Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Purification of tubulin with controlled post-translational modifications by polymerization–depolymerization cycles

Nature Protocols volume 14, pages 1634–1660 (2019)Cite this article

Subjects

Abstract

In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6–7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Flow scheme of the tubulin purification protocol.
Fig. 2: Growing HeLa S3 and HEK-293 cells in spinner cultures for tubulin purification.
Fig. 3: Tubulin purification from cells cultured in spinner bottles.
Fig. 4: Analyses of purified tubulin obtained from cell lines and mouse brains.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Borisy, G. G. & Taylor, E. W. The mechanism of action of colchicine. Binding of colchincine-3H to cellular protein. J. Cell Biol. 34, 525–533 (1967).

    Article  CAS  Google Scholar 

  2. Weisenberg, R. C. Microtubule formation in vitro in solutions containing low calcium concentrations. Science 177, 1104–1105 (1972).

    Article  CAS  Google Scholar 

  3. Borisy, G. G. & Olmsted, J. B. Nucleated assembly of microtubules in porcine brain extracts. Science 177, 1196–1197 (1972).

    Article  CAS  Google Scholar 

  4. Kirschner, M. W. & Williams, R. C. The mechanism of microtubule assembly in vitro. J. Supramol. Struct. 2, 412–428 (1974).

    Article  CAS  Google Scholar 

  5. Margolis, R. L. & Wilson, L. Addition of colchicine-tubulin complex to microtubule ends: the mechanism of substoichiometric colchicine poisoning. Proc. Natl. Acad. Sci. USA 74, 3466–3470 (1977).

    Article  CAS  Google Scholar 

  6. Murphy, D. B. & Borisy, G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc. Natl. Acad. Sci. USA 72, 2696–2700 (1975).

    Article  CAS  Google Scholar 

  7. Margolis, R. L. & Wilson, L. Opposite end assembly and disassembly of microtubules at steady state in vitro. Cell 13, 1–8 (1978).

    Article  CAS  Google Scholar 

  8. Mitchison, T. & Kirschner, M. Dynamic instability of microtubule growth. Nature 312, 237–242 (1984).

    Article  CAS  Google Scholar 

  9. Axelrod, D. Cell-substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89, 141–145 (1981).

    Article  CAS  Google Scholar 

  10. Dogterom, M. & Surrey, T. Microtubule organization in vitro. Curr. Opin. Cell Biol. 25, 23–29 (2013).

    Article  CAS  Google Scholar 

  11. Nedelec, F. J., Surrey, T., Maggs, A. C. & Leibler, S. Self-organization of microtubules and motors. Nature 389, 305–308 (1997).

    Article  CAS  Google Scholar 

  12. Bieling, P., Telley, I. A. & Surrey, T. A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps. Cell 142, 420–432 (2010).

    Article  CAS  Google Scholar 

  13. Roostalu, J. et al. Directional switching of the kinesin Cin8 through motor coupling. Science 332, 94–99 (2011).

    Article  CAS  Google Scholar 

  14. Hendricks, A. G., Goldman, Y. E. & Holzbaur, E. L. F. Reconstituting the motility of isolated intracellular cargoes. Methods Enzymol. 540, 249–262 (2014).

    Article  CAS  Google Scholar 

  15. Schaedel, L. et al. Microtubules self-repair in response to mechanical stress. Nat. Mater. 14, 1156–1163 (2015).

    Article  CAS  Google Scholar 

  16. Vallee, R. B. Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments. Methods Enzymol. 134, 89–104 (1986).

    Article  CAS  Google Scholar 

  17. Castoldi, M. & Popov, A. V. Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr. Purif. 32, 83–88 (2003).

    Article  CAS  Google Scholar 

  18. Wloga, D., Joachimiak, E., Louka, P. & Gaertig, J. Posttranslational modifications of tubulin and cilia. Cold Spring Harb. Perspect. Biol. 9, a028159 (2017).

    Article  Google Scholar 

  19. Janke, C. The tubulin code: molecular components, readout mechanisms, and functions. J. Cell Biol. 206, 461–472 (2014).

