Measuring Kinetochore–Microtubule Attachment Stability in Cultured Cells

  • Keith F. DeLuca
  • Jacob A. Herman
  • Jennifer G. DeLuca
Part of the Methods in Molecular Biology book series (MIMB, volume 1413)


Duplicated sister chromatids connect to the mitotic spindle through kinetochores, large proteinaceous structures built at sites of centromeric heterochromatin. Kinetochores are responsible for harnessing the forces generated by microtubule polymerization and depolymerization to power chromosome movements. The fidelity of chromosome segregation relies on proper kinetochore function, as precise regulation of the attachment between kinetochores and microtubules is essential to prevent mitotic errors, which are linked to the initiation and progression of cancer and the formation of birth defects (Godek et al., Nat Rev Mol Cell Biol 16(1):57–64, 2014; Ricke and van Deursen, Semin Cell Dev Biol 22(6):559–565, 2011; Holland and Cleveland, EMBO Rep 13(6):501–514, 2012). Here we describe assays to quantitatively measure kinetochore–microtubule attachment stability in cultured cells.

Key words

Kinetochore Microtubule Spindle Mitosis Chromosome 


  1. 1.
    Godek KM, Kabeche L, Compton DA (2014) Regulation of kinetochore-microtubule attachments through homeostatic control during mitosis. Nat Rev Mol Cell Biol 16(1):57–64CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ricke RM, van Deursen JM (2011) Correction of microtubule-kinetochore attachment errors: mechanisms and role in tumor suppression. Semin Cell Dev Biol 22(6):559–565CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Holland AJ, Cleveland DW (2012) Losing balance: the origin and impact of aneuploidy in cancer. EMBO Rep 13(6):501–514CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sarangapani KK, Asbury CL (2014) Catch and release: how do kinetochores hook the right microtubules during mitosis? Trends Genet 30(4):150–159CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Foley EA, Kapoor TM (2013) Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. Nat Rev Mol Cell Biol 14(1):25–37CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Caldas GV, DeLuca JG (2014) KNL1: bringing order to the kinetochore. Chromosoma 23:169–181CrossRefGoogle Scholar
  7. 7.
    Sacristan C, Kops GJ (2014) Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling. Trends Cell Biol 25(1):21–28CrossRefPubMedGoogle Scholar
  8. 8.
    Santaguida S, Musacchio A (2009) The life and miracles of kinetochores. EMBO J 28(17):2511–2531CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Fukagawa T, Earnshaw WC (2014) The centromere: chromatin foundation for the kinetochore machinery. Dev Cell 30(5):496–508CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hori T, Fukagawa T (2012) Establishment of the vertebrate kinetochores. Chromosome Res 20(5):547–561CrossRefPubMedGoogle Scholar
  11. 11.
    Ding Y, Hubert CG, Herman J, Corrin P, Toledo CM, Skutt-Kakaria K, Vazquez J, Basom R, Zhang B, Risler JK, Pollard SM, Nam DH, Delrow JJ, Zhu J, Lee J, DeLuca J, Olson JM, Paddison PJ (2012) Cancer specific requirement for BUB1B/BubR1 in human brain tumor isolates and genetically transformed cells. Cancer Discov 3(2):198–211CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bakhoum SF, Thompson SL, Manning AL, Compton DA (2009) Genome stability is ensured by temporal control of kinetochore-microtubule dynamics. Nat Cell Biol 11(1):27–35CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ertych N, Stolz A, Stenzinger A, Weichert W, Kaulfuß S, Burfeind P, Aigner A, Wordeman L, Bastians H (2014) Increased microtubule assembly rates influence chromosomal instability in colorectal cancer cells. Nat Cell Biol 16(8):779–791CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Wendell KL, Wilson L, Jordan MA (1993) Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes. J Cell Sci 104(Pt 2):261–274PubMedGoogle Scholar
  15. 15.
