Letter | Published:

Ki-67 acts as a biological surfactant to disperse mitotic chromosomes

Nature volume 535, pages 308312 (14 July 2016) | Download Citation

Abstract

Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis1,2,3. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes4 and the discovery of proteins at the chromosome surface3,5,6, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Shaping mitotic chromosomes: From classical concepts to molecular mechanisms. BioEssays 37, 755–766 (2015)

  2. 2.

    Condensin-based chromosome organization from bacteria to vertebrates. Cell 164, 847–857 (2016)

  3. 3.

    , , , & Building mitotic chromosomes. Curr. Opin. Cell Biol. 23, 114–121 (2011)

  4. 4.

    et al. Organization of the mitotic chromosome. Science 342, 948–953 (2013)

  5. 5.

    , & The perichromosomal layer. Chromosoma 114, 377–388 (2005)

  6. 6.

    et al. The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics. Cell 142, 810–821 (2010)

  7. 7.

    et al. Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient. Nature453, 1132–1136 (2008)

  8. 8.

    et al. Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature464, 721–727 (2010)

  9. 9.

    et al. Integration of biological data by kernels on graph nodes allows prediction of new genes involved in mitotic chromosome condensation. Mol. Biol. Cell 25, 2522–2536 (2014)

  10. 10.

    , , & Common markers of proliferation. Nature Rev. Cancer 6, 99–106 (2006)

  11. 11.

    , , , & Ki67 in breast cancer: prognostic and predictive potential. Lancet Oncol. 11, 174–183 (2010)

  12. 12.

    , , , & In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase. Exp. Cell Res. 308, 123–134 (2005)

  13. 13.

    et al. Ki-67 detects a nuclear matrix-associated proliferation-related antigen. II. Localization in mitotic cells and association with chromosomes. J. Cell Sci. 92, 531–540 (1989)

  14. 14.

    et al. Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery. eLife 3, e01641 (2014)

  15. 15.

    et al. The cell proliferation antigen Ki-67 organises heterochromatin. eLife 5, e13722 (2016)

  16. 16.

    , , & The role of Hklp2 in the stabilization and maintenance of spindle bipolarity. Curr. Biol. 19, 1712–1717 (2009)

  17. 17.

    , , , & Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108, 83–96 (2002)

  18. 18.

    et al. Ki-67 is required for maintenance of cancer stem cells but not cell proliferation. Oncotarget 7, 6281–6293 (2016)

  19. 19.

    & The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors. Trends Biochem. Sci. 20, 347–349 (1995)

  20. 20.

    et al. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J. Cell Biol. 123, 513–522 (1993)

  21. 21.

    , , & Chmadrin: a novel Ki-67 antigen-related perichromosomal protein possibly implicated in higher order chromatin structure. J. Cell Sci. 112, 2463–2472 (1999)

  22. 22.

    , , , & A novel nucleolar protein, NIFK, interacts with the forkhead associated domain of Ki-67 antigen in mitosis. J. Biol. Chem. 276, 25386–25391 (2001)

  23. 23.

    & Surfactants and Interfacial Phenomena 4th edn (John Wiley & Sons, 2012)

  24. 24.

    Polymer brushes. Science 251, 905–914 (1991)

  25. 25.

    , & Liquid–liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39–58 (2014)

  26. 26.

    , & Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc. Natl Acad. Sci. USA 108, 4334–4339 (2011)

  27. 27.

    , , , & Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles. Proc. Natl Acad. Sci. USA 111, E2636–E2645 (2014)

  28. 28.

