Skip to main content
SpringerLink
Log in

Assays for mitotic chromosome condensation in live yeast and mammalian cells

  • Published:
Chromosome Research Aims and scope Submit manuscript

Abstract

The dynamic reorganization of chromatin into rigid and compact mitotic chromosomes is of fundamental importance for faithful chromosome segregation. Owing to the difficulty of investigating this process under physiological conditions, the exact morphological transitions and the molecular machinery driving chromosome condensation remain poorly defined. Here, we review how imaging-based methods can be used to quantitate chromosome condensation in vivo, focusing on yeast and animal tissue culture cells as widely used model systems. We discuss approaches how to address structural dynamics of condensing chromosomes and chromosome segments, as well as to probe for mechanical properties of mitotic chromosomes. Application of such methods to systematic perturbation studies will provide a means to reveal the molecular networks underlying the regulation of mitotic chromosome condensation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

Abbreviations

DAPI:

4′,6-diamidino-2-phenylindole dihydrochloride

EGFP:

enhanced green fluorescent protein

GFP:

green fluorescent protein

Sir2:

silent information regulator 2

Yku 70:

70 kDa subunit of the DNA-end binding Ku complex

References

  • Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A (2002) An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci U S A 99:12651–12656

    Article  PubMed  CAS  Google Scholar 

  • Beaudouin J, Gerlich D, Daigle N, Eils R, Ellenberg J (2002) Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108:83–96

    Article  PubMed  CAS  Google Scholar 

  • Belmont AS (2006) Mitotic chromosome structure and condensation. Curr Opin Cell Biol 18:632–638

    Article  PubMed  CAS  Google Scholar 

  • Belmont AS, Bruce K (1994) Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure. J Cell Biol 127:287–302

    Article  PubMed  CAS  Google Scholar 

  • Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  PubMed  CAS  Google Scholar 

  • Bouck DC, Joglekar AP, Bloom KS (2008) Design features of a mitotic spindle: balancing tension and compression at a single microtubule kinetochore interface in budding yeast. Annu Rev Genet 135(5):894–906

    Google Scholar 

  • Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22:1435–1439

    Article  PubMed  CAS  Google Scholar 

  • Claussen U, Mazur A, Rubtsov N (1994) Chromosomes are highly elastic and can be stretched. Cytogenet Cell Genet 66:120–125

    Article  PubMed  CAS  Google Scholar 

  • Conrad C, Erfle H, Warnat P et al (2004) Automatic identification of subcellular phenotypes on human cell arrays. Genome Res 14:1130–1136

    Article  PubMed  CAS  Google Scholar 

  • Dietzel S, Belmont AS (2001) Reproducible but dynamic positioning of DNA in chromosomes during mitosis. Nat Cell Biol 3:767–770

    Article  PubMed  CAS  Google Scholar 

  • Freeman L, Aragon-Alcaide L, Strunnikov A (2000) The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J Cell Biol 149:811–824

    Article  PubMed  CAS  Google Scholar 

  • Gassmann R, Vagnarelli P, Hudson D, Earnshaw WC (2004) Mitotic chromosome formation and the condensin paradox. Exp Cell Res 296:35–42

    Article  PubMed  CAS  Google Scholar 

  • Gerlich D, Beaudouin J, Gebhard M, Ellenberg J, Eils R (2001) Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells. Nat Cell Biol 3:852–855

    Article  PubMed  CAS  Google Scholar 

  • Gerlich D, Beaudouin J, Kalbfuss B, Daigle N, Eils R, Ellenberg J (2003) Global chromosome positions are transmitted through mitosis in mammalian cells. Cell 112:751–764

    Article  PubMed  CAS  Google Scholar 

  • Gerlich D, Hirota T, Koch B, Peters JM, Ellenberg J (2006) Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells. Curr Biol 16:333–344

    Article  PubMed  CAS  Google Scholar 

  • Gittes F, Mickey B, Nettleton J, Howard J (1993) Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape. J Cell Biol 120:923–934

    Article  PubMed  CAS  Google Scholar 

  • Glory E, Murphy RF (2007) Automated subcellular location determination and high-throughput microscopy. Dev Cell 12:7–16

    Article  PubMed  CAS  Google Scholar 

  • Guacci V, Hogan E, Koshland D (1994) Chromosome condensation and sister chromatid pairing in budding yeast. J Cell Biol 125:517–530

    Article  PubMed  CAS  Google Scholar 

  • Habuchi S, Ando R, Dedecker P et al (2005) Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci U S A 102:9511–9516

    Article  PubMed  CAS  Google Scholar 

  • Hill A, Bloom K (1989) Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae. Mol Cell Biol 9:1368–1370

    PubMed  CAS  Google Scholar 

  • Hliscs R, Muhlig P, Claussen U (1997) The nature of G-bands analyzed by chromosome stretching. Cytogenet Cell Genet 79:162–166