    McEwen BF, Chan GK, Zubrowski B, Savoian MS, Sauer MT, Yen TJ (2001) 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(9):2776–2789CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    DeLuca JG, Dong Y, Hergert P, Strauss J, Hickey JM, Salmon ED, McEwen BF (2005) Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites. Mol Biol Cell 16(2):519–531CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zaytsev AV, Sundin LJR, DeLuca KF, Grishchuk EL, DeLuca JG (2014) Accurate phosphoregulation of kinetochore-microtubule affinity requires unconstrained molecular interactions. J Cell Biol 206:45–59CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hoffman DB, Pearson CG, Yen TJ, Howell BJ, Salmon ED (2001) Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores. Mol Biol Cell 12(7):1995–2009CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Waters JC, Chen RH, Murray AW, Salmon ED (1998) Localization of Mad2 to kinetochores depends on microtubule attachment, not tension. J Cell Biol 141(5):1181–1191CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    DeLuca JG, Moree B, Hickey JM, Kilmartin JV, Salmon ED (2002) hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells. J Cell Biol 159(4):549–555CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Brinkley BR, Cartwright J Jr (1975) Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells. Ann N Y Acad Sci 253:428–439CrossRefPubMedGoogle Scholar
  22. 22.
    Salmon ED, Goode D, Maugel TK, Bonar DB (1976) Pressure-induced depolymerization of spindle microtubules. III. Differential stability in HeLa cells. J Cell Biol 69(2):443–454CrossRefPubMedGoogle Scholar
  23. 23.
    Mitchison T, Evans L, Schulze E, Kirschner M (1986) Sites of microtubule assembly and disassembly in the mitotic spindle. Cell 45(4):515–527CrossRefPubMedGoogle Scholar
  24. 24.
    Cassimeris L, Rieder CL, Rupp G, Salmon ED (1990) Stability of microtubule attachment to metaphase kinetochores in PtK1 cells. J Cell Sci 96(Pt 1):9–15PubMedGoogle Scholar
  25. 25.
    Biggins S, Severin FF, Bhalla N, Sassoon I, Hyman AA, Murray AW (1999) The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast. Genes Dev 13(5):532–544CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lampson MA, Cheeseman IM (2011) Sensing centromere tension: aurora B and the regulation of kinetochore function. Trends Cell Biol 21(3):133–140CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Pinsky BA, Kung C, Shokat KM, Biggins S (2006) The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores. Nat Cell Biol 8(1):78–83CrossRefPubMedGoogle Scholar
  28. 28.
    Carmena M, Wheelock M, Funabiki H, Earnshaw WC (2012) The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nat Rev Mol Cell Biol 13(12):789–803CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Funabiki H, Wynne DJ (2013) Making an effective switch at the kinetochore by phosphorylation and dephosphorylation. Chromosoma 122(3):135–158CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    DeLuca KF, Lens SM, DeLuca JG (2011) Temporal changes in Hec1 phosphorylation control kinetochore-microtubule attachment stability during mitosis. J Cell Sci 124(Pt 4):622–634CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Caldas GV, DeLuca KF, DeLuca JG (2014) KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity. J Cell Biol 203(6):957–969CrossRefGoogle Scholar
  32. 32.
    Salmon ED, Cimini D, Cameron LA, DeLuca JG (2005) Merotelic kinetochores in mammalian tissue cells. Philos Trans R Soc Lond B Biol Sci 360(1455):553–568CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Gregan J, Polakova S, Zhang L, Tolić-Nørrelykke IM, Cimini D (2011) Merotelic kinetochore attachment: causes and effects. Trends Cell Biol 21(6):374–381CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Lampson MA, Renduchitala K, Khodjakov A, Kapoor TM (2004) Correcting improper chromosome-spindle attachments during cell division. Nat Cell Biol 6(3):232–237CrossRefPubMedGoogle Scholar
  35. 35.
