Strategies for Imaging Microtubules in Plant Cells

  • Andreas Holzinger
  • Eiko Kawamura
  • Geoffrey O. Wasteneys
Part of the Methods in Molecular Biology book series (MIMB, volume 586)


Microtubules are required throughout plant development for a wide variety of processes, and different strategies have been evolved to visualize them. This chapter summarizes the most effective of these methods and points out potential problems and pitfalls. We outline the freeze-shattering method for immunolabeling microtubules in aerial organs such as leaves that require mechanical permeabilization, discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide different fixation protocols for preserving MTs for transmission electron microscopy including chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining procedures for transmission electron microscopy.

Key words

Alexa Microtubules Dynein EB1 GFP, Kinesin MAP4 MBD mor1-1 



This chapter has been supported in part by a grant from the Universitätszentrum Obergurgl, University of Innsbruck, Austria to AH and funding from the Natural Sciences and Engineering Research Council and the Canadian Institutes of Health Research to GOW. The Thieme Verlag KG is acknowledged for their kind permission to reproduce Fig. 1b, c and Elsevier for reproduction of Fig. 7.


  1. 1.
    Wasteneys, G. O. (2002) Microtubule organization in the green kingdom: chaos or selforder? J. Cell Sci. 115, 1345–1354.Google Scholar
  2. 2.
    Otegui, M. S. and Austin, J. R. II. (2007) Visualization of membrane-cytoskeletal interactions during plant cytokinesis. Methods Cell Biol. 79, 221–240.CrossRefGoogle Scholar
  3. 3.
    Ambrose, J. C., Shoji, T., Kotzer, A. M., Pighin, J. A. and Wasteneys G. O. (2007) The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division. Plant Cell 19, 2763–2775.CrossRefGoogle Scholar
  4. 4.
    Green, P. B. (1962) Mechanisms for plant cellular morphogenesis. Science 138, 1404–1405.CrossRefGoogle Scholar
  5. 5.
    Kost, B., Bao, Y.-Q. and Chua, N.-H. (2002) Cytoskeleton and plant organogenesis. Philos. Trans. R. Soc. Lond. B 357, 777–789.CrossRefGoogle Scholar
  6. 6.
    Smith, L. G. (2003) Cytoskeletal control of plant cell shape: getting the fine points. Curr. Opin. Plant Biol. 6, 63–73.CrossRefGoogle Scholar
  7. 7.
    Kawamura, E., Himmelspach, R., Rashbrooke, M. C., Whittington, A. T., Gale, K. R., Collings, D. A. and Wasteneys, G. O. (2006) MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root. Plant Physiol. 140, 102–114.CrossRefGoogle Scholar
  8. 8.
    Ambrose, J. C. and Wasteneys, G. O. (2008) CLASP modulates microtubule-cortex interaction during self-organization of acentrosomal microtubules. Mol. Biol. Cell 19, 4730–4737.CrossRefGoogle Scholar
  9. 9.
    Bibikova, T. N., Blancaflor, E. B. and Gilroy, S. (1999) Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana. Plant J. 17, 657–665.CrossRefGoogle Scholar
  10. 10.
    Ketelaar, T. and Emons, A. M. C. (2001) The cytoskeleton in plant cell growth: lessons from root hairs. New Phytol. 152, 409–418.CrossRefGoogle Scholar
  11. 11.
    Yu, R., Huang, R.-F., Wang, X.-C. and Yuan, M. (2001) Microtubule dynamics are involved in stomatal movement of Vicia faba L. Protoplasma 216, 113–118.CrossRefGoogle Scholar
  12. 12.
    Holzinger, A. and Lütz-Meindl, U. (2002) Kinesin-like proteins are involved in postmitotic nuclear migration of the unicellular green alga Micrasterias denticulata. Cell Biol. Int. 26, 689–697.CrossRefGoogle Scholar
  13. 13.
    Holzinger, A. and Lütz-Meindl, U. (2003) Evidence for kinesin- and dynein-like protein function in circular nuclear migration in the green alga Pleurenterium tumidum: digital time lapse analysis of inhibitor effects. J. Phycol. 39, 106–114.CrossRefGoogle Scholar
  14. 14.
    Kandasamy, M. K. and Meagher, R. B. (1999) Actin-organelle interaction: association with chloroplast in Arabidopsis leaf mesophyll cells. Cell Motil. Cytoskeleton 44, 110–118.CrossRefGoogle Scholar
  15. 15.
    Holzinger, A., Wasteneys, G. and Lütz, C. (2007) Investigating cytoskeletal function in chloroplast protrusion formation in the arctic- alpine plant Oxyria digyna. Plant Biol. 9, 400–410.CrossRefGoogle Scholar
  16. 16.
    Holzinger, A., Kwok, E. Y. and Hanson, M. R. (2008) Effects of arc3, arc5 and arc6 mutations on plastid morphology and stromule formation in green and non-green tissues of Arabidopsis thaliana. Photochem. Photobiol. 84, 1324-1335.Google Scholar
  17. 17.
    Zaffryar, S., Zimerman, B., Abu-Abied, M., Belausov, E., Lurya, G., Vainstein, A., Kamenetsky, R. and Sadot, E. (2007) Development-specific association of amyloplasts with microtubules in scale cells of Narcissus tazetta. Protoplasma 230, 153–163.CrossRefGoogle Scholar
  18. 18.
    Gilroy, S. (1997) Fluorescence microscopy of living plant cells. Annu. Rev. Plant Mol. Biol. 48, 165–190.CrossRefGoogle Scholar
  19. 19.
    Cyr, R., Dixit, R. and Gilroy, S. (2006) Live cell imaging using GFPs. Plant J. 45, 599–615.CrossRefGoogle Scholar
  20. 20.
    Mathur, J. (2006) The illuminated plant cell. Trends Plant Sci. 12, 506–513.CrossRefGoogle Scholar
  21. 21.
    Kwok, E. Y. and Hanson, M. R. (2003) Microfilaments and microtubules control the morphology and movement of non-green plastids and stromules in Nicotiana tabacum. Plant J. 35, 16–26.CrossRefGoogle Scholar
  22. 22.
    Wasteneys, G. O., Willingale-Theune, J. and Menzel, D. (1997) Freeze shattering: a simple and effective method for permeabilizing higher plant cell walls. J. Microsc. 188, 51–61.CrossRefGoogle Scholar
  23. 23.
    Shaw, S. L., Kamyar, R. and Ehrhardt, D. W. (2003) Sustained microtubule treadmilling in Arabidopsis cortical arrays. Science 300, 1715–1718.CrossRefGoogle Scholar
  24. 24.
    Chan, J., Calder, G., Fox, S. and Lloyd, C. (2007) Cortical microtubule arrays undergo rotary movements in Arabidopsis hypocotyl epidermal cells. Nat. Cell Biol. 9, 171–175.CrossRefGoogle Scholar
  25. 25.
    Kawamura, E. and Wasteneys, G. O. (2008) MOR1, the Arabidopsis thaliana homologue of Xenopus MAP215, promotes rapid growth and shrinkage, and suppresses the pausing of microtubules in vivo. J. Cell Sci. 121, 4114–4123.Google Scholar
  26. 26.
    Pastuglia, M., Azimzadeh, J., Goussot, M., Camilleri, C., Belcram, K., Evrard, J. L., Schmit, A. C., Guerche, P. and Bouchez, D. (2006) Gamma-tubulin is essential for microtubule organization and development in Arabidopsis. Plant Cell 18, 1412–1425.CrossRefGoogle Scholar
  27. 27.
    Chang, H. Y., Smertenko, A. P., Igarashi, H., Dixon, D. P. and Hussey, P. J. (2005) Dynamic interaction of NtMAP65-1a with microtubules in vivo. J. Cell Sci. 118, 3195–3201.CrossRefGoogle Scholar
  28. 28.
    Shaw, S. L. (2006) Imaging the live plant cell. Plant J. 45, 573–598.CrossRefGoogle Scholar
  29. 29.
    Ledbetter, M. C. and Porter, K. R. (1963) A “microtubule” in plant fine structure. J. Cell Biol. 19, 239–250.CrossRefGoogle Scholar
  30. 30.
    Meindl, U., Lancelle, S. and Hepler, P. K. (1992) Vesicle production and fusion during lobe formation in Micrasterias denticulata visualized by high-pressure freeze fixation. Protoplasma 170, 104–114.CrossRefGoogle Scholar
  31. 31.
    Holzinger, A. (2000) Aspects of cell development in Micrasterias muricata (Desmidiaceae) revealed by cryofixation and freeze substitution. Nova Hedwigia 70, 275–288.Google Scholar
  32. 32.
    Seguí-Simarro, J. M., Austin, J. R. II, White, E. A. and Staehelin L. A. (2004) Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing. Plant Cell 16, 836–856.CrossRefGoogle Scholar
  33. 33.
    Eder, M. and Lütz-Meindl, U. (2008) Pectin-like carbohydrates in the green alga Micrasterias characterized by cytochemical analysis and energy filtering TEM. J. Microsc. 231, 201–214.CrossRefGoogle Scholar
  34. 34.
    Holzinger, A., Valenta, R. and Lütz-Meindl, U. (2000) Profilin is localized in the nucleus-associated microtubule and actin system and is evenly distributed in the cytoplasm of the green alga Micrasterias denticulata. Protoplasma 212, 197–205.CrossRefGoogle Scholar
  35. 35.
    Gaillard, J., Neumann, E., Van Damme, D., Stoppin-Mellet, V., Ebel, C., Barbier, E., Geelen, D. and Vantard, M. (2008) Two microtubule-associated proteins of Arabidopsis MAP65s promote anti-parallel microtubule bundling. Mol. Biol. Cell, 19, 4534-4544.Google Scholar
  36. 36.
