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Preparation and Characterization of Posttranslationally Modified Tubulins From Artemia franciscana

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Microtubule Protocols

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 137))

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Abstract

Tubulin heterogeneity within eukaryotic cells is generated by differential gene expression and posttranslational modification of α- and β-tubulin gene products, either as heterodimers or when polymerized into microtubules. The characterization of posttranslationally modified tubulins from the crustacean Artemia franciscana is presented, although tubulins from other sources can be studied with these procedures. Tubulin is prepared from cell free extracts by taxol-induced assembly and centrifugation of microtubules through sucrose cushions, which also yields microtubule-associated proteins, or it is purified to apparent homogeneity by relatively simple chromatographic procedures and assembly/disassembly steps. To detect posttranslationally modified tubulins protein samples are electrophoresed in sodium dodecyl sulfate (SDS) polyacrylamide gels, blotted to nitrocellulose membranes and probed with isoform-specific antibodies. Isotubulins, for which gene-encoded amino acid differences and post translational modifications generate charge variations, are resolved in two-dimensional gels using isoelectric focusing followed by SDS polyacrylamide gel electrophoresis, a procedure useful for resolution of microtubule-associated proteins. Isoforms patterns are visualized by Coomassie blue and/or silver staining and individual isoforms are identified by antibody reactivity on Western blots. Tubulin isoforms are localized in Artemia by immunofluorescent staining of larvae. The focus of this chapter is the purification of tubulin from a nonneural source and characterization of tyrosinated, detyrosinated, and nontyrosinatable α-tubulins using polyclonal antibodies made to carboxy-terminal peptides of each i

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References

  1. Krebs, A., Goldie, K. N., and Hoenger, A. (2005) Structural rearrangements in tubulin following microtubule formation. EMBO Rep. 6, 227–232.

    Article  CAS  PubMed  Google Scholar 

  2. Amos, L. A. (2004) Microtubule structure and its stabilization. Org. Biomol. Chem. 2, 2153–2160.

    Article  CAS  PubMed  Google Scholar 

  3. Löwe, J., Li, H., Downing, K. H., and Nogales, E. (2001) Refined structure of αβ-tubulin at 3.5 Å resolution. J. Mol. Biol. 313, 1045–1057.

    Article  PubMed  Google Scholar 

  4. Ludueña, R. F. (1998) Multiple forms of tubulin: different gene products and covalent modifications. Int. Rev. Cytol. 178, 207–275.

    Article  PubMed  Google Scholar 

  5. Dutcher, S. K. (2003) Long-lost relatives reappear: identification of new members of the tubulin superfamily. Curr. Opin. Microbiol. 6, 634–640.

    Article  CAS  PubMed  Google Scholar 

  6. McKean, P. G., Vaughan, S., and Gull, K. (2001) The extended tubulin superfamily. J. Cell Sci. 114, 2723–2733.

    CAS  PubMed  Google Scholar 

  7. Westermann, S. and Weber, K. (2003) Post-translational modifications regulate microtubule function. Nat. Rev. Mol. Cell Biol. 4, 938–947.

    Article  CAS  PubMed  Google Scholar 

  8. MacRae, T. H. (1997) Tubulin post-translational modifications. Enzymes and their mechanisms of action. Eur. J. Biochem. 244, 265–278.

    Article  CAS  PubMed  Google Scholar 

  9. Rosenbaum, J. (2000) Functions for tubulin modifications at last. Curr. Biol. 10, R801–R803.

    Article  CAS  PubMed  Google Scholar 

  10. Idriss, H. T. (2000) Man to trypanosome: the tubulin tyrosination/detyrosination cycle revisited. Cell Motil. Cytoskel. 45, 173–184.