    & Automated live microscopy to study mitotic gene function in fluorescent reporter cell lines. Methods Mol. Biol. 545, 113–134 (2009)

  29. 29.

    et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013)

  30. 30.

    et al. Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science 331, 1616–1620 (2011)

  31. 31.

    et al. SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nature Commun . 6, 8497 (2015)

  32. 32.

    et al. Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates. J. Biomol. Screen. 13, 575–580 (2008)

  33. 33.

    et al. CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging. Nature Methods 7, 747–754 (2010)

Download references

Acknowledgements

We thank the IMBA/IMP BioOptics microscopy facility and Molecular Biology Service, J. Meissner, and M. J. Hossain for technical support, H. Liu and S. Tietscher for generation of plasmids, C. Haering, M. Samwer, W. H. Gerlich, and O. Wueseke for comments on the manuscript, Life Science Editors for editing assistance, and U. Kutay for LAP2β-GFP/H2B–mRFP-expressing cells. D.W.G., A.A.H. and J.E. have received funding from the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 241548 (MitoSys), and A.Z.P., D.W.G. and J.E. under grant agreement no. 258068 (Systems Microscopy). D.G. has received funding from an ERC Starting Grant under agreement no. 281198 (DIVIMAGE), and from the Austrian Science Fund (FWF) project no. SFB F34-06 (Chromosome Dynamics). S.C. has received funding from a Human Frontier Science Program Long-Term Postdoctoral Fellowship and the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 330114 (IEF). T.M.R. was supported by Deutsche Forschungsgemeinschaft (DFG): SPP1384 ‘Mechanisms of Genome Haploidization’ MU 1423/3-2 and grant MU 1423/8-1.

Author information

Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria

    • Sara Cuylen
    • , Claudia Blaukopf
    •  & Daniel W. Gerlich
  2. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany

    • Antonio Z. Politi
    •  & Jan Ellenberg
  3. Medical Faculty Carl Gustav Carus, Experimental Center, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany

    • Thomas Müller-Reichert
  4. Advanced Light Microscopy Facility, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany

    • Beate Neumann
  5. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany

    • Ina Poser
    •  & Anthony A. Hyman

Authors

  1. Search for Sara Cuylen in:

  2. Search for Claudia Blaukopf in:

  3. Search for Antonio Z. Politi in:

  4. Search for Thomas Müller-Reichert in:

  5. Search for Beate Neumann in:

  6. Search for Ina Poser in:

  7. Search for Jan Ellenberg in:

  8. Search for Anthony A. Hyman in:

  9. Search for Daniel W. Gerlich in:

Contributions

D.W.G. and S.C. conceived the project and designed experiments. B.N. and J.E. generated siRNA library transfection plates. I.P. and A.A.H. generated Ki-67–EGFP BAC cell pools. A.Z.P. and J.E. performed FCS measurements. D.W.G. and T.M.-R. performed electron microscopy experiments. S.C. performed all other experiments and C.B. assisted with the RNAi screen, chromosome spreads, cell line generation and cloning. D.W.G., S.C. and A.A.H. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Daniel W. Gerlich.

Reviewer Information Nature thanks T. Mitchison and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figure 1 (uncropped Western blots), Supplementary Methods, Supplementary Tables 2 - 4 and Supplementary References.

Excel files

  1. 1.

    Supplementary Table 1

    SiRNAs used in the screen for mitotic chromosome surface adhesion regulators

Videos

  1. 1.

    Chromosome motility in Ki-67 and control depleted mitotic cells in the absence of the spindle

    Mitotic monoclonal fluorescent HeLa cell line expressing H2B-mCherry/CENPA-EGFP was imaged in presence of nocodazole 70 h after indicated siRNA transfection. Bar, 10 µm.

  2. 2.

    Nuclear envelope removal in Ki-67 and control depleted cells

    Monoclonal fluorescent HeLa cell line expressing H2B-mRFP/LAP2β-EGFP was imaged in the presence of nocodazole 48 h after indicated siRNA transfection. Bar, 10 µm.

  3. 3.

    Adhesion of chromosomes following nuclear envelope break-down in Ki-67 knockout cells

    Ki-67 knock-out HeLa cell line labeled with SiR-Hoechst was imaged in presence of nocodazole starting at prophase. Bar, 10 µm.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature18610

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.