Cell and Tissue Biology

, Volume 6, Issue 2, pp 128–136 | Cite as

Parameters that affect estimation of nucleolar proteins’ mobility in living cells by the FRAP method with the example of protein fibrillarin

  • V. V. Barygina
  • A. A. Mironova
  • O. V. Zatsepina


The fluorescence recovery after photobleaching (FRAP) method, in combination with confocal laser scanning microscopy, represents one of the basic approaches to studying the properties of proteins in living mammalian cells. However, the data of different authors on the dynamic properties of the same protein and even in cells of the same type can differ greatly. Until now, the reasons for such discrepancies have not been specifically analyzed. In the present work, using the example of nucleolar protein fibrillarin fused with EGFP, we studied the effect of the area of the irradiated region (the region of interest (ROI)) and temperature conditions of experiments on the main dynamic characteristics of the protein—the portion of the mobile fraction of protein and the half-recovery time of fluorescence after photobleaching (t 1/2). The obtained results have shown that both parameters affect markedly the estimation of the fibrillarin-EGFP mobility in HeLa cells. It was concluded that, in FRAP experiments the ROI area can be standardized and, where possible, minimized. In addition, when analyzing the dynamic characteristics of the nucleolar proteins, which participate in the temperature-dependent enzymatic reactions, it is necessary to maintain standard temperature conditions.


FRAP fibrillarin mobile protein fraction fluorescence half-recovery time (t1/2area of ROI photobleaching temperature conditions 