    Vader G, Cruijsen CW, van Harn T, Vromans MJ, Medema RH, Lens SM (2007) The chromosomal passenger complex controls spindle checkpoint function independent from its role in correcting microtubule kinetochore interactions. Mol Biol Cell 18(11):4553–4564CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Sundin LJ, Guimaraes GJ, Deluca JG (2011) The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis. Mol Biol Cell 22(6):759–768CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchison TJ (1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286(5441):971–974CrossRefPubMedGoogle Scholar
  38. 38.
    Kapoor TM, Mayer TU, Coughlin ML, Mitchison TJ (2000) Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5. J Cell Biol 150(5):975–988CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Skoufias DA, DeBonis S, Saoudi Y, Lebeau L, Crevel I, Cross R, Wade RH, Hackney D, Kozielski F (2006) S-trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression. J Biol Chem 281(26):17559–17569CrossRefPubMedGoogle Scholar
  40. 40.
    Kabeche L, Compton DA (2013) Cyclin A regulates kinetochore microtubules to promote faithful chromosome segregation. Nature 502(7469):110–113CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    DeLuca JG, Gall WE, Ciferri C, Cimini D, Musacchio A, Salmon ED (2006) Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell 127(5):969–982CrossRefPubMedGoogle Scholar
  42. 42.
    Cimini D, Wan X, Hirel CB, Salmon ED (2006) Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors. Curr Biol 16(17): 1711–1718CrossRefPubMedGoogle Scholar
  43. 43.
    Hsu TC (1954) Cytological studies on HeLa, a strain of human cervical carcinoma, I. Observations on mitosis and chromosomes. Tex Rep Biol Med 12(4):833–846PubMedGoogle Scholar
  44. 44.
    Moses MJ, Counce SJ (1974) Electron microscopy of kinetochores in whole mount spreads of mitotic chromosomes from hela cells. J Exp Zool 189(1):115–120CrossRefPubMedGoogle Scholar
  45. 45.
    Brinkley BR, Rao PN (1973) Nitrous oxide: effects on the mitotic apparatus and chromosome movement in HeLa cells. J Cell Biol 58(1):96–106CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Landry JJ, Pyl PT, Rausch T, Zichner T, Tekkedil MM, Stütz AM, Jauch A, Aiyar RS, Pau G, Delhomme N, Gagneur J, Korbel JO, Huber W, Steinmetz LM (2013) The genomic and transcriptomic landscape of a HeLa cell line. G3 (Bethesda) 3(8):1213–1224CrossRefGoogle Scholar
  47. 47.
    Orr B, Compton DA (2013) A double-edged sword: how oncogenes and tumor suppressor genes can contribute to chromosomal instability. Front Oncol 3:164CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Luo J, Emanuele MJ, Li D, Creighton CJ, Schlabach MR, Westbrook TF, Wong KK, Elledge SJ (2009) A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137(5):835–848CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Herman JA, Toledo CM, Olson JM, DeLuca JG, Paddison PJ (2015) Molecular pathways: regulation and targeting of kinetochore-microtubule attachment in cancer. Clin Cancer Res 21:233–239CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Thompson SL, Bakhoum SF, Compton DA (2010) Mechanisms of chromosomal instability. Curr Biol 20(6):R285–R295CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Salimian KJ, Ballister ER, Smoak EM, Wood S, Panchenko T, Lampson MA, Black BE (2011) Feedback control in sensing chromosome biorientation by the Aurora B kinase. Curr Biol 21(13):1158–1165CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, Beeche M, Bodnar AG, Wahl GM, Tlsty TD, Chiu CP (1999) Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet 21(1):111–114CrossRefPubMedGoogle Scholar
  53. 53.
    Mikhailov A, Cole RW, Rieder CL (2002) DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint. Curr Biol 12(21):1797–1806CrossRefPubMedGoogle Scholar
  54. 54.
    Uetake Y, Sluder G (2004) ll cycle progression after cleavage failure: mammalian somatic cells do not possess a “tetraploidy checkpoint”. J Cell Biol 165(5):609–615CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Walen KH, Brown SW (1962) Chromosomes in a marsupial (Potorous tridactylis) tissue culture. Nature 194:406CrossRefPubMedGoogle Scholar
  56. 56.
