thery, M. Quantification of MAP and molecular motor activities on geometrically controlled microtubule networks. Cytoskeleton 70, 12–23 (2012).

    Article  PubMed  Google Scholar 

  29. Pellman, D., Bagget, M., Tu, Y. H., Fink, G. R. & Tu, H. Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae. J. Cell Biol. 130, 1373–1385 (1995).

    CAS  Article  PubMed  Google Scholar 

  30. Roostalu, J., Schiebel, E. & Khmelinskii, A. Cell cycle control of spindle elongation. Cell Cycle 9, 1084–1090 (2010).

    CAS  Article  PubMed  Google Scholar 

  31. Miki, H., Okada, Y. & Hirokawa, N. Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol. 15, 467–476 (2005).

    CAS  Article  PubMed  Google Scholar 

  32. Klumpp, L. M., Mackey, A. T., Farrell, C. M., Rosenberg, J. M. & Gilbert, S. P. A kinesin switch I arginine to lysine mutation rescues microtubule function. J. Biol. Chem. 278, 39059–39067 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Gardner, M. K. et al. The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle. J. Cell Biol. 180, 91–100 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Subramanian, R. et al. Insights into antiparallel microtubule crosslinking byPRC1, a conserved nonmotor microtubule binding protein. Cell 142, 433–443 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Gandhi, S. R. et al. Kinetochore-dependent microtubule rescue ensurestheir efficient and sustained interactions in early mitosis. Dev. Cell 21, 920–933 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Hoyt, M. A., He, L., Loo, K. K. & Saunders, W. S. Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly. J. Cell Biol. 118, 109–120 (1992).

    CAS  Article  PubMed  Google Scholar 

  37. Cahu, J. & Surrey, T. Motile microtubule crosslinkers require distinct dynamic properties for correct functioning during spindle organization in Xenopus egg extract. J. Cell Sci. 122, 1295–1300 (2009).

    CAS  Article  PubMed  Google Scholar 

  38. Straight, A. F., Sedat, J. W. & Murray, A. W. Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast. J. Cell Biol. 143, 687–694 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Tanenbaum, M. E. et al. Kif15 cooperates with eg5 to promote bipolar spindle assembly. Curr. Biol. 19, 1703–1711 (2009).

    CAS  Article  PubMed  Google Scholar 

  40. Stumpff, J. et al. A tethering mechanism controls the processivity and kinetochore-microtubule plus-end enrichment of the kinesin-8 Kif18A. Mol. Cell 43, 764–775 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Weaver, L. N. et al. Kif18A uses a microtubule binding site in the tail for plus-end localization and spindle length regulation. Curr. Biol. 21, 1500–1506 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Mayr, M. I., Storch, M., Howard, J. & Mayer, T. U. A non-motor microtubule binding site is essential for the high processivity and mitotic function of kinesin-8 Kif18A. PLoS One 6, e27471 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Gatt, M. K. et al. Klp67A destabilises pre-anaphase microtubules butsubsequently is required to stabilise the central spindle. J. Cell Sci. 118, 2671–2682 (2005).

    CAS  Article  PubMed  Google Scholar 

  44. Savoian, M. S. & Glover, D. M. Drosophila Klp67A binds prophase kinetochores to subsequently regulate congression and spindle length. J. Cell Sci. 123, 767–776 (2010).

    CAS  Article  PubMed  Google Scholar 

  45. Hovland, P., Flick, J., Johnston, M. & Sclafani, R. A. Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose. Gene 83, 57–64 (1989).

    CAS  Article  PubMed  Google Scholar 

  46. Schuyler, S. C. & Pellman, D. Analysis of the size and shape of protein complexes from yeast. Methods Enzymol. 351, 150–168 (2002).

    CAS  Article  PubMed  Google Scholar 

  47. Hoskins, A. A. et al. Ordered and dynamic assembly of single spliceosomes. Science 331, 1289–1295 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are grateful to the Reck-Peterson Laboratory at Harvard Medical School for sharing the usage of their TIRF microscope. We appreciate H. Li’s help with FACS analysis. We thank M. Gupta and R. Ohi for suggestions and comments on the manuscript. D. Pellman is supported by Howard Hughes Medical Institute and a National Institute of Health grant (GM61345). M. Thery is supported by the Human Frontier Scientific Program (RGY0088/201). H. Arellano-Santoyo is an international fellow of the Howard Hughes Medical Institute.

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X.S. and D. Pellman conceived the project and wrote the manuscript. H.A-S. performed the assay measuring microtubule dynamics and analysed the data. D. Portran, J.G., M.V. and M.T. performed the micropattering assay and analysed the data. X.S. performed the remainder of the experiments and analysed the data.

Corresponding author

Correspondence to David Pellman.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Protein purification panel and demonstration of Kip3-CC dimer formation.

Coomassie staining after SDS-PAGE was used to visualize Kip3, Kip3ΔT-LZ (Kip3 without its tail domain), Kip3-CC (Kip3 with its neck replaced with leucine zipper), and Kip3-M (Kip3 motor domain) with or without EGS [ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester)] cross-linking. Note that after cross-linking Kip3-CC migrated at approximately the predicted size of a dimer, as Kip3 did. In contrast, Kip3-M remains at the similar position before and after cross-linking. Protein input: 1 μg.

Supplementary Figure 2 Comparable amounts of Kip3 and Kip3ΔT-LZ on track microtubules, related to the sliding assay in Fig. 1b.

(LEFT) TMR-labeled Kip3 or Kip3ΔT-LZ decorated track microtubules in the sliding assay. Scale bar: 5 μm. (RIGHT) Quantification of fluorescence intensity of Kip3 and Kip3ΔT-LZ along track microtubules at the beginning of sliding assays. Shown is mean±s.e.m. (N = 30 microtubules). Protein input: 150 nM.