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  1. Aris, J.P. and Blobel, G., cDNA Cloning and Sequencing of Human Fibrillarin, a Conserved Nucleolar Protein Recognized by Autoimmune Antisera, Cell Biol., 1991, vol. 88, pp. 931–935.Google Scholar
  2. Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E., and Webb, W.W., Mobility Measurement by Analysis of Fluorescence Photobleaching Recovery Kinetics, Biophysics, 1976, vol. J 16, pp. 1055–1069.Google Scholar
  3. Barygina, V.V., Veiko, V.P., and Zatsepina, O.V., Analysis of Nucleolar Protein Fibrillarin Mobility and Functional State in Living HeLa Cells, Biochemistry (Moscow), 2010, vol. 75, no. 8, pp. 979–988.CrossRefGoogle Scholar
  4. Braga, J., Desterro, J.M.P., and Carmo-Fonseca, M., Intracellular Macromolecular Mobility Measured by Fluorescence Recovery after Photobleaching with Confocal Laser Scanning Microscopes, Mol. Biol. Cell, 2004, vol. 15, pp. 4749–4760.PubMedCrossRefGoogle Scholar
  5. Carrero, G., McDonald, D., Crawford, E., de, Vries, G., and Hendzel, M.J., Using FRAP and Mathematical Modeling to Determine the in vivo Kinetics of Nuclear Proteins, Methods, 2003, vol. 29, pp. 14–28.PubMedCrossRefGoogle Scholar
  6. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C., Green Fluorescent Protein as a Marker for Gene Expression, Science, 1994, vol. 263, pp. 802–805.PubMedCrossRefGoogle Scholar
  7. Chen, D. and Huang, S., Nucleolar Components Involved in Ribosome Biogenesis Cycle between the Nucleolus and Nucleoplasm in Interphase Cells, J. Cell Biol., 2001, vol. 153, pp. 169–176.PubMedCrossRefGoogle Scholar
  8. Chudakov, D.M., Matz, M.V., Lukyanov, S., and Lukyanov, K.A., Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues, Physiol. Rev., 2010, vol. 90, pp. 1103–1163.PubMedCrossRefGoogle Scholar
  9. Dobrucki, J.W., Feret, D., and Noatynska, A., Scattering of Exciting Light by Live Cells in Fluorescence Confocal Imaging: Phototoxic Effects and Relevance for FRAP Studies, Biophysics, 2007, vol. 93, pp. 1778–1786.CrossRefGoogle Scholar
  10. Dundr, M., Misteli, T., and Olson, M.O.J., The Dynamics of Postmitotic Reassembly of the Nucleolus, J. Cell Biol., 2000, vol. 150, pp. 433–446.PubMedCrossRefGoogle Scholar
  11. Gerbi, S.A., Borovjagin, A.V., and Lange, T.S., The Nucleolus: A Site of Ribonucleoprotein Maturation, Curr. Opin. Cell Biol., 2003, vol. 15, pp. 318–325.PubMedCrossRefGoogle Scholar
  12. Gurskaya, N.G., Verkhusha, V.V., Shcheglov, A.S., Staroverov, D.B., Chepurnykh, T.V., Fradkov, A.F., Lukyanov, Sergey S., and Lukyanov, K.A., Engineering of a Monomeric Green-to-Red Photoactivatable Fluorescent Protein Induced by Blue Light, Nature Biotechnol., 2006, vol. 24, pp. 461–465.CrossRefGoogle Scholar
  13. Hernandez-Verdun, D., Nucleolus: From Structure to Dynamics, Histochem. Cell Biol., 2006, vol. 125, pp. 127–137.PubMedCrossRefGoogle Scholar
  14. Hoogstraten, D., Nigg, A.L., Heath, H., Mullenders, L.H., van, Driel, R., Hoeijmakers, J.H., Vermeulen, W., and Houtsmuller, A.B., Rapid Switching of TFIIH between RNA Polymerase I and II Transcription and DNA Repair in vivo, Mol. Cell., 2002, vol. 10, pp. 1163–1174.PubMedCrossRefGoogle Scholar
  15. Houtsmuller, A.B., Fluorescence Recovery after Photobleaching: Application to Nuclear Proteins, Adv. Biochem. Engin./Biotechnol., 2005, vol. 95, pp. 177–199.Google Scholar
  16. Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., and Tilley, L., Fluorescence Photobleaching Analysis for the Study of Cellular Dynamics, Eur. Biophys., 2002, vol. 31, pp. 36–51.CrossRefGoogle Scholar
  17. Lam, Y.W., Trinkle-Mulcahy, L., and Lamond, A.I., The Nucleolus, J. Cell Sci., 2005, vol. 118, pp. 1335–1337.PubMedCrossRefGoogle Scholar
  18. Leung, A.K.L., Andersen, J.S., Mann, M., and Lamond, A.I., Bioinformatic Analysis of the Nucleolus, Biochemistry, 2003, vol. J 376, pp. 553–569.Google Scholar
  19. Louvet, E., Junéra, H.R., Le Panse, S., and Danièle Hernandez-Verdun, D., Dynamics and Compartmentation of the Nucleolar Processing Machinery, Exper. Cell Res., 2005, vol. 304, pp. 457–470.CrossRefGoogle Scholar
  20. Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G.H., Photobleaching and Photoactivation: Following Protein Dynamics in Living Cells, Nat. Cell Biol., 2003, suppl., pp. S7–14.Google Scholar
  21. Lippincott-Schwartz, J., Snapp, E., and Kenworthy, A., Studying Protein Dynamics in Living Cells, Nat. Rev. Mol. Cell Biol., 2001, vol. 2, pp. 444–456.PubMedCrossRefGoogle Scholar
  22. Lischwe, M.A., Ochs, R.L., Reddy, R., Cook, R.G., Yeoman, L.C., Tan, E.M., Reichlin, M., and Busch, H., Purification and Partial Characterization of a Nucleolar Scleroderma Antigen (Mr = 34000; pI, 8.5) Rich in NG,NG-Dimethylarginine, J. Biol. Chem., 1985, vol. 260, pp. 14304–14310.PubMedGoogle Scholar
  23. Meyvis, T.K., De Smedt, S.C., Van, Oostveldt, P., and Demeester, J., Fluorescence Recovery after Photobleaching: A Versatile Tool for Mobility and Interaction Measurements in Pharmaceutical Research, Pharm. Res., 1999, vol. 16, pp. 1153–1162.PubMedCrossRefGoogle Scholar
  24. Müeller, F., Mazza, D., Stasevich, T.J., and McNally, J.G., FRAP and Kinetic Modeling in the Analysis of Nuclear Protein Dynamics: What Do We Really Know? Curr. Opin. Cell Biol., 2010, vol. 22, pp. 403–411.PubMedCrossRefGoogle Scholar