    Nakamura, M., Naoi, K., Shoji, T. and Hashimoto, T. (2004) Low concentrations of propyzamide and oryzalin alter microtubule dynamics in Arabidopsis epidermal cells. Plant Cell Physiol. 45, 1330–1334.CrossRefGoogle Scholar
  37. 37.
    Marc J., Granger, C. L., Brincat, J., Fisher, D. D., Kao, T.-H., McCubbin, A.-G. and Cyr, R. J. (1998) A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10, 1927–1939.CrossRefGoogle Scholar
  38. 38.
    Holzinger, A., Karsten, U., Lütz, C. and Wiencke, C. (2006) Ultrastructure and photosynthesis in the supralittoral green macroalga Prasiola crispa (Lightfoot) Kützing from Spitsbergen (Norway) under UV exposure. Phycologia 45, 168–177.CrossRefGoogle Scholar
  39. 39.
    Holzinger, A. and Meindl, U. (1997) Jasplakinolide, a novel actin targeting peptide, inhibits cell growth and induces actin filament polymerization in the green alga Micrasterias. Cell Motil. Cytoskeleton 38, 365–372.CrossRefGoogle Scholar
  40. 40.
    Ueda, K., Matsuyama, T. and Hashimoto, T. (1999) Visualization of microtubules in living cells of transgenic Arabidopsis thaliana. Protoplasma 206, 201–206.CrossRefGoogle Scholar
  41. 41.
    Abe, T. and Hashimoto, T. (2005) Altered microtubule dynamics by expression of modified alpha-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants. Plant J. 43, 191–204.CrossRefGoogle Scholar
  42. 42.
    Granger CL, Cyr RJ (2001) Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD. Protoplasma 216, 201-214CrossRefPubMedGoogle Scholar
  43. 43.
    Sakai, T., van der Honing, H., Nishioka, M., Uehara, Y., Takahashi, M., Fujisawa, N., Saji, K., Seki, M., Shinozaki, K., Jones, M. A., Smirnoff, N., Okada, K. and Wasteneys, G. O. (2007) Armadillo repeat-containing kinesins and a NIMA-related kinase are required for epidermal cell morphogenesis in Arabidopsis. Plant J. 53, 157–171.CrossRefGoogle Scholar
  44. 44.
    Van Bruaene, N., Joss, G. and Van Oostveldt, P. (2004) Reorganization and in vivo dynamics of microtubules during Arabidopsis root hair development. Plant Physiol. 136, 3905–3919.CrossRefGoogle Scholar
  45. 45.
    Dixit, R., Chang, E. and Cyr, R. (2006). Establishment of polarity during organization of the acentrosomal plant cortical microtubule array. Mol. Biol. Cell. 17, 1298–1305.CrossRefGoogle Scholar
  46. 46.
    Dixit, R. and Cyr, R. (2003) Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J. 36, 280–290.CrossRefGoogle Scholar
  47. 47.
    Chan J., Calder, G. M., Doonan, J. H. and Lloyd, C. W. (2003) EB1 reveals mobile microtubule nucleation sites in Arabidopsis. Nat. Cell Biol. 5, 967–971.CrossRefGoogle Scholar
  48. 48.
    Mathur, J., Mathur, N., Kernebeck, B., Srinivas, B. P. and Hulskamp, M. (2003) A novel localization pattern for an EB1-like protein links microtubule dynamics to endomembrane organization. Curr. Biol. 13, 1991–1997.CrossRefGoogle Scholar
  49. 49.
    DeBolt, S., Gutierrez, R., Ehrhardt, D. W., Melo, C. V., Ross, L., Cutler, S. R., Somerville, C. and Bonetta, D. (2007) Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement. Proc. Natl Acad. Sci. U. S. A. 104, 5854–5849.CrossRefGoogle Scholar
  50. 50.
    Stoppin-Mellet, V., Gaillard, J. and Vantard, M. (2006) Katanin’s severing activity favors bundling of cortical microtubules in plants. Plant J. 46, 1009–1017.CrossRefGoogle Scholar
  51. 51.
    Wightman, R. and Turner, S. R. (2007) Severing at sites of microtubule crossover contributes to microtubule alignment in cortical arrays. Plant J. 52, 742–751.CrossRefGoogle Scholar
  52. 52.
    Buser, C. and Walther, P. (2008) Freeze substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around −60°C. J. Microsc. 230, 268–277.CrossRefGoogle Scholar
  53. 53.
    Walther, P. and Ziegler, A. (2002) Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water J. Microsc. 208, 3–10.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Andreas Holzinger
    • 1
  • Eiko Kawamura
    • 2
  • Geoffrey O. Wasteneys
    • 2
  1. 1.Institute of Botany, Department of Physiology and Cell PhysiologyUniversity of InnsbruckInnsbruckAustria
  2. 2.Department of BiologyUniversity of British ColumbiaVancouverCanada

Personalised recommendations