Analysing Cellulose Biosynthesis with Confocal Microscopy

  • Meera Nair
  • Seth DeBoltEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 715)


Plant cells are delimited by a rigid cell wall that resists internal turgor pressure, but extends with a remarkable degree of control that allows the cell to grow and acquire specific shapes. Live cell fluorescence microscopy systems have allowed an amazing view into the complex and dynamic lives of individual proteins during cell morphogenesis. The current chapter will focus on methodology for live cell imaging of cellulose synthase (CESA) in Arabidopsis, which will also provide a launching pad to explore ones specific protein of interest.

Key words

Cellulose synthesis Live cell imaging Confocal microscopy CESA AFP YFP 


  1. 1.
    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.PubMedCrossRefGoogle Scholar
  2. 2.
    Cutler, S. R., Ehrhardt, D. W., Griffitts, J. S., and Somerville, C. (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci USA 97, 3718–3723.PubMedCrossRefGoogle Scholar
  3. 3.
    Tian, G. W., Mohanty, A., Chary, S. N., Li, S., Paap, B., Drakakaki, G., Kopec, C. D., Li, J., Ehrhardt, D., Jackson, D., et al (2004) ­High-throughput fluorescent tagging of ­full-length Arabidopsis gene products in planta. Plant Physiol 135, 25–38.PubMedCrossRefGoogle Scholar
  4. 4.
    Heazlewood, J. L., Verboom, R. E., Tonti-Filippini, J., Small, I., and Millar, A. H. (2007) SUBA: The Arabidopsis subcellular database. Nucleic Acids Res 35, D213–D218.PubMedCrossRefGoogle Scholar
  5. 5.
    Moore, I. and Murphy, A. (2009) Validating the location of fluorescent protein fusions in the endomembrane system. Plant Cell 21, 1632–1636.PubMedCrossRefGoogle Scholar
  6. 6.
    Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., and ­Tsien, R. Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, 1567–1572.PubMedCrossRefGoogle Scholar
  7. 7.
    Shaner, N. C., Steinbach, P. A., and Tsien, R. Y. (2005) A guide to choosing fluorescent proteins. Nat Methods 2, 905–909.PubMedCrossRefGoogle Scholar
  8. 8.
    Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3, 906–918.PubMedCrossRefGoogle Scholar
  9. 9.
    Habuchi, S., Ando, R., Dedecker, P., Verheijen, W., Mizuno, H., Miyawaki, A., and Hofkens, J. (2005) Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci USA 102, 9511–9516.PubMedCrossRefGoogle Scholar
  10. 10.
    Patterson, G. H., and Lippincott-Schwartz, J. (2004) Selective photolabeling of proteins using photoactivatable GFP. Methods 32, 445–450.PubMedCrossRefGoogle Scholar
  11. 11.
    Bates, I. R., Wiseman, P. W., and Hanrahan, J. W. (2006) Investigating membrane protein dynamics in living cells. Biochem Cell Biol 84, 825–831.PubMedCrossRefGoogle Scholar
  12. 12.
    Hink, M. A., Bisselin, T., and Visser, A. J. (2002) Imaging protein-protein interactions in living cells. Plant Mol Biol 50, 871–883.PubMedCrossRefGoogle Scholar
  13. 13.
    Bhat, R. A., Lahaye, T., and Panstruga, R. (2006) The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods. Plant Methods 2, 12.PubMedCrossRefGoogle Scholar
  14. 14.
    Goodin, M. M., Chakrabarty, R., Banerjee, R., Yelton, S., and DeBolt, S. (2007) New gateways to discovery. Plant Physiol 145, 1100–1109.PubMedCrossRefGoogle Scholar
  15. 15.
    Curtis, M. D. and Grossniklaus, U. (2003)A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133, 462–469.PubMedCrossRefGoogle Scholar
  16. 16.
    Alonso, J. M., Stepanova, A. N., Leisse, T.J., Kim, C. J., Chen, H., Shinn, P., Stevenson, D. K., Zimmerman, J., Barajas, P., Cheuk, R., et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657.PubMedCrossRefGoogle Scholar
  17. 17.
    Simpson, J. C., Neubrand, V. E., Wiemann, S., and Pepperkok, R. (2001) Illuminating the human genome. Histochem Cell Biol 115, 23–29.PubMedGoogle Scholar
  18. 18.
    Chakrabarty, R., Banerjee, R., Chung, S. M., Farman, M., Citovsky, V., Hogenhout, S. A., Tzfira, T., and Goodin, M. (2007) pSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions. Mol Plant Microbe Interact 20, 740–750.PubMedCrossRefGoogle Scholar
  19. 19.
    Earley, K. W., Haag, J. R., Pontes, O., Opper, K., Juehne, T., Song, K., and Pikaard, C. S. (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45, 616–629.PubMedCrossRefGoogle Scholar
  20. 20.
    Tzfira, T., Tian, G. W., Lacroix, B., Vyas, S., Li, J., Leitner-Dagan, Y., Krichevsky, A., Taylor, T., Vainstein, A., and Citovsky, V. (2005) pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants. Plant Mol Biol 57, 503–516.PubMedCrossRefGoogle Scholar
  21. 21.
    Matz, M. V., Fradkov, A. F., Labas, Y.A., Savitsky, A. P., Zaraisky, A. G., Markelov, M.L., and Lukyanov, S.A. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17, 969–973.PubMedCrossRefGoogle Scholar
  22. 22.
    Merzlyak, E. M., Goedhart, J., Shcherbo, D., Bulina, M. E., Shcheglov, A. S., Fradkov, A. F., Gaintzeva, A., Lukyanov, K. A., Lukyanov, S., Gadella, T. W., et al (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4, 555–557.PubMedCrossRefGoogle Scholar
  23. 23.
    Paredez, A. R., Somerville, C. R., and Ehrhardt, D. W. (2006) Visualization of cellulose synthase demonstrates functional association with microtubules Science 312, 1491–1495.PubMedCrossRefGoogle Scholar
  24. 24.
    Clough, S. J. and Bent, A. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.PubMedCrossRefGoogle Scholar
  25. 25.
    Lukowitz, W., Gillmor, C. S., and Scheible, W. R. (2000) Positional cloning in Arabidopsis Why it feels good to have a genome initiative working for you. Plant Physiol 123, 795–805.PubMedCrossRefGoogle Scholar
  26. 26.
    Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S., and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328, 575–592.PubMedCrossRefGoogle Scholar
  27. 27.
    Gutierrez, R., Lindeboom, J. J., Paredez, A. R., Emons, A. M. C., and Ehrhardt, D. W. (2009) Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments. Nat Cell Biol 11, 797–806.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of HorticultureUniversity of KentuckyLexingtonUSA

Personalised recommendations