Chromosome Research

, Volume 24, Issue 3, pp 339–353 | Cite as

Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines

  • Adrienne Eastland
  • Jessica Hornick
  • Ryo Kawamura
  • Dhaval Nanavati
  • John F. MarkoEmail author
Original Article


We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275–285, 1973) and Sumner (J Cell Sci 70:177–188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.


Metaphase chromosome Chromosome structure Disulfide Cysteine 



Chromosome-associated protein






Fetal bovine serum


Human embryonic kidney (cell)


Human serum albumin


N-ethyl maleimide


Phosphate-buffered saline


Polyacrylamide gel electrophoresis


Piconewton (10−12 N)


Regulator of chromosome condensation


Sodium dodecyl sulfate


Structural maintenance of chromosome (complex)





This work was supported by the NSF through grants MCB-1022117 and DMR-1206868 and by the NIH through grants R01-GM105847 and U54-CA193419 and by subcontract to U54-DK107980. Work at the Northwestern Proteomics Core was supported in part by the Northwestern Office of Research and the Feinberg School of Medicine.

Compliance with ethical standards

Ethical standards

The experiments described in this article comply with the current laws of the country where they were performed (USA). This article does not contain any studies with human or animal subjects performed by any of the authors.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Supplementary material

10577_2016_9528_MOESM1_ESM.pdf (620 kb)
ESM 1 (PDF 620 kb)
10577_2016_9528_MOESM2_ESM.xlsx (32 kb)
ESM 2 (XLSX 31 kb)
10577_2016_9528_MOESM3_ESM.xlsx (23 kb)
ESM 3 (XLSX 23 kb)
10577_2016_9528_MOESM4_ESM.xlsb (2.3 mb)
ESM 4 (XLSB 2347 kb)
10577_2016_9528_MOESM5_ESM.xlsx (51 kb)
ESM 5 (XLSX 51 kb)
10577_2016_9528_MOESM6_ESM.xlsx (21 kb)
ESM 6 (XLSX 20 kb)


  1. Brinkley M (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjug Chem 3:2–13CrossRefPubMedGoogle Scholar
  2. Bulleid NJ, Ellgaard L (2011) Multiple ways to make disulfides. Trends Biochem Sci 36:485–492CrossRefPubMedGoogle Scholar
  3. Chatterjee M, Paschal BM (2015) Disruption of the ran system by cysteine oxidation of the nucleotide exchange factor RCC1. Mol Cell Biol 35:566–581CrossRefPubMedGoogle Scholar
  4. Dounce AL, Chanda SK, Townes PL (1973) The structure of higher eukaryotic chromosomes. J Theor Biol 42:275–285CrossRefPubMedGoogle Scholar
  5. Goswami PC, Sheren J, Albee LD et al (2000) Cell cycle-coupled variation in topoisomerase IIalpha mRNA is regulated by the 3’-untranslated region. Possible role of redox-sensitive protein binding in mRNA accumulation. J Biol Chem 275:38384–38392CrossRefPubMedGoogle Scholar
  6. Hermanson GT (1996) Bioconjugate techniques. Academic, New YorkGoogle Scholar
  7. Hirano T, Mitchison TJ (1994) A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell 79:449–458CrossRefPubMedGoogle Scholar
  8. Hornick JE, Duncan FE, Sun M, Kawamura R, Marko JF, Woodruff TK (2015) Age-associated alterations in the micromechanical properties of chromosomes in the mammalian egg. J Assist Reprod Genet 32:765–769CrossRefPubMedPubMedCentralGoogle Scholar
  9. Hudson DA, Gannon SA, Thorpe C (2015) Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum. Free Radic Biol Med 80:171–182CrossRefPubMedGoogle Scholar
  10. Hwang NR, Yim SH, Kim YM et al (2009) Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions. Biochem J 423:253–264CrossRefPubMedGoogle Scholar
  11. Jeppesen P, Morten H (1985) Effects of sulphydryl reagents on the structure of dehistonized metaphase chromosomes. J Cell Sci 73:245–260PubMedGoogle Scholar
  12. Kawamura N, Dan K (1958) A cytochemical study of the sulfhydryl groups of sea urchin eggs during the first cleavage. J Biophys Biochem Cytol 4:615–619CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kawamura R, Pope LH, Christensen MO et al (2010) Mitotic chromosomes are constrained by topoisomerase II-sensitive DNA entanglements. J Cell Biol 188:653–663CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kuga T, Nakayama Y, Iwamatsu A, Fukumoto Y, Yokomori K, Yamaguchi N (2007) Separation of a disulfide-linked phosphoprotein by diagonal SDS-PAGE with optimized gel crosslinking. Anal Biochem 370:252–254CrossRefPubMedGoogle Scholar
  15. Lewis CD, Laemmli UK (1982) Higher order metaphase chromosome structure: evidence for metalloprotein interactions. Cell 29:171–181CrossRefPubMedGoogle Scholar
  16. Maeshima K, Laemmli UK (2003) A two-step scaffolding model for mitotic chromosome assembly. Dev Cell 4:467–480CrossRefPubMedGoogle Scholar
  17. Makde RD, England JR, Yennawar HP, Tan S (2010) Structure of RCC1 chromatin factor bound to the nucleosome core particle. Nature 467:562–566CrossRefPubMedPubMedCentralGoogle Scholar
  18. Marko JF (2008) Micromechanical studies of mitotic chromosomes. Chromosom Res 16:469–497CrossRefGoogle Scholar
  19. Meikrantz W, Suprynowicz FA, Halleck MS, Schlegel RA (1990) Identification of mitosis-specific p65 dimer as a component of human M phase-promoting factor. Proc Natl Acad Sci U S A 87:9600–9604CrossRefPubMedPubMedCentralGoogle Scholar
  20. Menon SG, Goswami PC (2007) A redox cycle within the cell cycle: ring in the old with the new. Oncogene 26:1101–1109CrossRefPubMedGoogle Scholar
  21. Menon SG, Sarsour EH, Spitz DR et al (2003) Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle. Cancer Res 63:2109–2117PubMedGoogle Scholar
  22. Nicklas RB (1963) A quantitative study of chromosomal elasticity and its influence on chromosome movement. Chromosoma 14:276–295CrossRefPubMedGoogle Scholar
  23. Nicklas RB (1983) Measurements of the force produced by the mitotic spindle in anaphase. J Cell Biol 97:542–548CrossRefPubMedGoogle Scholar
  24. Ohta S, Bukowski-Wills JC, Sanchez-Pulido L et al (2010) The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics. Cell 142:810–821CrossRefPubMedPubMedCentralGoogle Scholar
  25. Olszewska MJ, Marciniak K, Kuran H (1990) The timing of synthesis of proteins required for mitotic spindle and phragmoplast in partially synchronized root meristems of Vicia faba L. Eur J Cell Biol 53:89–92PubMedGoogle Scholar
  26. Ono T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T (2003) Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115:109–121CrossRefPubMedGoogle Scholar
  27. Patil NA, Tailhades J, Hughes RA, Separovic F, Wade JD, Hossain MA (2015) Cellular disulfide bond formation in bioactive peptides and proteins. Int J Mol Sci 16:1791–1805CrossRefPubMedPubMedCentralGoogle Scholar
  28. Poirier MG, Marko JF (2002) Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold. Proc Natl Acad Sci U S A 99:15393–15397CrossRefPubMedPubMedCentralGoogle Scholar
  29. Poirier M, Eroglu S, Chatenay D, Marko JF (2000) Reversible and irreversible unfolding of mitotic newt chromosomes by applied force. Mol Biol Cell 11:269–276CrossRefPubMedPubMedCentralGoogle Scholar
  30. Pope LH, Xiong C, Marko JF (2006) Proteolysis of mitotic chromosomes induces gradual and anisotropic decondensation correlated with a reduction of elastic modulus and structural sensitivity to rarely cutting restriction enzymes. Mol Biol Cell 17:104–113CrossRefPubMedPubMedCentralGoogle Scholar
  31. Rapkine L (1931) Su les processus chimiques au cours de la division cellulaire. Ann Physiol Physiochem Biol 7:382–418Google Scholar
  32. Samejima K, Samejima I, Vagnarelli P et al (2012) Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα. J Cell Biol 199:755–770CrossRefPubMedPubMedCentralGoogle Scholar
  33. Sarsour EH, Kumar MG, Chaudhuri L, Kalen AL, Goswami PC (2009) Redox control of the cell cycle in health and disease. Antioxid Redox Signal 11:2985–3011CrossRefPubMedPubMedCentralGoogle Scholar
  34. Sarsour EH, Kalen AL, Goswami PC (2014) Manganese superoxide dismutase regulates a redox cycle within the cell cycle. Antioxid Redox Signal 20:1618–1627CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sevier CS, Kaiser CA (2002) Formation and transfer of disulphide bonds in living cells. Nat Rev Mol Cell Biol 3:836–847CrossRefPubMedGoogle Scholar
  36. Shintomi K, Takahashi TS, Hirano T (2015) Reconstitution of mitotic chromatids with a minimum set of purified factors. Nat Cell Biol 17:1014–1023CrossRefPubMedGoogle Scholar
  37. Sone T, Iwano M, Kobayashi S et al (2002) Changes in chromosomal surface structure by different isolation conditions. Arch Histol Cytol 65:445–455CrossRefPubMedGoogle Scholar
  38. Struchkov VA, Strazhevskaia NB (1989) Specific disulfide links of the DNA residual protein complex. Dokl Akad Nauk SSSR 307:755–758PubMedGoogle Scholar
  39. Struchkov VA, Strazhevskaya NB (1994) Significance of specific protein S-S bonds in the structural-functional organization of DNA. Bull Exp Biol Med 118:875–878CrossRefGoogle Scholar
  40. Struchkov VA, Strazhevskaya NB, Blokhin DY (1992) Thiol-Induced fragmentation of chromosomal DNA. Bull Exp Biol Med 113:716–719Google Scholar
  41. Struchkov VA, Strazhevskaya NB, Blokhin DY (1995) Role of the disulfide bridges of the residual protein in the structure of chromosomal DNA. Biofizika 40:296–316PubMedGoogle Scholar
  42. Sumner AT (1984a) Distribution of protein sulphydryls and disulphides in fixed mammalian chromosomes, and their relationship to banding. J Cell Sci 70:177–188PubMedGoogle Scholar
  43. Sumner AT (1984b) X-ray microanalysis of protein sulfhydryl-groups in chromatin. Scan Electron Microsc, PT 2:897–904Google Scholar
  44. Sun M, Kawamura R, Marko JF (2011) Micromechanics of human mitotic chromosomes. Phys Biol 8:015003CrossRefPubMedPubMedCentralGoogle Scholar
  45. Takata H, Uchiyama S, Nakamura N et al (2007) A comparative proteome analysis of human metaphase chromosomes isolated from two different cell lines reveals a set of conserved chromosome-associated proteins. Genes Cells 12:269–284CrossRefPubMedGoogle Scholar
  46. Uchiyama S, Kobayashi S, Takata H et al (2005) Proteome analysis of human metaphase chromosomes. J Biol Chem 280:16994–17004CrossRefPubMedGoogle Scholar
  47. Vagnarelli P, Hudson DF, Ribeiro SA et al (2006) Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis. Nat Cell Biol 8:1133–1142CrossRefPubMedPubMedCentralGoogle Scholar
  48. Yang J, Carroll KS, Liebler DC (2016) The expanding landscape of the thiol redox proteome. Mol Cell Proteomics 15:1–11CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Adrienne Eastland
    • 1
  • Jessica Hornick
    • 1
  • Ryo Kawamura
    • 1
    • 2
  • Dhaval Nanavati
    • 3
  • John F. Marko
    • 1
    • 2
    Email author
  1. 1.Department of Molecular BiosciencesNorthwestern UniversityEvanstonUSA
  2. 2.Department of Physics and AstronomyNorthwestern UniversityEvanstonUSA
  3. 3.Proteomics Core, Chemistry of Life Processes InstituteNorthwestern UniversityEvanstonUSA

Personalised recommendations