How to Measure Microtubule Dynamics?

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

Abstract

Microtubules are one of the most spectacular features in the cell: long, fairly rigid tubules that provide physical strength while at the same time serving as tracks of the intracellular transport network. In addition, they are the main constituents of the cell division machinery, and guide axonal growth and the direction of cell migration. To be able to fulfil such diverse functions, microtubules have to be arranged into suitable patterns and remodelled according to extra- and intracellular cues. Moreover, the delicate regulation of microtubule dynamics and the dynamic interactions with subcellular structures, such as kinetochores or cell adhesion sites, appear to be of crucial importance to microtubule functions. It is, therefore, important to understand microtubule dynamics and its spatiotemporal regulation at the molecular level. In this chapter, I introduce the concept of microtubule dynamics and discuss the techniques that can be employed to study microtubule dynamics in vitro and in cells, for many of which detailed protocols can be found in this volume. Microtubule dynamics is traditionally assessed by the four parameters of dynamic instability: growth and shrinkage rates, rescue and catastrophe frequencies, sometimes supplemented by pause duration. I discuss emerging issues with and alternatives to this parameter description of microtubule dynamics.

Key words

Microtubule Dynamic instability Microtubule assembly Catastrophe Rescue GTP cap GFP-Tubulin EB3 CLIP-170 

References

  1. 1.
    Inoué, S. and Sato, H. (1967), Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement.J Gen Physiol 50: Suppl:259–92.Google Scholar
  2. 2.
    Borisy, G. G. and Taylor, E. W. (1967), The mechanism of action of colchicine. Binding of colchincine-3H to cellular protein.The Journal of Cell Biology 34: 525–33.Google Scholar
  3. 3.
    Shelanski, M. L. and Taylor, E. W. (1967), Isolation of a protein subunit from microtubules.The Journal of Cell Biology 34: 549–54.PubMedCrossRefGoogle Scholar
  4. 4.
    Mohri, H. (1968), Amino-acid composition of “Tubulin” constituting microtubules of sperm flagella.Nature 217: 1053–4.PubMedCrossRefGoogle Scholar
  5. 5.
    Weisenberg, R. C. (1972), Microtubule formation in vitro in solutions containing low calcium concentrations.Science 177: 1104–5.PubMedCrossRefGoogle Scholar
  6. 6.
    Margolis, R. L. and Wilson, L. (1981), Microtubule treadmills--possible molecular machinery.Nature 293: 705–11.PubMedCrossRefGoogle Scholar
  7. 7.
    Mitchison, T. and Kirschner, M. (1984), Dynamic instability of microtubule growth.Nature 312: 237–42.PubMedCrossRefGoogle Scholar
  8. 8.
    Horio, T. and Hotani, H. (1986), Visualization of the dynamic instability of individual microtubules by dark-field microscopy.Nature 321: 605–7.PubMedCrossRefGoogle Scholar
  9. 9.
    Walker, R., O’brien, E., et al. (1988), Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. The Journal of Cell Biology 107: 1437.PubMedCrossRefGoogle Scholar
  10. 10.
    Cassimeris, L., Pryer, N. K., and Salmon, E. D. (1988), Real-time observations of microtubule dynamic instability in living cells.J Cell Biol 107: 2223–31.PubMedCrossRefGoogle Scholar
  11. 11.
    Sammak, P. J. and Borisy, G. G. (1988), Direct observation of microtubule dynamics in living cells.Nature 332: 724–6.PubMedCrossRefGoogle Scholar
  12. 12.
    Howard, J. and Hyman, A. A. (2009), Growth, fluctuation and switching at microtubule plus ends.Nat Rev Mol Cell Biol 10: 569–74.PubMedCrossRefGoogle Scholar
  13. 13.
    Hyman, A. A., Chrétien, D., et al. (1995), Structural changes accompanying GTP hydrolysis in microtubules: information from a slowly hydrolyzable analogue guanylyl-(alpha,beta)-methylene-diphosphonate.The Journal of Cell Biology 128: 117–25.PubMedCrossRefGoogle Scholar
  14. 14.
