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

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

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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.

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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.

Data availability

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

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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.

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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

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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.

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The authors declare no competing interests.

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

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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

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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

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