    CAS  Google Scholar 

  11. Erck, C., Peris, L., Andrieux, A., et al. (2005) A vital role of tubulin-tyrosineligase for neuronal organization. Proc. Natl. Acad. Sci. USA 102, 7853–7858

    Article  CAS  PubMed  Google Scholar 

  12. Hubbert, C., Guardlola, A., Shao, R., et al. (2002) HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458.

    Article  CAS  PubMed  Google Scholar 

  13. Redeker, V., Levilliers, N., Vinolo, E., et al. (2005) Mutations of tubulin glycylation sites reveal cross-talk between the C termini of α-and β-tubulin and affect the ciliary matrix in Tetrahymena. J. Biol. Chem. 280, 596–606.

    CAS  PubMed  Google Scholar 

  14. Thazhath, R., Jerka-Dziadosz, M., Duan, J., et al. (2004) Cell context-specific effects of the β-tubulin glycylation domain on assembly and size of microtubular organelles. Mol. Biol. Cell 15, 4136–4147.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  16. Infante, A. S., Stein, M. S., Zhai, Y., Borisy, G. G., and Gundersen, G. G. (2000) Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap. J. Cell Sci. 113, 3907–3919.

    CAS  PubMed  Google Scholar 

  17. Kreitzer, G., Liao, G., and Gundersen, G. G. (1999) Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism. Mol. Biol. Cell 10, 1105–1118.

    CAS  PubMed  Google Scholar 

  18. Lin, S. X., Gundersen, G. G., and Maxfield, F. R. (2002) Export from pericentriolar endocytic recycling compartment to cell surface depends on stable, detyrosinated (Glu) microtubules and kinesin. Mol. Biol. Cell 13, 96–109.

    Article  CAS  PubMed  Google Scholar 

  19. Bonnet, C., Boucher, D., Lazereg, S., et al. (2001) Differential binding regulation of microtubule-associated proteins MAP1A, MAP1B, and MAP2 by tubulin polyglutamylation. J. Biol. Chem. 276, 12,839–12,848.

    Article  CAS  PubMed  Google Scholar 

  20. MacRae, T. H. (2003) Molecular chaperones, stress resistance and development in Artemia franciscana. Semin. Cell Develop. Biol. 14, 251–258.

    Article  CAS  Google Scholar 

  21. Liang, P. and MacRae, T. H. (1999) The synthesis of a small heat shock/α-crystallin protein in Artemia and its relationship to stress tolerance during development. Dev. Biol. 207, 445–456.

    Article  CAS  PubMed  Google Scholar 

  22. Bulinski, J. C., Kumar, S., Titani, K., and Hauschka, S. D. (1983) Peptide antibody specific for the amino terminus of skeletal muscle α-actin. Proc. Natl. Acad. Sci. USA 80, 1506–1510.

    Article  CAS  PubMed  Google Scholar 

  23. Gundersen, G. G., Kalnoski, M. H., and Bulinski, J. C. (1984) Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell 38, 779–789.

    Article  CAS  PubMed  Google Scholar 

  24. Woods, A., Sherwin, T., Sasse, R., MacRae, T. H., Baines, A. J., and Gull, K. (1989) Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci. 93, 491–500.

    PubMed  Google Scholar 

  25. Xiang, H. and MacRae, T. H. (1995) Production and utilization of detyrosinated tubulin in developing Artemia larvae: evidence for a tubulin-reactive carboxypeptidase. Biochem. Cell Biol. 73, 673–685.

    CAS  PubMed  Google Scholar 

  26. Criel, G. R. J., Van Oostveldt, P., and MacRae, T. H. (2005) Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscana. J. Morph. 262, 203–215.

    Article  Google Scholar 

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© 2007 Humana Press Inc.

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O’Connell, P.A., MacRae, T.H. (2007). Preparation and Characterization of Posttranslationally Modified Tubulins From Artemia franciscana . In: Zhou, J. (eds) Microtubule Protocols. Methods in Molecular Medicine™, vol 137. Humana Press. https://doi.org/10.1007/978-1-59745-442-1_4

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  • DOI: https://doi.org/10.1007/978-1-59745-442-1_4

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-642-9

  • Online ISBN: 978-1-59745-442-1

  • eBook Packages: Springer Protocols

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