    Levan A, Nichols WW, Peluse M, Coriell LL (1966) The stemline chromosomes of three cell lines representing different vertebrate classes. Chromosoma 18(2):343–358CrossRefPubMedGoogle Scholar
  57. 57.
    Levan G (1970) Contributions to the chromosomal characterization of the PTK 1 rat-kangaroo cell line. Hereditas 64(1):85–96CrossRefPubMedGoogle Scholar
  58. 58.
    Stout JR, Rizk RS, Kline SL, Walczak CE (2006) Deciphering protein function during mitosis in PtK cells using RNAi. BMC Cell Biol 23:7–26Google Scholar
  59. 59.
    Guimaraes GJ, Dong Y, McEwen BF, Deluca JG (2008) Kinetochore-microtubule attachment relies on the disordered N-terminal tail domain of Hec1. Curr Biol 18(22):1778–1784CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7):397–405CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188(4):773–782CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Joung JK, Sander JD (2013) TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14(1):49–55CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096CrossRefPubMedGoogle Scholar
  64. 64.
    Song M, Kim YH, Kim JS, Kim H (2014) Genome engineering in human cells. Methods Enzymol 546:93–118CrossRefPubMedGoogle Scholar
  65. 65.
    Kitajima TS, Hauf S, Ohsugi M, Yamamoto T, Watanabe Y (2005) Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization. Curr Biol 15(4):353–359CrossRefPubMedGoogle Scholar
  66. 66.
    Ritchie K, Seah C, Moulin J, Isaac C, Dick F, Bérubé NG (2008) Loss of ATRX leads to chromosome cohesion and congression defects. J Cell Biol 180(2):315–324CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Daum JR, Potapova TA, Sivakumar S, Daniel JJ, Flynn JN, Rankin S, Gorbsky GJ (2011) Cohesion fatigue induces chromatid separation in cells delayed at metaphase. Curr Biol 21(12):1018–1024CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Sonoda E, Matsusaka T, Morrison C, Vagnarelli P, Hoshi O, Ushiki T, Nojima K, Fukagawa T, Waizenegger IC, Peters JM, Earnshaw WC, Takeda S (2001) Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells. Dev Cell 1(6):759–770CrossRefPubMedGoogle Scholar
  69. 69.
    Tang Z, Sun Y, Harley SE, Zou H, Yu H (2004) Human Bub1 protects centromeric sister-chromatid cohesion through Shugoshin during mitosis. Proc Natl Acad Sci U S A 101(52):18012–18017CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Maresca TJ, Salmon ED (2009) Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity. J Cell Biol 184(3):373–381CrossRefPubMedPubMedCentralGoogle Scholar
  71. 71.
    Uchida KS, Takagaki K, Kumada K, Hirayama Y, Noda T, Hirota T (2009) Kinetochore stretching inactivates the spindle assembly checkpoint. J Cell Biol 184(3):383–390CrossRefPubMedPubMedCentralGoogle Scholar
  72. 72.
    Suzuki A, Badger BL, Wan X, DeLuca JG, Salmon ED (2014) The architecture of CCAN proteins creates a structural integrity to resist spindle forces and achieve proper Intrakinetochore stretch. Dev Cell 30(6):717–730CrossRefPubMedPubMedCentralGoogle Scholar
  73. 73.
    Wan X, O’Quinn RP, Pierce HL, Joglekar AP, Gall WE, DeLuca JG, Carroll CW, Liu ST, Yen TJ, McEwen BF, Stukenberg PT, Desai A, Salmon ED (2009) Protein architecture of the human kinetochore microtubule attachment site. Cell 137(4):672–684CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Keith F. DeLuca
    • 1
  • Jacob A. Herman
    • 1
  • Jennifer G. DeLuca
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyColorado State UniversityFort CollinsUSA

Personalised recommendations