  25. Mukhar’yamova, K.Sh. and Zatsepina, O.V., Visualization of Ribosomal Genes Transcription in SPEV Culture Cells Using Bromouridine Triphosphate, Tsitologiya, 2001, vol. 43, no. 8, pp. 792–795.Google Scholar
  26. Negi, S.S. and Olson, M.O., Effects of Interphase and Mitotic Phosphorylation on the Mobility and Location of Nucleolar Protein B23, J. Cell Sci., 2006, vol. 119, pp. 3676–3685.PubMedCrossRefGoogle Scholar
  27. Olson, M.O., Dundr, M., and Szebeni, A., The Nucleolus: An Old Factory with Unexpected Capabilities, Trends Cell Biol., 2000, vol. 10, pp. 189–196.PubMedCrossRefGoogle Scholar
  28. Phair, R.D. and Misteli, T., High Mobility of Proteins in the Mammalian Cell Nucleus, Nature, 2000, vol. 404, pp. 604–609.PubMedCrossRefGoogle Scholar
  29. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J., Primary Structure of the Aequorea Victoria Green-Fluorescent Protein, Gene, 1992, vol. 111, pp. 229–233.PubMedCrossRefGoogle Scholar
  30. Pucadyil, T.J. and Chattopadhyay, A., Cholesterol Depletion Induces Dynamic Confinement of the G-protein Coupled Serotonin (1A) Receptor in the Plasma Membrane of Living Cells, Biochim. Biophys. Acta, 2007, vol. 1768, pp. 655–668.PubMedCrossRefGoogle Scholar
  31. Reits, E.A. and Neefjes, J.J., From Fixed to FRAP: Measuring Protein Mobility and Activity in Living Cells, Nat. Cell Biol., 2001, vol. 3, pp. E145–E147.PubMedCrossRefGoogle Scholar
  32. Rizzuto, R., Brini, M., Pizzo, P., Murgia, M., and Pozzan, T., Chimeric Green Fluorescent Protein as a Tool for Visualizing Subcellular Organelles in Living Cells, Current Biol., 1995, vol. 5, pp. 635–642.CrossRefGoogle Scholar
  33. Saxena, R., and Chattopadhyay, A., Membrane Organization and Dynamics of the Serotonin(1A) Receptor in Live Cells, J. Neurochem., 2011, vol. 116, pp. 726–733.PubMedCrossRefGoogle Scholar
  34. Snaar, S., Wiesmeijer, K., Jochemsen, A.G., Tanke, H.J., and Dirks, R.W., Mutational Analysis of Fibrillarin and Its Mobility in Living Human Cells, J. Cell Biol., 2000, vol. 151, pp. 653–662.PubMedCrossRefGoogle Scholar
  35. Sprague, B.L., Pego, R.L., Stavreva, D.A., and McNally, J.G., Analysis of Binding Reactions by Fluorescence Recovery after Photobleaching, Biophysics, 2004, vol. J 86, pp. 3473–3495.Google Scholar
  36. Sprague, B.L. and McNally, J.G., FRAP Analysis of Binding: Proper and Fitting, Trends Cell Biol., 2005, vol. 15, pp. 84–91.PubMedCrossRefGoogle Scholar
  37. Stasevich, T.J., Mueller, F., Michelman-Ribeiro, A., Rosales, T., Knutson, J.R., and McNally, J.G., Cross-Validating FRAP and FCS to Quantify the Impact of Photobleaching on in vivo Binding Estimates, Biophysics, 2010, vol. J 99, pp. 3093–3101.Google Scholar
  38. Tollervey, D., Temperature-Sensitive Mutations Demonstrate Roles for Yeast Fibrillarin in Pre-rRNA Processing, Pre-rRNA Methylation, and Ribosome Assembly, Cell, 1993, vol. 72, pp. 443–457.PubMedCrossRefGoogle Scholar
  39. Tripathi, K. and Parnaik, V.K., Differential Dynamics of Splicing Factor SC35 during the Cell Cycle, J. Biosci., 2008, vol. 33, pp. 345–354.PubMedCrossRefGoogle Scholar
  40. Tsien, R.Y., The Green Fluorescent Protein, Annu. Rev. Biochem., 1998, vol. 67, pp. 509–544.PubMedCrossRefGoogle Scholar
  41. Tsien, R.Y., Ernst, L., and Waggoner, A., Fluorophores for Confocal Microscopy: Photophysics and Photochemistry, in Handbook of Biological Confocal Microscopy, New York: Springer Science, Business Media, 2006.Google Scholar
  42. Van Royen, M.E., Dinant, C., Farla, P., Trapman, J., and Houtsmuller, A.B., FRAP and FRET Methods to Study Nuclear Receptors in Living Cells, in Methods in Molecular Biology: the Nuclear Receptor Superfamily, New York: Humana Press, 2009, pp. 69–96.CrossRefGoogle Scholar
  43. Van Royen, M.E., Farla, P., Mattern, K.A., Geverts, B., Trapman, J., and Houtsmuller, A.B., Fluorescence Recovery after Photobleaching (FRAP) to Study Nuclear Protein Dynamics in Living Cells, in The Nucleus, Vol. 2: Chromatin, Transcription, Envelope, Proteins, Dynamics, and Imaging, New York: Humana Press, 2008, pp. 363–385.Google Scholar
  44. Verkman, A.S., Solute and Macromolecule Diffusion in Cellular Aqueous Compartments, Trends Biochem. Sci., 2002, vol. 27, pp. 27–33.PubMedCrossRefGoogle Scholar
  45. Wang, H., Boisvert, D., Kim, K.K., Kim, R., and Kim, S.-H., Crystal Structure of a Fibrillarin Homologue from Methanococcus jannaschii, a Hyperthermophile, at 1.6 — tion, EMBO, 2000, vol. J 19, pp. 317–323.Google Scholar
  46. White, J. and Stelzer, E., Photobleaching GFP Reveals Protein Dynamics Inside live Cells, Trends Cell Biol., 1999, vol. 9, pp. 61–65.PubMedCrossRefGoogle Scholar
  47. Yang, T.T., Cheng, L., and Kain, S.R., Optimized Codon Usage and Chromophore Mutations Provide Enhanced Sensitivity with the Green Fluorescent Protein, Nucleic Acids Res., 1996, vol. 24, pp. 4592–4593.PubMedCrossRefGoogle Scholar
  48. Zubova, N.N., Bulavina, A.Yu., and Savitskii, A.P., The Spectral and Physicochemical Properties of Green (GFP) and Red (drFP583) Fluorescent Proteins, Usp. Biol. Khim., 2003, vol. 43, pp. 163–224.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Faculty of BiologyMoscow State UniversityMoscowRussia
  3. 3.Faculty of Bioengineering and BioinformaticsMoscow State UniversityMoscowRussia

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