Supplementary Figure 3 Categories of possible microtubule movements mediated by Kip3.

In each of the six situations, the bottom microtubule is immobilized to coverslip (blue bar). Kip3 motors, upright or inverted, slide the top cargo microtubules. The sliding direction was described as “ +”, “ −”, or “ +/−”, which indicates plus end-leading, minus end-leading, or tug-of-war, respectively. The microtubule orientation is indicated as being ‘p’ (parallel) or ‘a’ (anti-parallel). The observed frequencies for each type of movements was listed (N = 110 microtubule pairs).

Supplementary Figure 4 Kip3 slides anti-parallel microtubules and occasionally parallel microtubules.

Polarity-labeled microtubules (bright red for the plus end of cargo microtubules and bright green for the plus end of track microtubules) show an anti-parallel orientation (#1–#3) and a parallel orientation (#4). “ +” and “ −” indicate the microtubule plus end and minus end, respectively. Scale bar: 5 μm. Kymographs at the bottom show sliding movement of the numbered cargo microtubules. Scale bar: 2 μm (horizontal) and 2 min (vertical). Kip3 input: 150 nM.

Supplementary Figure 5 Kip3 induces the formation of curvy microtubule bundles.

Image panels show examples of curvy microtubule bundles in the presence of 10 μM tubulin together with indicated proteins (50 nM of Kip3 or/and 33 nM of Ase1). The images were taken 30 min after the reaction was started. Note that this panel does not include the substantial amount of asters pairs that were either non-connected or connected by straight bundles. The quantification of overall bundle curvature (including straight bundles) is shown in Fig. 2c. Scale bar: 10 μm.

Supplementary Figure 6 Comparable fluorescence intensities of motors along microtubules in the dynamic microtubule assay in Fig. 3.

(a) Kymographs show microtubule dynamics in the presence of Kip3 or Kip3-CC. (TOP) HiLyte 647-labeled tubulin. (BOTTOM) TMR-labeled motor. Input: 150 nM Kip3, 6 nM Kip3-CC. Scale bar: 5 μm (horizontal) and 1 min (vertical). b) Quantification of fluorescence intensity of Kip3 and Kip3-CC along microtubules. Shown is mean±s.e.m. (N = 47 and 50 microtubules for Kip3 and Kip3-CC, respectively). c) Quantification of the growth and shrinkage rates, related to Fig. 3e. Shown is mean±s.e.m. (N = 71, 46, and 63 growth events for Kip3, Kip3-CC, and control, respectively; N = 164, 25, and 23 shrinkage events for Kip3, Kip3-CC, and control, respectively). P value was obtained from a two-tailed Student’s T test.

Supplementary Figure 7 The spindle localization of Ase1 and Stu2 in cells overexpressing Kip3-CC.

(a) Representative images of a control cell and a cell overexpressing Kip3-CC. These cells were synchronized in S phase by hydroxyurea. Microtubules were labeled with CFP-Tub1. Ase1 was visualized with a 3xYFP tag. Scale bar: 2 μm. b) Comparison of spindle-associated Ase1 and spindle length between control cells and cells overexpressing Kip3-CC. Shown is mean+s.e.m. (N = 50 cells). c) Representative images of a control cell and a cell overexpressing Kip3-CC. Microtubules were labeled with CFP-Tub1. Stu2 was tagged with EYFP. Scale bar: 2 μm. d) Comparisons of the fluorescence intensity of Stu2 at mid region of pre-anaphase spindles (arrows in Supplementary Fig. S7c) between control cells and cells overexpressing Kip3-CC. Shown is mean+s.e.m. (N = 64 cells).

Supplementary Table 1 Dynamic instability of yeast cytoplasmic microtubules.
Supplementary Table 2 Strains used in this study.

Supplementary information

Supplementary Information

Supplementary Information (PDF 664 kb)

Kip3-driven sliding and ‘tug-of-war’ movements of stabilized microtubules.

Cargo microtubules (red) move along track microtubules (green) in the presence of 150 nM Kip3. Videos play at 300× speed. Field view: 27×60 μm. (MOV 3247 kb)

Rare flipping of cargo microtubules in the sliding assay.

Cargo microtubules (red) move along track microtubules (green) in the presence of 150 nM Kip3. Videos play at 300× speed. Shown is sliding followed by flipping over and sliding in the opposite direction. This is a rare event, which was found in 4 out of 300 microtubule pairs. Scale bar: 1 μm. (MOV 77 kb)

A control for micropatterned sliding assay.

Dynamic microtubules (green) were growing from seeds (red) adsorbed on micropatterned sites. Scale bar: 10 μm. (MOV 130 kb)

Kip3-mediated buckling of dynamic microtubules.

Dynamic microtubules (green) were growing from seeds (red) adsorbed on micropatterned sites in the presence of 50 nM Kip3. One microtubule bundle started to buckle around 13:00. Scale bar: 10 μm. (MOV 127 kb)

Ase1-induced microtubule bundles.

Dynamic microtubules (green) were growing from seeds (red) adsorbed on micropatterned sites in the presence of 33 nM Ase1. Straight and stable bundles were formed. Scale bar: 10 μm. (MOV 124 kb)

Kip3-mediated buckling of Ase1 bundled microtubules.

Dynamic microtubules (green) were growing from seeds (red) adsorbed on micropatterned sites in the presence of 50 nM Kip3 and 33 nM Ase1. A stable microtubule bundle steadily buckled and elongated. Scale bar: 10 μm. (MOV 103 kb)

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Su, X., Arellano-Santoyo, H., Portran, D. et al. Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control. Nat Cell Biol 15, 948–957 (2013). https://doi.org/10.1038/ncb2801

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