Photoactivatable-GFP-α-Tubulin as a Tool to Study Microtubule Plus-End Turnover in Living Human Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 777)

Abstract

The development of photactivatable (PA) variants of Green fluorescent protein (GFP) has allowed the dynamics of spatially restricted protein pools within living cells to be determined. Over the last 5 years, experiments utilizing PA-GFP fused to α-tubulin have provided important insights into the mechanisms that control microtubule dynamics in living cells. In this chapter, we describe the methodology required to generate stable cell lines expressing photoactivatable-GFP-α-tubulin and to derive quantitative measurements of tubulin turnover at microtubules plus-ends in living cells.

Key words

Microtubule α-Tubulin Photoactivatable-GFP Plus-end turnover Dynamics Kinetochore 

References

  1. 1.
    Mitchison, T. & Kirschner, M. (1984) Dynamic instability of microtubule growth. Nature 312:237–242.PubMedCrossRefGoogle Scholar
  2. 2.
    Kueh HY, Mitchison TJ. (2009) Stuctural plasticity in actin and tubulin polymer dynamics. Science. 325.5943:960–3.Google Scholar
  3. 3.
    Brust-Mascher I, et al (2009) Kinesin-5–dependent Poleward Flux and Spindle Length Control in Drosophila Embryo Mitosis Molecular Biology of the Cell Vol. 20, 1749–1762.Google Scholar
  4. 4.
    Maddox P, et al (2002) Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos. Curr Biol. 12(19):1670–4.PubMedCrossRefGoogle Scholar
  5. 5.
    Brust-Mascher I, Scholey JM. (2002) Microtubule flux and sliding in mitotic spindles of Drosophila embryos. Mol Biol Cell. 3967–75.Google Scholar
  6. 6.
    Buster DW, Zhang D, Sharp DJ. (2007) Poleward tubulin flux in spindles: regulation and function in mitotic cells. Mol Biol Cell. 18(8):3094–104.PubMedCrossRefGoogle Scholar
  7. 7.
    Matos I, et al (2009) Synchronizing chromosome segregation by flux-dependent force equalization at kinetochores. J Cell Biol. 186(1):11–26.PubMedCrossRefGoogle Scholar
  8. 8.
    Uteng M, et al (2008) Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles. J Cell Biol. 182(4):715–26.PubMedCrossRefGoogle Scholar
  9. 9.
    Mitchison TJ. (2005) Mechanism and function of poleward flux in Xenopus extract meiotic spindles. Philos Trans R Soc Lond B Biol Sci. 360(1455):623–9.PubMedCrossRefGoogle Scholar
  10. 10.
    Miyamoto DT, et al (2004) The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles. J Cell Biol. 167(5):813–8.PubMedCrossRefGoogle Scholar
  11. 11.
    Shirasu-Hiza M, et al (2004) Eg5 causes elongation of meiotic spindles when flux-associated microtubule depolymerization is blocked. Curr Biol. 14(21):1941–5.PubMedCrossRefGoogle Scholar
  12. 12.
    Mitchison TJ. (1989) Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J Cell Biol. 109(2):637–52.PubMedCrossRefGoogle Scholar
  13. 13.
    Vallotton P, et al (2003) Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study. Biophys J. 85(2):1289–306.PubMedCrossRefGoogle Scholar
  14. 14.
    G.H. Patterson, J. Lippincott-Schwartz (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science. 297:1873–1877.PubMedCrossRefGoogle Scholar
  15. 15.
    Zhai, Y., Kronebusch, P.J. & Borisy, G.G. (1995) Kinetochore microtubule dynamics and the metaphase-anaphase transition. J Cell Biol. 131:721–734.PubMedCrossRefGoogle Scholar
  16. 16.
    DeLuca, J.G. et al. (2006) Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell. 127:969–982.PubMedCrossRefGoogle Scholar
  17. 17.
    Ganem, N.J., Upton, K. & Compton, D.A. (2005) Efficient mitosis in human cells lacking poleward microtubule flux. Curr Biol. 15:1827–1832.PubMedCrossRefGoogle Scholar
  18. 18.
    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):1711–8.PubMedCrossRefGoogle Scholar
  19. 19.
    Cameron LA, et al. (2006) Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells. J Cell Biol.173(2):173–9.PubMedCrossRefGoogle Scholar
  20. 20.
    Amaro AC, et al (2010) Molecular control of kinetochore-microtubule dynamics and chromosome oscillations Nat Cell Biol. 12(4): 319–29.Google Scholar
  21. 21.
    Maffini S, et al (2009) Motor-independent targeting of CLASPs to kinetochores by CENP-E promotes microtubule turnover and poleward flux. Current Biology 19(18):1566–72.PubMedCrossRefGoogle Scholar
  22. 22.
    Rizk RS, et al (2009) MCAK and paclitaxel have differential effects on spindle microtubule organization and dynamics. Mol Biol Cell. 20(6):1639–51.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Centre for Mechanochemical Cell Biology, Warwick Medical SchoolUniversity of WarwickCoventryUK

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