Chromosome Research

, 17:145

Assays for mitotic chromosome condensation in live yeast and mammalian cells

Article

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.

Keywords

chromosome condensation cell division condensin live cell imaging quantitative image analysis GFP 

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

  1. 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 99:12651–12656PubMedCrossRefGoogle Scholar
  2. Beaudouin J, Gerlich D, Daigle N, Eils R, Ellenberg J (2002) Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108:83–96PubMedCrossRefGoogle Scholar
  3. Belmont AS (2006) Mitotic chromosome structure and condensation. Curr Opin Cell Biol 18:632–638PubMedCrossRefGoogle Scholar
  4. Belmont AS, Bruce K (1994) Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure. J Cell Biol 127:287–302PubMedCrossRefGoogle Scholar
  5. Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645PubMedCrossRefGoogle Scholar
  6. 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):894–906Google Scholar
  7. Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22:1435–1439PubMedCrossRefGoogle Scholar
  8. Claussen U, Mazur A, Rubtsov N (1994) Chromosomes are highly elastic and can be stretched. Cytogenet Cell Genet 66:120–125PubMedCrossRefGoogle Scholar
  9. Conrad C, Erfle H, Warnat P et al (2004) Automatic identification of subcellular phenotypes on human cell arrays. Genome Res 14:1130–1136PubMedCrossRefGoogle Scholar
  10. Dietzel S, Belmont AS (2001) Reproducible but dynamic positioning of DNA in chromosomes during mitosis. Nat Cell Biol 3:767–770PubMedCrossRefGoogle Scholar
  11. Freeman L, Aragon-Alcaide L, Strunnikov A (2000) The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J Cell Biol 149:811–824PubMedCrossRefGoogle Scholar
  12. Gassmann R, Vagnarelli P, Hudson D, Earnshaw WC (2004) Mitotic chromosome formation and the condensin paradox. Exp Cell Res 296:35–42PubMedCrossRefGoogle Scholar
  13. 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 3:852–855PubMedCrossRefGoogle Scholar
  14. Gerlich D, Beaudouin J, Kalbfuss B, Daigle N, Eils R, Ellenberg J (2003) Global chromosome positions are transmitted through mitosis in mammalian cells. Cell 112:751–764PubMedCrossRefGoogle Scholar
  15. 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 16:333–344PubMedCrossRefGoogle Scholar
  16. 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 120:923–934PubMedCrossRefGoogle Scholar
  17. Glory E, Murphy RF (2007) Automated subcellular location determination and high-throughput microscopy. Dev Cell 12:7–16PubMedCrossRefGoogle Scholar
  18. Guacci V, Hogan E, Koshland D (1994) Chromosome condensation and sister chromatid pairing in budding yeast. J Cell Biol 125:517–530PubMedCrossRefGoogle Scholar
  19. 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 102:9511–9516PubMedCrossRefGoogle Scholar
  20. Hill A, Bloom K (1989) Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae. Mol Cell Biol 9:1368–1370PubMedGoogle Scholar
  21. Hliscs R, Muhlig P, Claussen U (1997) The nature of G-bands analyzed by chromosome stretching. Cytogenet Cell Genet 79:162–166PubMedGoogle Scholar
  22. Houchmandzadeh B, Dimitrov S (1999) Elasticity measurements show the existence of thin rigid cores inside mitotic chromosomes. J Cell Biol 145:215–223PubMedCrossRefGoogle Scholar
  23. Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813PubMedCrossRefGoogle Scholar
  24. 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 12:798–812PubMedCrossRefGoogle Scholar
  25. Kanda T, Sullivan KF, Wahl GM (1998) Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol 8:377–385PubMedCrossRefGoogle Scholar
  26. 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 166:775–785PubMedCrossRefGoogle Scholar
  27. Kuroiwa T, Kojima H, Miyakawa I, Sando N (1984) Meiotic karyotype of the yeast Saccharomyces cerevisiae. Exp Cell Res 153:259–65PubMedCrossRefGoogle Scholar
  28. 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 18:76–87PubMedCrossRefGoogle Scholar
  29. Li X, Nicklas RB (1995) Mitotic forces control a cell-cycle checkpoint. Nature 373:630–632PubMedCrossRefGoogle Scholar
  30. 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 168:209–219PubMedCrossRefGoogle Scholar
  31. Manders EM, Kimura H, Cook PR (1999) Direct imaging of DNA in living cells reveals the dynamics of chromosome formation. J Cell Biol 144:813–821PubMedCrossRefGoogle Scholar
  32. Marko JF (2008) Micromechanical studies of mitotic chromosomes. Chromosome Res 16:469–497PubMedCrossRefGoogle Scholar
  33. 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 11:569–578PubMedCrossRefGoogle Scholar