    Müller-Reichert, T., Chrétien, D., et al. (1998), Structural changes at microtubule ends accompanying GTP hydrolysis: information from a slowly hydrolyzable analogue of GTP, guanylyl (alpha,beta)methylenediphosphonate.Proc Natl Acad Sci USA 95: 3661–6.PubMedCrossRefGoogle Scholar
  15. 15.
    Mandelkow, E. M., Mandelkow, E., and Milligan, R. A. (1991), Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study.J Cell Biol 114: 977–91.PubMedCrossRefGoogle Scholar
  16. 16.
    Drechsel, D. N. and Kirschner, M. W. (1994), The minimum GTP cap required to stabilize microtubules.Curr Biol 4: 1053–61.PubMedCrossRefGoogle Scholar
  17. 17.
    Schek, H. T., 3rd, Gardner, M. K., et al. (2007), Microtubule assembly dynamics at the nanoscale.Curr Biol 17: 1445–55.PubMedCrossRefGoogle Scholar
  18. 18.
    Walker, R. A., Pryer, N. K., and Salmon, E. D. (1991), Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.J Cell Biol 114: 73–81.PubMedCrossRefGoogle Scholar
  19. 19.
    Bieling, P., Laan, L., et al. (2007), Reconstitution of a microtubule plus-end tracking system in vitro.Nature 450: 1100–5.PubMedCrossRefGoogle Scholar
  20. 20.
    Tirnauer, J. S., Grego, S., et al. (2002), EB1-microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules.Mol Biol Cell 13: 3614–26.PubMedCrossRefGoogle Scholar
  21. 21.
    Zanic, M., Stear, J. H., et al. (2009), EB1 recognizes the nucleotide state of tubulin in the microtubule lattice.PLoS One 4: e7585.PubMedCrossRefGoogle Scholar
  22. 22.
    Grego, S., Cantillana, V., and Salmon, E. D. (2001), Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.Biophys J 81: 66–78.PubMedCrossRefGoogle Scholar
  23. 23.
    Rusan, N. M., Fagerstrom, C. J., et al. (2001), Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin.Mol Biol Cell 12: 971–80.PubMedGoogle Scholar
  24. 24.
    Shelden, E. and Wadsworth, P. (1993), Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific.J Cell Biol 120: 935–45.PubMedCrossRefGoogle Scholar
  25. 25.
    Waterman-Storer, C. M. and Salmon, E. D. (1997), Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling.J Cell Biol 139: 417–34.PubMedCrossRefGoogle Scholar
  26. 26.
    Keller, P. J., Pampaloni, F., and Stelzer, E. H. K. (2007), Three-dimensional preparation and imaging reveal intrinsic microtubule properties.Nature Methods 4: 843–6.PubMedCrossRefGoogle Scholar
  27. 27.
    VanBuren, V., Cassimeris, L., and Odde, D. J. (2005), Mechanochemical model of microtubule structure and self-assembly kinetics.Biophys J 89: 2911–26.PubMedCrossRefGoogle Scholar
  28. 28.
    Straube, A. and Merdes, A. (2007), EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and fusion.Curr Biol 17: 1318–25.PubMedCrossRefGoogle Scholar
  29. 29.
    Komarova, Y. A., Vorobjev, I. A., and Borisy, G. G. (2002), Life cycle of MTs: persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary.J Cell Sci 115: 3527–39.PubMedGoogle Scholar
  30. 30.
    Amaro, A. C., Samora, C. P., et al. (2010), Molecular control of kinetochore-microtubule dynamics and chromosome oscillations.Nat Cell Biol 12: 319–29.PubMedCrossRefGoogle Scholar
  31. 31.
    van der Vaart, B., Akhmanova, A., and Straube, A. (2009), Regulation of microtubule dynamic instability.Biochem Soc Trans 37: 1007–13.PubMedCrossRefGoogle Scholar
  32. 32.
    Maiato, H., DeLuca, J., et al. (2004), The dynamic kinetochore-microtubule interface.J Cell Sci 117: 5461–77.PubMedCrossRefGoogle Scholar
  33. 33.
    Needleman, D. J., Groen, A., et al. (2010), Fast microtubule dynamics in meiotic spindles measured by single molecule imaging: evidence that the spindle environment does not stabilize microtubules.Mol Biol Cell 21: 323–33.PubMedCrossRefGoogle Scholar
  34. 34.
    Katsuki, M., Drummond, D. R., et al. (2009), Mal3 masks catastrophe events in Schizosaccharomyces pombe microtubules by inhibiting shrinkage and promoting rescue.J Biol Chem 284: 29246–50.PubMedCrossRefGoogle Scholar
  35. 35.
    Vigers, G. P., Coue, M., and McIntosh, J. R. (1988), Fluorescent microtubules break up under illumination.The Journal of Cell Biology 107: 1011–24.PubMedCrossRefGoogle Scholar
  36. 36.
    Bormuth, V., Howard, J., and Schäffer, E. (2007), LED illumination for video-enhanced DIC imaging of single microtubules.J Microsc 226: 1–5.PubMedCrossRefGoogle Scholar
  37. 37.
    Allen, R. D., Allen, N. S., and Travis, J. L. (1981), Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris.Cell Motil 1: 291–302.PubMedCrossRefGoogle Scholar
  38. 38.
    Salmon, E. D. and Tran, P. (2007), High-resolution video-enhanced differential interference contrast light microscopy.Methods Cell Biol 81: 335–64.PubMedCrossRefGoogle Scholar
  39. 39.
    Komarova, Y., De Groot, C. O., et al. (2009), Mammalian end binding proteins control persistent microtubule growth.J Cell Biol 184: 691–706.PubMedCrossRefGoogle Scholar
  40. 40.
    Smal, I., Grigoriev, I., et al. (2010), Microtubule Dynamics Analysis Using Kymographs And Variable-Rate Particle Filters.IEEE Trans Image Process Google Scholar
  41. 41.
    Montenegro Gouveia, S., Leslie, K., et al. (2010), In Vitro Reconstitution of the Functional Interplay between MCAK and EB3 at Microtubule Plus Ends.Curr Biol 20: 1717–22.PubMedCrossRefGoogle Scholar
  42. 42.
    Axelrod, D., Thompson, N. L., and Burghardt, T. P. (1983), Total internal inflection fluorescent microscopy.J Microsc 129: 19–28.PubMedCrossRefGoogle Scholar
  43. 43.
    Kerssemakers, J. W. J., Munteanu, E. L., et al. (2006), Assembly dynamics of microtubules at molecular resolution.Nature 442: 709–12.PubMedCrossRefGoogle Scholar
  44. 44.
    Carter, N. J. and Cross, R. A. (2005), Mechanics of the kinesin step.Nature 435: 308–12.PubMedCrossRefGoogle Scholar
  45. 45.
    Felgner, H., Frank, R., and Schliwa, M. (1996), Flexural rigidity of microtubules measured with the use of optical tweezers.J Cell Sci 109 ( Pt 2): 509–16.Google Scholar
  46. 46.
    Venier, P., Maggs, A. C., et al. (1994), Analysis of microtubule rigidity using hydrodynamic flow and thermal fluctuations.J Biol Chem 269: 13353–60.PubMedGoogle Scholar
  47. 47.
    Gittes, F., Mickey, B., et al. (1993), Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape.J Cell Biol 120: 923–34.PubMedCrossRefGoogle Scholar
  48. 48.
    Mickey, B. and Howard, J. (1995), Rigidity of microtubules is increased by stabilizing agents.J Cell Biol 130: 909–17.PubMedCrossRefGoogle Scholar
  49. 49.
    Kikumoto, M., Kurachi, M., et al. (2006), Flexural rigidity of individual microtubules measured by a buckling force with optical traps.Biophys J 90: 1687–96.PubMedCrossRefGoogle Scholar
  50. 50.
