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.

Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces

Abstract

Accurate chromosome segregation during cell division in metazoans relies on proper chromosome congression at the equator. Chromosome congression is achieved after bi-orientation to both spindle poles shortly after nuclear envelope breakdown, or by the coordinated action of motor proteins that slide misaligned chromosomes along pre-existing spindle microtubules1. These proteins include the minus-end-directed kinetochore motor dynein2,3,4,5, and the plus-end-directed motors CENP-E at kinetochores6,7 and chromokinesins on chromosome arms8,9,10,11. However, how these opposite and spatially distinct activities are coordinated to drive chromosome congression remains unknown. Here we used RNAi, chemical inhibition, kinetochore tracking and laser microsurgery to uncover the functional hierarchy between kinetochore and arm-associated motors, exclusively required for congression of peripheral polar chromosomes in human cells. We show that dynein poleward force counteracts chromokinesins to prevent stabilization of immature/incorrect end-on kinetochore–microtubule attachments and random ejection of polar chromosomes. At the poles, CENP-E becomes dominant over dynein and chromokinesins to bias chromosome ejection towards the equator. Thus, dynein and CENP-E at kinetochores drive congression of peripheral polar chromosomes by preventing arm-ejection forces mediated by chromokinesins from working in the wrong direction.

Your institute does not have access to this article

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: Only a subgroup of chromosomes rely on dynein and CENP-E to congress to the metaphase plate.
Figure 2: Kinetochore motors are exclusively required to align peripheral chromosomes that are closer to one of the spindle poles.
Figure 3: Dynein counteracts PEFs to bring peripheral chromosomes to the poles and prevent stabilization of end-on kinetochore–microtubule attachments.
Figure 4: Stabilization of kinetochore–microtubule attachments on polar chromosomes leads to random ejection.
Figure 5: CENP-E is critical to bias polar chromosome movement exclusively towards the cell equator.

References

  1. Walczak, C. E., Cai, S. & Khodjakov, A. Mechanisms of chromosome behaviour during mitosis. Nat. Rev. 11, 91–102 (2010).

    CAS  Article  Google Scholar 

  2. Rieder, C. L. & Alexander, S. P. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J. Cell Biol. 110, 81–95 (1990).

    CAS  Article  Google Scholar 

  3. Li, Y., Yu, W., Liang, Y. & Zhu, X. Kinetochore dynein generates a poleward pulling force to facilitate congression and full chromosome alignment. Cell Res. 17, 701–712 (2007).

    CAS  Article  Google Scholar 

  4. Yang, Z., Tulu, U. S., Wadsworth, P. & Rieder, C. L. Kinetochore dynein is required for chromosome motion and congression independent of the spindle checkpoint. Curr. Biol. 17, 973–980 (2007).

    CAS  Article  Google Scholar 

  5. Vorozhko, V. V., Emanuele, M. J., Kallio, M. J., Stukenberg, P. T. & Gorbsky, G. J. Multiple mechanisms of chromosome movement in vertebrate cells mediated through the Ndc80 complex and dynein/dynactin. Chromosoma 117, 169–179 (2008).

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  7. Cai, S., O’Connell, C. B., Khodjakov, A. & Walczak, C. E. Chromosome congression in the absence of kinetochore fibres. Nat. Cell Biol. 11, 832–838 (2009).

    CAS  Article  Google Scholar 

  8. Antonio, C. et al. Xkid, a chromokinesin required for chromosome alignment on the metaphase plate. Cell 102, 425–435 (2000).

    CAS  Article  Google Scholar 

  9. Funabiki, H. & Murray, A. W. The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement. Cell 102, 411–424 (2000).

    CAS  Article  Google Scholar 

  10. Rieder, C. L., Davison, E. A., Jensen, L. C., Cassimeris, L. & Salmon, E. D. Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle. J. Cell Biol. 103, 581–591 (1986).

    CAS  Article  Google Scholar 

  11. Wandke, C. et al. Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis. J. Cell Biol. 198, 847–863 (2012).

    CAS  Article  Google Scholar 

  12. Poirier, C. C., Zheng, Y. & Iglesias, P. A. Mitotic membrane helps to focus and stabilize the mitotic spindle. Biophys. J. 99, 3182–3190 (2010).

    CAS  Article  Google Scholar 

  13. Civelekoglu-Scholey, G., Tao, L., Brust-Mascher, I., Wollman, R. & Scholey, J. M. Prometaphase spindle maintenance by an antagonistic motor-dependent force balance made robust by a disassembling lamin-B envelope. J. Cell Biol. 188, 49–68 (2010).