    Article  CAS  Google Scholar 

  20. Barisic, M. et al. Microtubule detyrosination guides chromosomes during mitosis. Science 348, 799–803 (2015).

    Article  CAS  Google Scholar 

  21. Nirschl, J. J., Magiera, M. M., Lazarus, J. E., Janke, C. & Holzbaur, E. L. F. Alpha-tubulin tyrosination and CLIP-170 phosphorylation regulate the initiation of dynein-driven transport in neurons. Cell Rep. 14, 2637–2652 (2016).

    Article  CAS  Google Scholar 

  22. Guedes-Dias, P. et al. Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse. Curr. Biol. 29, 268–282.e8 (2019).

    Article  CAS  Google Scholar 

  23. Hiller, G. & Weber, K. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell 14, 795–804 (1978).

    Article  CAS  Google Scholar 

  24. Bulinski, J. C. & Borisy, G. G. Self-assembly of microtubules in extracts of cultured HeLa cells and the identification of HeLa microtubule-associated proteins. Proc. Natl. Acad. Sci. USA 76, 293–297 (1979).

    Article  CAS  Google Scholar 

  25. Farrell, K. W. Isolation of tubulin from nonneural sources. Methods Enzymol. 85(Pt B), 385–393 (1982).

    Article  CAS  Google Scholar 

  26. Banerjee, A., Roach, M. C., Trcka, P. & Ludueña, R. F. Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain. J. Biol. Chem. 267, 5625–5630 (1992).

    CAS  PubMed  Google Scholar 

  27. Newton, C. N. et al. Intrinsically slow dynamic instability of HeLa cell microtubules in vitro. J. Biol. Chem. 277, 42456–42462 (2002).

    Article  CAS  Google Scholar 

  28. Lacroix, B. & Janke, C. Generation of differentially polyglutamylated microtubules. Methods Mol. Biol. 777, 57–69 (2011).

    Article  CAS  Google Scholar 

  29. Widlund, P. O. et al. One-step purification of assembly-competent tubulin from diverse eukaryotic sources. Mol. Biol. Cell 23, 4393–4401 (2012).

    Article  CAS  Google Scholar 

  30. Howes, S. C. et al. Structural differences between yeast and mammalian microtubules revealed by cryo-EM. J. Cell Biol. 216, 2669–2677 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Pamula, M. C., Ti, S.-C. & Kapoor, T. M. The structured core of human beta tubulin confers isotype-specific polymerization properties. J. Cell Biol. 213, 425–433 (2016).

    Article  CAS  Google Scholar 

  32. Vemu, A., Atherton, J., Spector, J. O., Moores, C. A. & Roll-Mecak, A. Tubulin isoform composition tunes microtubule dynamics. Mol. Biol. Cell 28, 3564–3572 (2017).

    Article  CAS  Google Scholar 

  33. von Loeffelholz, O. et al. Nucleotide- and Mal3-dependent changes in fission yeast microtubules suggest a structural plasticity view of dynamics. Nat. Commun. 8, 2110 (2017).

    Article  Google Scholar 

  34. Chaaban, S. et al. The structure and dynamics of C. elegans tubulin reveals the mechanistic basis of microtubule growth. Dev. Cell 47, 191–204.e8 (2018).

    Article  CAS  Google Scholar 

  35. Alper, J. D., Decker, F., Agana, B. & Howard, J. The motility of axonemal dynein is regulated by the tubulin code. Biophys. J. 107, 2872–2880 (2014).

    Article  CAS  Google Scholar 

  36. Minoura, I. et al. Overexpression, purification, and functional analysis of recombinant human tubulin dimer. FEBS Lett. 587, 3450–3455 (2013).

    Article  CAS  Google Scholar 

  37. Vemu, A. et al. Structure and dynamics of single-isoform recombinant neuronal human tubulin. J. Biol. Chem. 291, 12907–12915 (2016).