    Zovko, S., Abrahams, J. P., et al. (2008), Microtubule plus-end conformations and dynamics in the periphery of interphase mouse fibroblasts.Mol Biol Cell 19: 3138–46.PubMedCrossRefGoogle Scholar
  51. 51.
    Hoog, J. L., Schwartz, C., et al. (2007), Organization of interphase microtubules in fission yeast analyzed by electron tomography.Dev Cell 12: 349–61.PubMedCrossRefGoogle Scholar
  52. 52.
    Arnal, I., Karsenti, E., and Hyman, A. A. (2000), Structural transitions at microtubule ends correlate with their dynamic properties in Xenopus egg extracts.J Cell Biol 149: 767–74.PubMedCrossRefGoogle Scholar
  53. 53.
    Chrétien, D., Fuller, S. D., and Karsenti, E. (1995), Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates.The Journal of Cell Biology 129: 1311–28.PubMedCrossRefGoogle Scholar
  54. 54.
    Simon, J. R. and Salmon, E. D. (1990), The structure of microtubule ends during the elongation and shortening phases of dynamic instability examined by negative-stain electron microscopy.J Cell Sci 96 ( Pt 4): 571–82.Google Scholar
  55. 55.
    Vitre, B., Coquelle, F. M., et al. (2008), EB1 regulates microtubule dynamics and tubulin sheet closure in vitro.Nat Cell Biol 10: 415–21.PubMedCrossRefGoogle Scholar
  56. 56.
    Keith, C. H., Feramisco, J. R., and Shelanski, M. (1981), Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts.The Journal of Cell Biology 88: 234–40.PubMedCrossRefGoogle Scholar
  57. 57.
    Saxton, W. M., Stemple, D. L., et al. (1984), Tubulin dynamics in cultured mammalian cells.J Cell Biol 99: 2175–86.PubMedCrossRefGoogle Scholar
  58. 58.
    Waterman-Storer, C. M., Desai, A., et al. (1998), Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.Curr Biol 8: 1227–30.PubMedCrossRefGoogle Scholar
  59. 59.
    Carminati, J. L. and Stearns, T. (1997), Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex.The Journal of Cell Biology 138: 629–41.PubMedCrossRefGoogle Scholar
  60. 60.
    Davidson, M. W. and Campbell, R. E. (2009), Engineered fluorescent proteins: innovations and applications.Nature Methods 6: 713–7.PubMedCrossRefGoogle Scholar
  61. 61.
    Ding, D. Q., Chikashige, Y., et al. (1998), Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells.J Cell Sci 111 ( Pt 6): 701–12.Google Scholar
  62. 62.
    Steinberg, G., Wedlich-Söldner, R., et al. (2001), Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity.J Cell Sci 114: 609–22.PubMedGoogle Scholar
  63. 63.
    Shaner, N. C., Campbell, R. E., et al. (2004), Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.Nat Biotechnol 22: 1567–72.Google Scholar
  64. 64.
    Jankovics, F. and Brunner, D. (2006), Transiently reorganized microtubules are essential for zippering during dorsal closure in Drosophila melanogaster.Dev Cell 11: 375–85.PubMedCrossRefGoogle Scholar
  65. 65.
    Faire, K., Waterman-Storer, C. M., et al. (1999), E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics.J Cell Sci 112 ( Pt 23): 4243–55.Google Scholar
  66. 66.
    Olson, K. R. and Olmsted, J. B. (1999), Analysis of microtubule organization and dynamics in living cells using green fluorescent protein-microtubule-associated protein 4 chimeras.Methods Enzymol 302: 103–20.PubMedCrossRefGoogle Scholar
  67. 67.
    Perez, F., Diamantopoulos, G. S., et al. (1999), CLIP-170 highlights growing microtubule ends in vivo.Cell 96: 517–27.PubMedCrossRefGoogle Scholar
  68. 68.