    PubMed  CAS  Google Scholar 

  • Houchmandzadeh B, Dimitrov S (1999) Elasticity measurements show the existence of thin rigid cores inside mitotic chromosomes. J Cell Biol 145:215–223

    Article  PubMed  CAS  Google Scholar 

  • Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813

    Article  PubMed  CAS  Google Scholar 

  • Kaitna S, Pasierbek P, Jantsch M, Loidl J, Glotzer M (2002) The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous chromosomes during meiosis. Curr Biol 12:798–812

    Article  PubMed  CAS  Google Scholar 

  • Kanda T, Sullivan KF, Wahl GM (1998) Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol 8:377–385

    Article  PubMed  CAS  Google Scholar 

  • Kireeva N, Lakonishok M, Kireev I, Hirano T, Belmont AS (2004) Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure. J Cell Biol 166:775–785

    Article  PubMed  CAS  Google Scholar 

  • Kuroiwa T, Kojima H, Miyakawa I, Sando N (1984) Meiotic karyotype of the yeast Saccharomyces cerevisiae. Exp Cell Res 153:259–65

    Article  PubMed  CAS  Google Scholar 

  • Lavoie BD, Hogan E, Koshland D (2004) In vivo requirements for rDNA chromosome condensation reveal two cell-cycle-regulated pathways for mitotic chromosome folding. Genes Dev 18:76–87

    Article  PubMed  CAS  Google Scholar 

  • Li X, Nicklas RB (1995) Mitotic forces control a cell-cycle checkpoint. Nature 373:630–632

    Article  PubMed  CAS  Google Scholar 

  • Machin F, Torres-Rosell J, Jarmuz A, Aragon L (2005) Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase. J Cell Biol 168:209–219

    Article  PubMed  CAS  Google Scholar 

  • Manders EM, Kimura H, Cook PR (1999) Direct imaging of DNA in living cells reveals the dynamics of chromosome formation. J Cell Biol 144:813–821

    Article  PubMed  CAS  Google Scholar 

  • Marko JF (2008) Micromechanical studies of mitotic chromosomes. Chromosome Res 16:469–497

    Article  PubMed  CAS  Google Scholar 

  • Marshall WF, Marko JF, Agard DA, Sedat JW (2001) Chromosome elasticity and mitotic polar ejection force measured in living Drosophila embryos by four-dimensional microscopy-based motion analysis. Curr Biol 11:569–578

    Article  PubMed  CAS  Google Scholar 

  • Matsuda T, Miyawaki A, Nagai T (2008) Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein. Nat Methods 5:339–345

    PubMed  CAS  Google Scholar 

  • Michaelis C, Ciosk R, Nasmyth K (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91:35–45

    Article  PubMed  CAS  Google Scholar 

  • Mikhailov A, Shinohara M, Rieder CL (2004) Topoisomerase II and histone deacetylase inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway. J Cell Biol 166:517–526

    Article  PubMed  CAS  Google Scholar 

  • Mo YY, Ameiss KA, Beck WT (1998) Overexpression of human DNA topoisomerase II alpha by fusion to enhanced green fluorescent protein. Biotechniques 25:1052–1057

    PubMed  CAS  Google Scholar 

  • Mora-Bermudez F, Ellenberg J (2007) Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy. Methods 41:158–167

    Article  PubMed  CAS  Google Scholar 

  • Mora-Bermudez F, Gerlich D, Ellenberg J (2007) Maximal chromosome compaction occurs by axial shortening in anaphase and depends on Aurora kinase. Nat Cell Biol 9:822–831

    Article  PubMed  CAS  Google Scholar 

  • Nicklas RB (1963) A quantitative study of chromosomal elasticity and its influence on chromosome movement. Chromosoma 14:276–295

    Article  PubMed  CAS  Google Scholar 

  • Nicklas RB (1983) Measurements of the force produced by the mitotic spindle in anaphase. J Cell Biol 97:542–548

    Article  PubMed  CAS  Google Scholar 

  • Oliveira RA, Coelho PA, Sunkel CE (2005) The condensin I subunit Barren/CAP-H is essential for the structural integrity of centromeric heterochromatin during mitosis. Mol Cell Biol 25:8971–8984

    Article  PubMed  CAS  Google Scholar 

  • Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877

    Article  PubMed  CAS  Google Scholar 

  • Pearson CG, Maddox PS, Salmon ED, Bloom K (2001) Budding yeast chromosome structure and dynamics during mitosis. J Cell Biol 152:1255–1266

    Article  PubMed  CAS  Google Scholar 

  • Poirier MG, Marko JF (2002) Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold. Proc Natl Acad Sci U S A 99:15393–15397

    Article  PubMed  CAS  Google Scholar 

  • Poirier MG, Marko JF (2003) Micromechanical studies of mitotic chromosomes. Curr Top Dev Biol 55:75–141

