Abstract
Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fluorescence recovery after photobleaching (FRAP) has been shown to be a strong approach to study exactly these properties, i.e. mobility and (transient) immobilisation of the proteins under investigation. Here, we provide and discuss detailed protocols for some of the FRAP procedures that we have used to study protein behaviour in living cell nuclei. In addition, we provide examples of their application in the investigation of the androgen receptor (AR), a hormone-inducible transcription factor, and of two DNA-maintenance factors, the telomere binding proteins TRF1 and TRF2. We also provide protocols for qualitative FRAP analysis and a general scheme for computer modelling of the presented FRAP procedures that can be used to quantitatively analyse experimental FRAP curves.
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References
Tsien, R. Y. (1998) The green fluorescent protein. Ann. Rev. Biochem. 67, 509-544
Lippincott-Schwartz, J. and Patterson, G. H. (2003) Development and use of fluorescent protein markers in living cells. Science 300, 87-91
Shaner, N. C., Steinbach, P. A., and Tsien, R. Y. (2005) A guide to choosing fluorescent proteins. Nat. Meth. 2, 905-909
4.Heim, R. and Tsien, R. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178-182
Giepmans, B. N. G., Adams, S. R., Ellisman, M. H., and Tsien, R. Y. (2006) The fluorescent toolbox for assessing protein location and function. Science 312, 217-224
Farla, P., Hersmus, R., Geverts, B., Mari, P. O., Nigg, A. L., Dubbink, H. J., Trapman, J., and Houtsmuller, A. B. (2004) The androgen receptor ligand-binding domain stabilizes DNA binding in living cells. J. Struct. Biol. 147, 50-61
Farla, P., Hersmus, R., Trapman, J., and Houtsmuller, A. B. (2005) Antiandrogens prevent stable DNA-binding of the androgen receptor. J. Cell Sci. 118, 4187-4198
Rayasam, G. V., Elbi, C., Walker, D. A., Wolford, R., Fletcher, T. M., Edwards, D. P., and Hager, G. L. (2005) Ligand-specific dynamics of the progesterone receptor in living cells and during chromatin remodeling in vitro. Mol. Cell. Biol. 25, 2406-2418
McNally, J. G., Müller, W. G., Walker, D., Wolford, R., and Hager, G. L. (2000) The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287, 1262-1265
Schaaf, M. J. and Cidlowski, J. A. (2003) Molecular determinants of glucocorticoid receptor mobility in living cells: the importance of ligand affinity. Mol. Cell. Biol. 23, 1922-1934
Stenoien, D. L., Patel, K., Mancini, M. G., Dutertre, M., Smith, C. L., O’Malley, B. W., and Mancini, M. A. (2001) FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and proteasome-dependent. Nat. Cell Biol. 3, 15-23
Agresti, A., Scaffidi, P., Riva, A., Caiolfa, V. R., and Bianchi, M. E. (2005) GR and HMGB1 interact only within chromatin and influence each other’s residence time. Mol. Cell 18, 109-121
Houtsmuller, A. B. (2005) Fluorescence recovery after photobleaching: application to nuclear proteins. In: Advances in biochemical engineering Rietdorf, J., Ed.), Vol. 95, SpringerVerlag, Berlin, pp. 177-199
Axelrod D, K. D., Schlessinger J., Elson E., and Webb WW. (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055-1069
Fukano, T., Hama, H., and Miyawaki, A. (2004) Similar diffusibility of membrane proteins across the axon-soma and dendrite-soma boundaries revealed by a novel FRAP technique. J. Struct. Biol. 147, 12-18
Houtsmuller, A. B. and Vermeulen, W. (2001) Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching. Histochem. Cell Biol. 115, 13-21
Hoogstraten, D., Nigg, A. L., Heath, H., Mullenders, L. H. F., van Driel, R., Hoeijmakers, J. H. J., Vermeulen, W., and Houtsmuller, A. B. (2002) Rapid switching of TFIIH between RNA polymerase I and II transcription and DNA repair in vivo. Mol. Cell 10, 1163-1174
Mattern, K. A., Swiggers, S. J., Nigg, A. L., Lowenberg, B., Houtsmuller, A. B., and Zijlmans, J. M. (2004) Dynamics of protein binding to telomeres in living cells: implications for telomere structure and function. Mol. Cell. Biol. 24, 5587-5594
Dundr, M., Hoffmann-Rohrer, U., Hu, Q., Grummt, I., Rothblum, L. I., Phair, R. D., and Misteli, T. (2002) A kinetic framework for a mammalian RNA polymerase in vivo. Science 298, 1623-1626
Houtsmuller, A. B., Rademakers, S., Nigg, A. L., Hoogstraten, D., Hoeijmakers, J. H. J., and Vermeulen, W. (1999) Action of DNA repair endonuclease ERCC1/XPF in living cells. Science 284, 958-961
Rademakers, S., Volker, M., Hoogstraten, D., Nigg, A. L., Mone, M. J., van Zeeland, A. A., Hoeijmakers, J. H. J., Houtsmuller, A. B., and Vermeulen, W. (2003) Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions. Mol. Cell. Biol. 23, 5755-5767
Sporbert, A., Gahl, A., Ankerhold, R., Leonhardt, H., and Cardoso, M. C. (2002) DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters. Mol. Cell 10, 1355-1365
Essers, J., Theil, A. F., Baldeyron, C., van Cappellen, W. A., Houtsmuller, A. B., Kanaar, R., and Vermeulen, W. (2005) Nuclear dynamics of PCNA in DNA replication and repair. Mol. Cell. Biol. 25, 9350-9359
Zotter, A., Luijsterburg, M. S., Warmerdam, D. O., Ibrahim, S., Nigg, A., van Cappellen, W. A., Hoeijmakers, J. H. J., van Driel, R., Vermeulen, W., and Houtsmuller, A. B. (2006) Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced DNA damage depends on functional TFIIH. Mol. Cell. Biol. 26, 8868-8879
Phair, R. D. and Misteli, T. (2000) High mobility of proteins in the mammalian cell nucleus. Nature 404, 604-609
Phair, R. D., Scaffidi, P., Elbi, C., Vecerova, J., Dey, A., Ozato, K., Brown, D. T., Hager, G., Bustin, M., and Misteli, T. (2004) Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins. Mol. Cell. Biol. 24, 6393-6402
Essers, J., Houtsmuller, A. B., van Veelen, L., Paulusma, C., Nigg, A. L., Pastink, A., Vermeulen, W., Hoeijmakers, J. H., Kanaar, R. (2002) Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage. EMBO J. 21, 2030-2037
Lukas, C., Falck, J., Bartkova, J., Bartek, J., and Lukas, J. (2003) Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat. Cell Biol. 5, 255-260
Lukas, C., Melander, F., Stucki, M., Falck, J., Bekker-Jensen, S., Goldberg, M., Lerenthal, Y., Jackson, S., Bartek, J., and Lukas, J. (2004) Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J. 23, 2674-2683
Bekker-Jensen, S., Lukas, C., Melander, F., Bartek, J., and Lukas, J. (2005) Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are coordinated by Mdc1/ NFBD1. J. Cell Biol. 170, 201-211
Leonhardt, H., Rahn, H. P., Weinzierl, P., Sporbert, A., Cremer, T., Zink, D., Cardoso, M. C. (2000) Dynamics of DNA replication factories in living cells. J. Cell Biol. 149, 271-280
Kimura, H. and Cook, P. R. (2001) Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. J. Cell Biol. 153, 1341-1354
Kimura, H. (2005) Histone dynamics in living cells revealed by photobleaching. DNA Rep. (Amst.) 4, 939-950
Chen, D., Dundr, M., Wang, C., Leung, A., Lamond, A., Misteli, T., and Huang, S. (2005) Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins. J. Cell Biol. 168, 41-54
Kruhlak, M. J., Lever, M. A., Fischle, W., Verdin, E., Bazett-Jones, D. P., and Hendzel, M. J. (2000) Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments. J. Cell Biol. 150, 41-52
Kimura, H., Sugaya, K., and Cook, P. R. (2002) The transcription cycle of RNA polymerase II in living cells. J. Cell Biol. 159, 777-782
Trapman, J. (2001) Molecular mechanisms of prostate cancer. Eur. J. Cancer 37, S119-125
Feldman, B. J. and Feldman, D. (2001) The development of androgen-independent prostate cancer. Nat. Rev. Cancer 1, 34-45