    Article  CAS  Google Scholar 

  38. Ti, S.-C., Alushin, G. M. & Kapoor, T. M. Human beta-tubulin isotypes can regulate microtubule protofilament number and stability. Dev. Cell 47, 175–190.e5 (2018).

    Article  CAS  Google Scholar 

  39. Uchimura, S. et al. A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation. J. Cell Biol. 208, 211–222 (2015).

    Article  CAS  Google Scholar 

  40. Denoulet, P., Eddé, B. & Gros, F. Differential expression of several neurospecific beta-tubulin mRNAs in the mouse brain during development. Gene 50, 289–297 (1986).

    Article  CAS  Google Scholar 

  41. Song, Y. et al. Transglutaminase and polyamination of tubulin: posttranslational modification for stabilizing axonal microtubules. Neuron 78, 109–123 (2013).

    Article  CAS  Google Scholar 

  42. Belvindrah, R. et al. Mutation of the alpha-tubulin Tuba1a leads to straighter microtubules and perturbs neuronal migration. J. Cell Biol. 216, 2443–2461 (2017).

    Article  CAS  Google Scholar 

  43. Breuss, M. et al. Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53 associated apoptosis. Development 143, 1126–33 (2016).

    Article  CAS  Google Scholar 

  44. Latremoliere, A. et al. Neuronal-specific TUBB3 is not required for normal neuronal function but is essential for timely axon regeneration. Cell Rep. 24, 1865–1879.e9 (2018).

    Article  CAS  Google Scholar 

  45. Magiera, M. M. et al. Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport. EMBO J. 37, e100440 (2018).

    Article  Google Scholar 

  46. Morley, S. J. et al. Acetylated tubulin is essential for touch sensation in mice. eLife 5, e20813 (2016).

    Article  Google Scholar 

  47. Kalebic, N. et al. Tubulin acetyltransferase alphaTAT1 destabilizes microtubules independently of its acetylation activity. Mol. Cell. Biol. 33, 1114–1123 (2013).

    Article  CAS  Google Scholar 

  48. Maurer, S. P., Bieling, P., Cope, J., Hoenger, A. & Surrey, T. GTPγS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs). Proc. Natl. Acad. Sci. USA 108, 3988–3993 (2011).

    Article  CAS  Google Scholar 

  49. Sandblad, L. et al. The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam. Cell 127, 1415–1424 (2006).

    Article  CAS  Google Scholar 

  50. Janke, C. et al. Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science 308, 1758–1762 (2005).

    Article  CAS  Google Scholar 

  51. van Dijk, J. et al. A targeted multienzyme mechanism for selective microtubule polyglutamylation. Mol. Cell 26, 437–448 (2007).

    Article  Google Scholar 

  52. Rogowski, K. et al. Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation. Cell 137, 1076–1087 (2009).

    Article  CAS  Google Scholar 

  53. Rogowski, K. et al. A family of protein-deglutamylating enzymes associated with neurodegeneration. Cell 143, 564–578 (2010).

    Article  CAS  Google Scholar 

  54. Aillaud, C. et al. Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation. Science 358, 1448–1453 (2017).

    Article  CAS  Google Scholar 

  55. Nieuwenhuis, J. et al. Vasohibins encode tubulin detyrosinating activity. Science 358, 1453–1456 (2017).

    Article  CAS  Google Scholar 

  56. Akella, J. S. et al. MEC-17 is an alpha-tubulin acetyltransferase. Nature 467, 218–222 (2010).

    Article  CAS  Google Scholar 

  57. Shida, T., Cueva, J. G., Xu, Z., Goodman, M. B. & Nachury, M. V. The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation. Proc. Natl. Acad. Sci. USA 107, 21517–21522 (2010).

    Article  CAS  Google Scholar 

  58. Magiera, M. M. & Janke, C. in Methods in Cell Biology Vol. 115 (eds Correia, J. J. & Wilson, L.) 247–267 (Academic Press, Burlington, MA, 2013).