    Stepanova, T., Slemmer, J., et al. (2003), Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).J Neurosci 23: 2655–64.PubMedGoogle Scholar
  69. 69.
    Akhmanova, A., Mausset-Bonnefont, A.-L., et al. (2005), The microtubule plus-end-tracking protein CLIP-170 associates with the spermatid manchette and is essential for spermatogenesis.Genes Dev 19: 2501–15.PubMedCrossRefGoogle Scholar
  70. 70.
    Matov, A., Applegate, K., et al. (2010), Analysis of microtubule dynamic instability using a plus-end growth marker.Nature Methods 7: 761–8.PubMedCrossRefGoogle Scholar
  71. 71.
    Piehl, M. and Cassimeris, L. (2003), Organization and dynamics of growing microtubule plus ends during early mitosis.Mol Biol Cell 14: 916–25.PubMedCrossRefGoogle Scholar
  72. 72.
    Stepanova, T., Smal, I., et al. (2010), History-dependent catastrophes regulate axonal microtubule behavior.Curr Biol 20: 1023–8.PubMedCrossRefGoogle Scholar
  73. 73.
    Efimov, A., Kharitonov, A., et al. (2007), Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network.Dev Cell 12: 917–30.PubMedCrossRefGoogle Scholar
  74. 74.
    Srayko, M., Kaya, A., et al. (2005), Identification and characterization of factors required for microtubule growth and nucleation in the early C. elegans embryo.Dev Cell 9: 223–36.Google Scholar
  75. 75.
    Straube, A., Brill, M., et al. (2003), Microtubule organization requires cell cycle-dependent nucleation at dispersed cytoplasmic sites: polar and perinuclear microtubule organizing centers in the plant pathogen Ustilago maydis.Mol Biol Cell 14: 642–57.PubMedCrossRefGoogle Scholar
  76. 76.
    Mitchison, T. J. (1989), Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence.J Cell Biol 109: 637–52.PubMedCrossRefGoogle Scholar
  77. 77.
    McKinney, S., Murphy, C., et al. (2009), A bright and photostable photoconvertible fluorescent protein.Nature Methods 6: 131–3.PubMedCrossRefGoogle Scholar
  78. 78.
    Patterson, G. H. and Lippincott-Schwartz, J. (2002), A photoactivatable GFP for selective photolabeling of proteins and cells.Science 297: 1873–7.PubMedCrossRefGoogle Scholar
  79. 79.
    Subach, F. V., Patterson, G. H., et al. (2009), Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.Nature Methods 6: 153–9.PubMedCrossRefGoogle Scholar
  80. 80.
    Subach, F. V., Patterson, G. H., et al. (2010), Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.J Am Chem Soc 132: 6481–91.PubMedCrossRefGoogle Scholar
  81. 81.
    Drummond, D. R. and Cross, R. A. (2000), Dynamics of interphase microtubules in Schizosaccharomyces pombe.Curr Biol 10: 766–75.PubMedCrossRefGoogle Scholar
  82. 82.
    Gildersleeve, R. F., Cross, A. R., et al. (1992), Microtubules grow and shorten at intrinsically variable rates.J Biol Chem 267: 7995–8006.PubMedGoogle Scholar
  83. 83.
    Yvon, A. M. and Wadsworth, P. (1997), Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells.J Cell Sci 110 ( Pt 19): 2391–401.Google Scholar
  84. 84.
    Efimov, A., Schiefermeier, N., et al. (2008), Paxillin-dependent stimulation of microtubule catastrophes at focal adhesion sites.Journal of Cell Science 121: 196–204.PubMedCrossRefGoogle Scholar
  85. 85.
    Grigoriev, I., Gouveia, S. M., et al. (2008), STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER.Curr Biol 18: 177–82.PubMedCrossRefGoogle Scholar
  86. 86.
    Mimori-Kiyosue, Y., Grigoriev, I., et al. (2005), CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex.J Cell Biol 168: 141–53.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

Personalised recommendations