    CAS  Article  Google Scholar 

  14. Magidson, V. et al. The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly. Cell 146, 555–567 (2011).

    CAS  Article  Google Scholar 

  15. Brouhard, G. J. & Hunt, A. J. Microtubule movements on the arms of mitotic chromosomes: polar ejection forces quantified in vitro. Proc. Natl Acad. Sci. USA 102, 13903–13908 (2005).

    CAS  Article  Google Scholar 

  16. Levesque, A. A. & Compton, D. A. The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles. J. Cell Biol. 154, 1135–1146 (2001).

    CAS  Article  Google Scholar 

  17. Rieder, C. L. & Salmon, E. D. Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle. J. Cell Biol. 124, 223–233 (1994).

    CAS  Article  Google Scholar 

  18. Wood, K. W. et al. Antitumor activity of an allosteric inhibitor of centromere-associated protein-E. Proc. Natl Acad. Sci. USA 107, 5839–5844 (2010).

    CAS  Article  Google Scholar 

  19. Gassmann, R. et al. Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells. Genes Dev. 24, 957–971 (2010).

    CAS  Article  Google Scholar 

  20. Lombillo, V. A., Nislow, C., Yen, T. J., Gelfand, V. I. & McIntosh, J. R. Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro. J. Cell Biol. 128, 107–115 (1995).

    CAS  Article  Google Scholar 

  21. Gudimchuk, N. et al. Kinetochore kinesin CENP-E is a processive bi-directional tracker of dynamic microtubule tips. Nat. Cell Biol. 15, 1079–1088 (2013).

    CAS  Article  Google Scholar 

  22. Cane, S., Ye, A. A., Luks-Morgan, S. J. & Maresca, T. J. Elevated polar ejection forces stabilize kinetochore-microtubule attachments. J. Cell Biol. 200, 203–218 (2013).

    CAS  Article  Google Scholar 

  23. Barisic, M. et al. Spindly/CCDC99 is required for efficient chromosome congression and mitotic checkpoint regulation. Mol. Biol. Cell 21, 1968–1981 (2010).

    CAS  Article  Google Scholar 

  24. 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  Google Scholar 

  25. Stumpff, J., Wagenbach, M., Franck, A., Asbury, C. L. & Wordeman, L. Kif18A and chromokinesins confine centromere movements via microtubule growth suppression and spatial control of kinetochore tension. Dev. Cell 22, 1017–1029 (2012).

    CAS  Article  Google Scholar 

  26. Putkey, F. R. et al. Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E. Dev. Cell 3, 351–365 (2002).

    CAS  Article  Google Scholar 

  27. McEwen, B. F. et al. CENP-E is essential for reliable bioriented spindle attachment, but chromosome alignment can be achieved via redundant mechanisms in mammalian cells. Mol. Biol. Cell 12, 2776–2789 (2001).

    CAS  Article  Google Scholar 

  28. Yang, Z., Kenny, A. E., Brito, D. A. & Rieder, C. L. Cells satisfy the mitotic checkpoint in Taxol, and do so faster in concentrations that stabilize syntelic attachments. J. Cell Biol. 186, 675–684 (2009).

    CAS  Article  Google Scholar 

  29. Vasquez, R. J., Howell, B., Yvon, A. M., Wadsworth, P. & Cassimeris, L. Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro. Mol. Biol. Cell 8, 973–985 (1997).

    CAS  Article  Google Scholar 

  30. Cimini, D., Wan, X., Hirel, C. B. & Salmon, E. D. Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors. Curr. Biol. 16, 1711–1718 (2006).

    CAS  Article  Google Scholar 

  31. Hauf, S. et al. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J. Cell Biol. 161, 281–294 (2003).

    CAS  Article  Google Scholar 

  32. Bakhoum, S. F., Kabeche, L., Murnane, J. P., Zaki, B. I. & Compton, D. A. DNA damage response during mitosis induces whole chromosome mis-segregation. Cancer Discov. 10.1158/2159-8290.CD-14-0403 (2014).

  33. Kim, Y., Holland, A. J., Lan, W. & Cleveland, D. W. Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E. Cell 142, 444–455 (2010).

    CAS  Article  Google Scholar 

  34. Silkworth, W. T., Nardi, I. K., Paul, R., Mogilner, A. & Cimini, D. Timing of centrosome separation is important for accurate chromosome segregation. Mol. Biol. Cell 23, 401–411 (2012).

    CAS  Article  Google Scholar 

  35. Kaseda, K., McAinsh, A. D. & Cross, R. A. Dual pathway spindle assembly increases both the speed and the fidelity of mitosis. Biol. Open 1, 12–18 (2012).

    Article  Google Scholar 

  36. Lancaster, O. M. et al. Mitotic rounding alters cell geometry to ensure efficient bipolar spindle formation. Dev. Cell 25, 270–283 (2013).

    CAS  Article  Google Scholar 

  37. Hoffman, D. B., Pearson, C. G., Yen, T. J., Howell, B. J. & Salmon, E. D. Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores. Mol. Biol. Cell 12, 1995–2009 (2001).

    CAS  Article  Google Scholar 

  38. Cheerambathur, D. K., Gassmann, R., Cook, B., Oegema, K. & Desai, A. Crosstalk between microtubule attachment complexes ensures accurate chromosome segregation. Science 342, 1239–1242 (2013).

    CAS  Article  Google Scholar 

  39. Shrestha, R. L. & Draviam, V. M. Lateral to end-on conversion of chromosome-microtubule attachment requires kinesins CENP-E and MCAK. Curr. Biol. 23, 1514–1526 (2013).

    CAS  Article  Google Scholar 

  40. Takagi, J., Itabashi, T., Suzuki, K. & Ishiwata, S. Chromosome position at the spindle equator is regulated by chromokinesin and a bipolar microtubule array. Sci. Rep. 3, 2808 (2013).

    Article  Google Scholar 

  41. Wolf, F., Wandke, C., Isenberg, N. & Geley, S. Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells. EMBO J. 25, 2802–2813 (2006).

    CAS  Article  Google Scholar 

  42. Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T. & Weber, K. Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci. 114, 4557–4565 (2001).

    CAS  PubMed  Google Scholar 

  43. Draviam, V. M., Shapiro, I., Aldridge, B. & Sorger, P. K. Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells. EMBO J. 25, 2814–2827 (2006).

    CAS  Article  Google Scholar 

  44. Wandke, C. & Geley, S. Generation and characterization of an hKid-specific monoclonal antibody. Hybridoma (Larchmt) 25, 41–43 (2006).

    CAS  Article  Google Scholar 

  45. Maffini, S. et al. Motor-independent targeting of CLASPs to kinetochores by CENP-E promotes microtubule turnover and poleward flux. Curr. Biol. 19, 1566–1572 (2009).

    CAS  Article  Google Scholar 

  46. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  Article  Google Scholar 

  47. Pereira, A. J., Matos, I., Lince-Faria, M. & Maiato, H. Dissecting mitosis with laser microsurgery and RNAi in Drosophila cells. Methods Mol. Biol. 545, 145–164 (2009).

    Article  Google Scholar 

  48. Pereira, A. J. & Maiato, H. Improved kymography tools and its applications to mitosis. Methods 51, 214–219 (2010).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A. Pereira for the development of image analysis tools, M. Barisic for exceptional technical help and R. Gassmann for the critical reading of this manuscript S.G. is supported by FWF DK W 1101 ‘MCBO’. H.M. is financially supported by FEDER through the Operational Competitiveness Programme—COMPETE and by National Funds through FCT—Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-015941 (PTDC/SAU-ONC/112917/2009), the Human Frontier Science Program and the 7th framework program grant PRECISE from the European Research Council.

Author information

Authors and Affiliations

Authors

Contributions

M.B. designed, performed and analysed experiments. P.A. performed image analysis. S.G. provided reagents. H.M. designed and analysed experiments, and coordinated the work. H.M. and M.B. wrote the manuscript.

Corresponding author

Correspondence to Helder Maiato.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 RNAi and live cell imaging phenotypes.

(a) Protein lysates obtained 48 h after siRNA transfection (50 nM) were immunoblotted for the target proteins and α-tubulin was used as loading control. Due to the large size of DHC (500 kDa), CLASP1 (160 kDa) was used as a loading control in the lowest panel. n(Kid RNAi) = 1 experiment; n(Kif4A RNAi) = 1 experiment; n(DHC RNAi) = 1 experiment (due to limited antibody supply); n(KK RNAi) = 2 experiments. (b) Chromokinesins Kid and Kif4A were depleted by RNAi in U2OS-H2B-GFP/mCherry-α-tubulin cells with or without the presence of CENP-E inhibitor. Data was then acquired by live-cell imaging. n(control) = 4 cells imaged with 10 s interval (2 experiments) plus 9 cells imaged with 2 min interval (3 experiments); n(KK RNAi) = 16 cells (3 experiments); n(KK RNAi + CENP-E inh) = 5 cells (1 experiment). Scale bar = 5 μm. Time = h:min.

Supplementary Figure 2 Preventing loading of Dynein to kinetochores stabilizes microtubule attachments and leads to ejection of polar chromosomes.

RNAi resistant GFP-Spindly wild-type (WT) or F258A mutant were expressed in Spindly depleted U2OS cells stably expressing mCherry-α-tubulin. Data was then acquired by live-cell imaging. There were 5.1 ± 2.5 (mean ± s.d.) chromosomes ejected from the spindle in cells expressing GFP-Spindly (F258A) (n = 16 cells), as oposed to none in cells expressing GFP-Spindly (WT) (n = 5 cells). Both conditions were reproduced in 2 independent experiments. Scale bar, 5 μm, except in inset = 1 μm. Time = h:min.

Supplementary Figure 3 Both CENP-E and Dynein remain on kinetochores from polar chromosomes after CENP-E inhibition.

(a) U2OS cells immuno-stained for DNA (DAPI), kinetochores (ACA), α-tubulin and CENP-E. Maximum intensity projection images of representative examples are shown. n(CENP-E inh) = 5 cells (1 experiment). Scale bar, 5 μm. (b) U2OS cells immuno-stained for DNA (DAPI), kinetochores (ACA), α-tubulin and DHC. Maximum intensity projection images of representative examples in each indicated condition are shown. n(CENP-E inh) = 5 cells (1 experiment); n(CENP-E inh + DHC RNAi) = 5 cells (1 experiment; due to limited antibody supply). Scale bar, 5 μm.

Supplementary Figure 4 Original Western blot scans.

Full scans of Western blots corresponding to Supplementary Fig. 1. White lines represents positions where the original membrane was cut. Dashed white lines represent the areas used in Supplementary Fig. 1.

Supplementary information

Supplementary Information

Supplementary Information (PDF 736 kb)

Congression of only a subgroup of chromosomes depends on Dynein and CENP-E.

Spinning disk confocal time-lapse imaging of chromosome congression in a control U2OS H2B-GFP/mCherry-α-tubulin cell recorded every 10 s (video on the left) and compared to a cell treated with 20 nM of CENP-E inhibitor GSK923295 (added prior to imaging and before mitotic entry; video in the middle) and to a cell transfected with DHC specific siRNA for 48 h (video on the right), both recorded every 2 min. Spinning-disk confocal time-lapse imaging was performed every 10 s. Time = min:sec. (MOV 5562 kb)

Kinetochore tracking of peripheral chromosomes relying on CENP-E motor activity for congression.

Representative videos of chromosome congression and respective kinetochore tracking in a control U2OS GFP-CENP-A/mCherry-α-tubulin cell (on the left), compared to a cell treated with 20 nM of CENP-E inhibitor GSK923295 prior to imaging and before mitotic entry (on the right). Spinning-disk confocal time-lapse imaging was performed every 10 s. Each tracked kinetochore pair is indicated with a different color. Time = min:sec. (MOV 15999 kb)

Dynein counteracts PEFs to prevent stabilization of end-on kinetochore-microtubule attachments and random ejection of polar chromosomes.

Representative videos of a U2OS H2B-GFP/mCherry-α-tubulin cell transfected with DHC specific siRNA for 48 h and treated with 20 nM of CENP-E inhibitor GSK923295 prior to imaging and before mitotic entry (on the left), compared to a similarly treated cell, which was additionally transfected with chromokinesins (Kid + Kif4a) specific siRNAs for 48 h (on the right). Spinning-disk confocal time-lapse imaging was performed every 2 min. Time = h:min. (MOV 3375 kb)

Preventing Dynein localization at kinetochores leads to random ejection of polar chromosomes.

Representative videos of a U2OS cell expressing mCherry-α-tubulin and either RNAi-resistant GFP-Spindly wild-type (on the left) or GFP-Spindly F258A mutant (on the right), transfected with Spindly specific siRNA for 48 h and treated with 20 nM of CENP-E inhibitor GSK923295 prior to imaging and before mitotic entry. Spinning-disk confocal time-lapse imaging was performed every 2 min. Time = h:min. (MOV 6241 kb)

The motion of acentric fragments from polar chromosomes depends on chromokinesins-mediated PEFs and can be directed both towards the cell equator and the cortex.

Laser microsurgery on chromosome arms of CENP-E inhibited (20 nM GSK923295) U2OS-H2B-GFP/mCherry-α-tubulin cells (on the left and in the middle) compared to similarly treated cells that were additionally depleted of chromokinesins Kid and Kif4A by 48 h RNAi (on the right). Spinning-disk confocal time-lapse imaging was performed every 1 min. Time = h:min. (MOV 1287 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Barisic, M., Aguiar, P., Geley, S. et al. Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces. Nat Cell Biol 16, 1249–1256 (2014). https://doi.org/10.1038/ncb3060

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3060

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