  59. Hubbert, C. et al. HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458 (2002).

    Article  CAS  Google Scholar 

  60. Matsuyama, A. et al. In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J. 21, 6820–6831 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank all members of the Janke lab for help during the establishment of the protocol. We thank the animal facility of the Institut Curie for help with mouse breeding and care. This work received support under the ‘Investissementsd’Avenir’ program launched by the French government and implemented by the French National Research Agency (ANR; award nos. ANR-10-LBX-0038 and ANR-10-IDEX-0001-02 PSL). The work of C.J. was supported by the Institut Curie, the ANR (award no. ANR-12-BSV2-0007), the Institut National du Cancer (INCA; grant nos. 2013-1-PL BIO-02-ICR-1 and 2014-PL BIO-11-ICR-1), the Fondation pour la Recherche Medicale (FRM; grant no.DEQ20170336756), and the CEFIPRA research project 5703-1. M.M.M. was supported by an EMBO short-term fellowship (ASTF 148-2015) and by the FondationVaincre Alzheimer (grant no. FR-16055p). J.S. was supported by an FRM fellowship (no. SPF20120523942) and the EMBO (grant nos. ALTF 638-2010 and EMBO ASTF 445-2012). S.B. was supported by the FRM (grant no. FDT201805005465). J.A.S. was supported by the European Union’s Horizon 2020 research and innovation programme under a Marie Skłodowska-Curie grant agreement (no. 675737). The antibody 12G10, developed by J. Frankel and M. Nelson, was obtained from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the NICHD and is maintained by the University of Iowa.

Author information

Author notes

  1. Goran Lakisic

    Present address: Institut MICALIS, AgroParisTech, Université Paris Saclay, INRA, Jouy-en-Josas, France

  2. These authors contributed equally: Judith Souphron, Satish Bodakuntla, A. S. Jijumon.

Authors and Affiliations

  1. Institut Curie, PSL Research University, CNRS UMR 3348, Orsay, France

    Judith Souphron, Satish Bodakuntla, A. S. Jijumon, Carsten Janke & Maria M. Magiera

  2. Université Paris Sud, Université Paris-Saclay, CNRS UMR 3348, Orsay, France

    Judith Souphron, Satish Bodakuntla, A. S. Jijumon, Carsten Janke & Maria M. Magiera

  3. BIOC, CNRS, Ecole Polytechnique, IP Paris, Palaiseau, France

    Goran Lakisic & Alexis M. Gautreau

Authors
  1. Judith Souphron
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Satish Bodakuntla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. S. Jijumon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Goran Lakisic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alexis M. Gautreau
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carsten Janke
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maria M. Magiera
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.S. and M.M.M. established the spinner cultures for various cell lines (with the help of G.L. and A.M.G.) and adapted the tubulin purification protocol to spinner-grown cells. S.B. and M.M.M. established the tubulin prep from adherent cells. S.B., M.M.M. and A.S.J. contributed to improving the protocol for the tubulin prep from large cell quantities. M.M.M. established the tubulin prep protocol from single mouse brains. M.M.M. and C.J. supervised the development of this method and wrote the manuscript. All the authors contributed to corrections of the text and figures.

Corresponding authors

Correspondence to Carsten Janke or Maria M. Magiera.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Journal peer review information: Nature Protocols thanks Manuel Théry and Maria Zanic for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Barisic, M. et al. Science 348, 799–803 (2015): http://science.sciencemag.org/content/348/6236/799

Nirschl, J. J., Magiera, M. M., Lazarus, J. E., Janke, C. & Holzbaur, E. L. F. Cell Rep. 14, 2637–2652 (2016): https://www.cell.com/cell-reports/fulltext/S2211-1247(16)30167-X

Guedes-Dias P. et al. Curr. Biol. 29 268–282.e8 (2019): https://www.cell.com/current-biology/fulltext/S0960-9822(18)31595-1

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Souphron, J., Bodakuntla, S., Jijumon, A.S. et al. Purification of tubulin with controlled post-translational modifications by polymerization–depolymerization cycles. Nat Protoc 14, 1634–1660 (2019). https://doi.org/10.1038/s41596-019-0153-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0153-7

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing