Characterization of Microtubule-Associated Proteins (MAPs) and Tubulin Interactions by Isothermal Titration Calorimetry (ITC)

  • Philipp O. Tsvetkov
  • Romain La Rocca
  • Soazig Malesinski
  • François DevredEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1964)


Microtubules are highly dynamic structures which play a central role in many cellular processes such as cell division, intracellular transport, and migration. Their dynamics is tightly regulated by stabilizing and destabilizing microtubule-associated proteins (MAPs), such as tau and stathmin. Many approaches have been developed to study interactions between tubulin and MAPs. However, isothermal titration calorimetry (ITC) is the only direct thermodynamic method that enables a full thermodynamic characterization of the interaction after a single titration experiment. We provide here the protocols to apply ITC to tubulin interaction with either stathmin or tau, which will help to avoid the common pitfalls in this very powerful and sensitive method.

Key words

Tubulin Microtubule Isothermal titration calorimetry Stathmin Tau 


  1. 1.
    Mitchison T, Kirschner M (1984) Dynamic instability of microtubule growth. Nature 312:237–242CrossRefGoogle Scholar
  2. 2.
    Larsson N, Marklund U, Gradin HM, Brattsand G, Gullberg M (1997) Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis. Mol Cell Biol 17:5530–5539CrossRefGoogle Scholar
  3. 3.
    Jameson L, Frey T, Zeeberg B, Dalldorf F, Caplow M (1980) Inhibition of microtubule assembly by phosphorylation of microtubule-associated proteins. Biochemistry 19:2472–2479CrossRefGoogle Scholar
  4. 4.
    Kiris E, Ventimiglia D, Feinstein SC (2010) Quantitative analysis of MAP-mediated regulation of microtubule dynamic instability in vitro. Methods Cell Biol 95:481–503CrossRefGoogle Scholar
  5. 5.
    Ross JL, Dixit R (2010) Multiple color single molecule TIRF imaging and tracking of MAPs and motors. Methods Cell Biol 95:521–542CrossRefGoogle Scholar
  6. 6.
    Devred F, Barbier P, Lafitte D, Landrieu I, Lippens G, Peyrot V (2010) Microtubule and MAPs: thermodynamics of complex formation by AUC, ITC, fluorescence, and NMR. Methods Cell Biol 95:449–480CrossRefGoogle Scholar
  7. 7.
    Kellogg EH, Hejab NMA, Poepsel S, Downing KH, DiMaio F, Nogales E (2018) Atomic model of microtubule-bound tau. Science, in pressGoogle Scholar
  8. 8.
    Kar S, Fan J, Smith MJ, Goedert M, Amos LA (2003) Repeat motifs of tau bind to the insides of microtubules in the absence of taxol. EMBO J 22:70–77CrossRefGoogle Scholar
  9. 9.
    Tsvetkov PO, Makarov AA, Malesinski S, Peyrot V, Devred F (2012) New insights into tau-microtubules interaction revealed by isothermal titration calorimetry. Biochimie 94:916–919CrossRefGoogle Scholar
  10. 10.
    Schiff PB, Fant J, Horwitz SB (1979) Promotion of microtubule assembly in vitro by taxol. Nature 277:665–667CrossRefGoogle Scholar
  11. 11.
    Weisenberg RC, Timasheff SN (1970) Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine and vinblastine. Biochemistry 9:4110–4116CrossRefGoogle Scholar
  12. 12.
    Calligaris D, Verdier-Pinard P, Devred F, Villard C, Braguer D, Lafitte D (2010) Microtubule targeting agents: from biophysics to proteomics. Cell Mol Life Sci 67:1089–1104CrossRefGoogle Scholar
  13. 13.
    Devred F, Tsvetkov PO, Barbier P, Allegro D, Horwitz SB, Makarov AA, Peyrot V (2008) Stathmin/Op18 is a novel mediator of vinblastine activity. FEBS Lett 582:2484–2488CrossRefGoogle Scholar
  14. 14.
    Barbier P, Tsvetkov PO, Breuzard G, Devred F (2013) Deciphering the molecular mechanisms of anti-tubulin plant derived drugs. Phytochem Rev 13:157–169CrossRefGoogle Scholar
  15. 15.
    Malesinski S, Tsvetkov PO, Kruczynski A, Peyrot V, Devred F (2015) Stathmin potentiates vinflunine and inhibits Paclitaxel activity. PLoS One 10:e0128704CrossRefGoogle Scholar
  16. 16.
    Honnappa S, Cutting B, Jahnke W, Seelig J, Steinmetz MO (2003) Thermodynamics of the Op18/stathmin-tubulin interaction. J Biol Chem 278:38926–38934CrossRefGoogle Scholar
  17. 17.
    Honnappa S, Jahnke W, Seelig J, Steinmetz MO (2006) Control of intrinsically disordered stathmin by multisite phosphorylation. J Biol Chem 281:16078–16083CrossRefGoogle Scholar
  18. 18.
    Devred F, Barbier P, Douillard S, Monasterio O, Andreu JM, Peyrot V (2004) Tau induces ring and microtubule formation from alphabeta-tubulin dimers under nonassembly conditions. Biochemistry 43:10520–10531CrossRefGoogle Scholar
  19. 19.
    Tsvetkov PO, Barbier P, Breuzard G, Peyrot V, Devred F (2013) Microtubule-associated proteins and tubulin interaction by isothermal titration calorimetry. Methods Cell Biol 115:283–302CrossRefGoogle Scholar
  20. 20.
    Tsvetkov FO, Kulikova AA, Devred F, Zerniĭ EI, Lafitte D, Makarov AA (2011) Thermodynamics of calmodulin and tubulin binding to the vinca-alkaloid vinorelbine. Mol Biol 45:697–702CrossRefGoogle Scholar
  21. 21.
    De Bessa T, Breuzard G, Allegro D, Devred F, Peyrot V, Barbier P (2017) Tau interaction with tubulin and microtubules: from purified proteins to cells. Methods Mol Biol 1523:61–85CrossRefGoogle Scholar
  22. 22.
    Tsvetkov PO Protein sequence analysis tool.
  23. 23.
    AFFINImeter for Isothermal Titration Calorimetry.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Philipp O. Tsvetkov
    • 1
  • Romain La Rocca
    • 1
  • Soazig Malesinski
    • 1
  • François Devred
    • 1
    Email author
  1. 1.Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Fac PharmMarseilleFrance

Personalised recommendations