    Article  PubMed  CAS  Google Scholar 

  • Poirier M, Eroglu S, Chatenay D, Marko JF (2000) Reversible and irreversible unfolding of mitotic newt chromosomes by applied force. Mol Biol Cell 11:269–276

    PubMed  CAS  Google Scholar 

  • Poirier MG, Eroglu S, Marko JF (2002) The bending rigidity of mitotic chromosomes. Mol Biol Cell 13:2170–2179

    PubMed  CAS  Google Scholar 

  • Robinett CC, Straight A, Li G et al (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol 135:1685–1700

    Article  PubMed  CAS  Google Scholar 

  • Rohner S, Gasser SM, Meister P (2008) Modules for cloning-free chromatin tagging in Saccharomyces cerevisae. Yeast 25:235–239

    Article  PubMed  CAS  Google Scholar 

  • Sarkar A, Eroglu S, Poirier MG, Gupta P, Nemani A, Marko JF (2002) Dynamics of chromosome compaction during mitosis. Exp Cell Res 277:48–56

    Article  PubMed  CAS  Google Scholar 

  • Schermelleh L, Carlton PM, Haase S et al (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332–1336

    Article  PubMed  CAS  Google Scholar 

  • Scherthan H, Loidl J, Schuster T, Schweizer D (1992) Meiotic chromosome condensation and pairing in Saccharomyces cerevisiae studied by chromosome painting. Chromosoma 101:590–595

    Article  PubMed  CAS  Google Scholar 

  • Shelby RD, Hahn KM, Sullivan KF (1996) Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells. J Cell Biol 135:545–557

    Article  PubMed  CAS  Google Scholar 

  • Straight AF, Belmont AS, Robinett CC, Murray AW (1996) GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6:1599–1608

    Article  PubMed  CAS  Google Scholar 

  • Strukov YG, Wang Y, Belmont AS (2003) Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes. J Cell Biol 162:23–35

    Article  PubMed  CAS  Google Scholar 

  • Sullivan M, Higuchi T, Katis VL, Uhlmann F (2004) Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast anaphase. Cell 117:471–482

    Article  PubMed  CAS  Google Scholar 

  • Swedlow JR, Hirano T (2003) The making of the mitotic chromosome: modern insights into classical questions. Mol Cell 11:557–569

    Article  PubMed  CAS  Google Scholar 

  • Thrower DA, Bloom K (2001) Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast. Mol Biol Cell 12:2800–2812

    PubMed  CAS  Google Scholar 

  • Toda T, Yamamoto M, Yanagida M (1981) Sequential alterations in the nuclear chromatin region during mitosis of the fission yeast Schizosaccharomyces pombe: video fluorescence microscopy of synchronously growing wild-type and cold-sensitive cdc mutants by using a DNA-binding fluorescent probe. J Cell Sci 52:271–287

    PubMed  CAS  Google Scholar 

  • Uemura T, Ohkura H, Adachi Y, Morino K, Shiozaki K, Yanagida M (1987) DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S pombe. Cell 50:917–925

    Article  PubMed  CAS  Google Scholar 

  • Vas AC, Andrews CA, Kirkland Matesky K, Clarke DJ (2007) In vivo analysis of chromosome condensation in Saccharomyces cerevisiae. Mol Biol Cell 18:557–568

    Article  PubMed  CAS  Google Scholar 

  • Walter J, Schermelleh L, Cremer M, Tashiro S, Cremer T (2003) Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages. J Cell Biol 160:685–697

    Article  PubMed  CAS  Google Scholar 

  • Westphal V, Rizzoli SO, Lauterbach MA, Kamin D, Jahn R, Hell SW (2008) Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320:246–249

    Article  PubMed  CAS  Google Scholar 

  • Wiedenmann J, Ivanchenko S, Oswald F et al (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci U S A 101:15905–15910

    Article  PubMed  CAS  Google Scholar 

  • Win TZ, Goodwin A, Hickson ID, Norbury CJ, Wang SW (2004) Requirement for Schizosaccharomyces pombe Top3 in the maintenance of chromosome integrity. J Cell Sci 117:4769–4778

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a European Young Investigator Award of the European Science Foundation to D.G.

Author information

Authors and Affiliations

  1. Institute of Biochemistry, Swiss Institute of Technology Zurich (ETHZ), Schafmattstr. 18, CH-8093, Zurich, Switzerland

    Gabriel Neurohr & Daniel W. Gerlich

Authors
  1. Gabriel Neurohr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel W. Gerlich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel W. Gerlich.

Additional information

Responsible Editor: Christian Haering.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Neurohr, G., Gerlich, D.W. Assays for mitotic chromosome condensation in live yeast and mammalian cells. Chromosome Res 17, 145–154 (2009). https://doi.org/10.1007/s10577-008-9010-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10577-008-9010-1

Keywords

Search

Navigation