  34. Matsuda T, Miyawaki A, Nagai T (2008) Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein. Nat Methods 5:339–345PubMedGoogle Scholar
  35. Michaelis C, Ciosk R, Nasmyth K (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91:35–45PubMedCrossRefGoogle Scholar
  36. 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 166:517–526PubMedCrossRefGoogle Scholar
  37. Mo YY, Ameiss KA, Beck WT (1998) Overexpression of human DNA topoisomerase II alpha by fusion to enhanced green fluorescent protein. Biotechniques 25:1052–1057PubMedGoogle Scholar
  38. Mora-Bermudez F, Ellenberg J (2007) Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy. Methods 41:158–167PubMedCrossRefGoogle Scholar
  39. 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 9:822–831PubMedCrossRefGoogle Scholar
  40. Nicklas RB (1963) A quantitative study of chromosomal elasticity and its influence on chromosome movement. Chromosoma 14:276–295PubMedCrossRefGoogle Scholar
  41. Nicklas RB (1983) Measurements of the force produced by the mitotic spindle in anaphase. J Cell Biol 97:542–548PubMedCrossRefGoogle Scholar
  42. 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 25:8971–8984PubMedCrossRefGoogle Scholar
  43. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877PubMedCrossRefGoogle Scholar
  44. Pearson CG, Maddox PS, Salmon ED, Bloom K (2001) Budding yeast chromosome structure and dynamics during mitosis. J Cell Biol 152:1255–1266PubMedCrossRefGoogle Scholar
  45. Poirier MG, Marko JF (2002) Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold. Proc Natl Acad Sci U S A 99:15393–15397PubMedCrossRefGoogle Scholar
  46. Poirier MG, Marko JF (2003) Micromechanical studies of mitotic chromosomes. Curr Top Dev Biol 55:75–141PubMedCrossRefGoogle Scholar
  47. Poirier M, Eroglu S, Chatenay D, Marko JF (2000) Reversible and irreversible unfolding of mitotic newt chromosomes by applied force. Mol Biol Cell 11:269–276PubMedGoogle Scholar
  48. Poirier MG, Eroglu S, Marko JF (2002) The bending rigidity of mitotic chromosomes. Mol Biol Cell 13:2170–2179PubMedGoogle Scholar
  49. 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 135:1685–1700PubMedCrossRefGoogle Scholar
  50. Rohner S, Gasser SM, Meister P (2008) Modules for cloning-free chromatin tagging in Saccharomyces cerevisae. Yeast 25:235–239PubMedCrossRefGoogle Scholar
  51. Sarkar A, Eroglu S, Poirier MG, Gupta P, Nemani A, Marko JF (2002) Dynamics of chromosome compaction during mitosis. Exp Cell Res 277:48–56PubMedCrossRefGoogle Scholar
  52. Schermelleh L, Carlton PM, Haase S et al (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332–1336PubMedCrossRefGoogle Scholar
  53. Scherthan H, Loidl J, Schuster T, Schweizer D (1992) Meiotic chromosome condensation and pairing in Saccharomyces cerevisiae studied by chromosome painting. Chromosoma 101:590–595PubMedCrossRefGoogle Scholar
  54. 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 135:545–557PubMedCrossRefGoogle Scholar
  55. 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 6:1599–1608PubMedCrossRefGoogle Scholar
  56. 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 162:23–35PubMedCrossRefGoogle Scholar
  57. Sullivan M, Higuchi T, Katis VL, Uhlmann F (2004) Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast anaphase. Cell 117:471–482PubMedCrossRefGoogle Scholar
  58. Swedlow JR, Hirano T (2003) The making of the mitotic chromosome: modern insights into classical questions. Mol Cell 11:557–569PubMedCrossRefGoogle Scholar
  59. Thrower DA, Bloom K (2001) Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast. Mol Biol Cell 12:2800–2812PubMedGoogle Scholar
  60. 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 52:271–287PubMedGoogle Scholar
  61. 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 50:917–925PubMedCrossRefGoogle Scholar
  62. Vas AC, Andrews CA, Kirkland Matesky K, Clarke DJ (2007) In vivo analysis of chromosome condensation in Saccharomyces cerevisiae. Mol Biol Cell 18:557–568PubMedCrossRefGoogle Scholar
  63. 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 160:685–697PubMedCrossRefGoogle Scholar
  64. Westphal V, Rizzoli SO, Lauterbach MA, Kamin D, Jahn R, Hell SW (2008) Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320:246–249PubMedCrossRefGoogle Scholar
  65. 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 101:15905–15910PubMedCrossRefGoogle Scholar
  66. 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 117:4769–4778PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Institute of BiochemistrySwiss Institute of Technology Zurich (ETHZ)ZurichSwitzerland

Personalised recommendations