Brinkmann, A. O., Faber, P. W., van Rooij, H. C. J., Kuiper, G. G. J. M., Ris, C., Klaassen, P., van der Korput, J. A. G. M., Voorhorst, M. M., van Laar, J. H., Mulder, E., and Trapman, J. (1989) The human androgen receptor: domain structure, genomic organization and regulation of expression. J. Steroid Biochem. 34, 307-310
Claessens, F., Verrijdt, G., Schoenmakers, E., Haelens, A., Peeters, B., Verhoeven, G., and Rombauts, W. (2001) Selective DNA binding by the androgen receptor as a mechanism for hormone-specific gene regulation. J. Steroid Biochem. Mol. Biol. 76, 23-30
Cleutjens, K. B. J. M., van der Korput, J. A. G. M., van Eekelen, C. C. E. M., van Rooij, H. C. J., Faber, P. W., and Trapman, J. (1997) An androgen response element in a far upstream enhancer region is essential for high, androgen-regulated activity of the prostate-specific antigen promoter. Mol. Endocrinol. 11, 148-161
Schaaf, M. J. M., Lewis-Tuffin, L. J., and Cidlowski, J. A. (2005) Ligand-selective targeting of the glucocorticoid receptor to nuclear subdomains is associated with decreased receptor mobility. Mol. Endocrinol. 19, 1501-1515
Van Royen, M. E., Cunha, S. M., Brink, M. C., Mattern, K. A., Nigg, A. L., Dubbink, H. J., Verschure, P. J., Trapman, J., and Houtsmuller, A. B. (2007) Compartmentalization of androgen receptor protein-protein interactions in living cells. J. Cell Biol. 177, 63-72
Bruggenwirth, H. T., Boehmer, A. L. M., Lobaccaro, J. M., Chiche, L., Sultan, C., Trapman, J., and Brinkmann, A. O. (1998) Substitution of Ala564 in the first zinc cluster of the deoxyribonucleic acid (DNA)-binding domain of the androgen receptor by Asp, Asn, or Leu exerts differential effects on DNA binding. Endocrinology 139, 103-110
Ellenberg, J., Siggia, E. D., Moreira, J. E., Smith, C. L., Presley, J. F., Worman, H. J., and Lippincott-Schwartz, J. (1997) Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol. 138, 1193-1206
Blonk, J. C. G., A. Don, H. Van Aalst, and J. J. Birmingham (1993) Fluorescence photobleaching recovery in the confocal scanning light microscope. J. Micros. 169, 363-374
Braga, J., Desterro, J., and Carmo-Fonseca, M. (2004) Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes. Mol. Biol. Cell 15, 4749-4760
Braga, J., McNally, J. G., and Carmo-Fonseca, M. (2007) A reaction-diffusion model to study RNA motion by quantitative fluorescence recovery after photobleaching. Biophys. J. 92, 2694-2703
Sprague, B. L., and McNally, J. G. (2005) FRAP analysis of binding: proper and fitting. Trends Cell Biol. 15, 84-91
Sprague, B. L., Pego, R. L., Stavreva, D. A., and McNally, J. G. (2004) Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys. J. 86, 3473-3495
Braeckmans, K., Peeters, L., Sanders, N. N., De Smedt, S. C., and Demeester, J. (2003) Threedimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope. Biophys. J. 85, 2240-2252
Carrero, G., McDonald, D., Crawford, E., de Vries, G., and Hendzel, M. J. (2003) Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. Methods 29, 14-28
Marcelli, M., Stenoien, D. L., Szafran, A. T., Simeoni, S., Agoulnik, I. U., Weigel, N. L., Moran, T., Mikic, I., Price, J. H., and Mancini, M. A. (2006) Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility. J. Cell. Biochem. 98, 770-788
Garcia-Parajo, M. F., Segers-Nolten, G. M. J., Veerman, J. A., Greve, J., and van Hulst, N. F. (2000) Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules. Proc. Natl. Acad. Sci. USA 97, 7237-7242
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van Royen, M.E., Farla, P., Mattern, K.A., Geverts, B., Trapman, J., Houtsmuller, A.B. (2008). Fluorescence Recovery After Photobleaching (FRAP) to Study Nuclear Protein Dynamics in Living Cells. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 464. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-461-6_20
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DOI: https://doi.org/10.1007/978-1-60327-461-6_20
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