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Imaging Protein Interactions in Living Cells Using the Fluorescent Proteins

  • Richard N. Day
  • Ammasi Periasamy
  • Ignacio Demarco
Part of the Reviews in Fluorescence 2008 book series (RFLU, volume 2008)

Abstract

Over the past decade, the visible fluorescent proteins (VFPs) have become an essential tool for the study of cell biology and physiology. The goal of this chapter is to describe the characteristics of the FPs and discuss the limits of their use as probes for noninvasive imaging of protein behavior in living cells. We also described live-cell imaging techniques used to monitor the subcellular dynamics and interactions of proteins in living cells. We then focused on the method of fluorescence resonance energy transfer (FRET) microscopy to quantify the interactions of proteins inside living cells and consider some of the factors that limit this approach.

Keywords

Fluorescence Resonance Energy Transfer Fluorescence Recovery After Photobleaching Fluorescence Lifetime Imaging Microscopy Fluorescence Resonance Energy Transfer Efficiency Fluorescence Resonance Energy Transfer Signal 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Shimomura, O., Johnson, F.H., and Saiga, Y., 1962, Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59:223–239.CrossRefPubMedGoogle Scholar
  2. 2.
    Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J., 1992, Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229–233.CrossRefPubMedGoogle Scholar
  3. 3.
    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.CrossRefPubMedGoogle Scholar
  4. 4.
    Inouye, S. and Tsuji, F.I., 1994, Aequorea green fluorescent protein, expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett. 341:277–280.CrossRefPubMedGoogle Scholar
  5. 5.
    Plautz, J.D., Day, R.N., Dailey, G.M., Welsh, S.B., Hall, J.C., Halpain, S., and Kay, S.A., 1996, Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster, plant and mammalian cells. Gene 173:83–87.CrossRefPubMedGoogle Scholar
  6. 6.
    Hadjantonakis, A.K. and Nagy, A., 2001, The color of mice: in the light of GFP-variant reporters, Histochem. Cell. Biol. 115:49–58.PubMedGoogle Scholar
  7. 7.
    Feng, G., Mellor, R.H., Bernstein, M., Keller-Peck, C., Nguyen, Q.T., Wallace, M., Nerbonne, J.M., Lichtman, J.W., and Sanes, J.R., 2000, Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41–51.CrossRefPubMedGoogle Scholar
  8. 8.
    Trachtenberg, J.T., Chen, B.E., Knott, G.W., Feng, G., Sanes, J.R., Welker, E., and Svoboda, K., 2002, Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex, Nature 420:788–794.CrossRefPubMedGoogle Scholar
  9. 9.
    Walsh, M.K. and Lichtman, J.W., 2003, In vivo time-lapse imaging of synaptic takeover associated with naturally occurring synapse elimination, Neuron 37:67–73.CrossRefPubMedGoogle Scholar
  10. 10.
    van Roessel, P., and Brand, A.H., 2001, Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nat. Cell Biol. 4:E15–E20.CrossRefGoogle Scholar
  11. 11.
    Lippincott-Schwartz, J., Snapp, E., and Kenworthy, A., 2001, Studying protein dynamics in living cells, Nat. Rev. Mol. Cell Biol. 2:444–456.CrossRefPubMedGoogle Scholar
  12. 12.
    Tsien, R.Y., 1998, The green fluorescent protein, Annu. Rev. Biochem. 67:509–544.CrossRefPubMedGoogle Scholar
  13. 13.
    Patterson, G., Day, R.N., and Piston, D., 2001, Fluorescent protein spectra. J. Cell Sci. 114:837–838.PubMedGoogle Scholar
  14. 14.
    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.CrossRefPubMedGoogle Scholar
  15. 15.
    Heim, R., Prasher, D.C., and Tsien, R.Y., 1994, Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. U S A 91:12501–12504.CrossRefPubMedGoogle Scholar
  16. 16.
    Brejc, K., Sixma, T.K., Kitts, P.A., Kain, S.R., Tsien, R.Y., Ormo, M., and Remington, S.J., 1997, Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein, Proc. Natl. Acad. Sci. U S A 94:2306–2311.CrossRefPubMedGoogle Scholar
  17. 17.
    Cubitt, A.B., Woollenweber, L.A., and Heim, R., 1999, Understanding structure-function relationships in the Aequorea victoria green fluorescent protein., Methods Cell Biol. 58:19–30.CrossRefPubMedGoogle Scholar
  18. 18.
    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.CrossRefPubMedGoogle Scholar
  19. 19.
    Day, R.N., Nordeen, S.K., and Wan, Y., 1999, Minireview: visualizing nuclear protein interactions in living cells, Mol. Endocrinol. 13:517–526.CrossRefPubMedGoogle Scholar
  20. 20.
    Day, R.N., Periasamy, A., and Schaufele, F., 2001, Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods 25:4–18.CrossRefPubMedGoogle Scholar
  21. 21.
    Ormö, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y., and Remington, S.J., 1996, Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395.CrossRefPubMedGoogle Scholar
  22. 22.
    Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., and Miyawaki, A., 2002, A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20:87–90.CrossRefPubMedGoogle Scholar
  23. 23.
    Karasawa, S., Araki, T., Nagai, T., Mizuno, H., and Miyawaki, A., 2004, Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem. J. 381:307–312.CrossRefPubMedGoogle Scholar
  24. 24.
    Shagin, D.A., Barsova, E.V., Yanushevich, Y.G., Fradkov, A.F., Lukyanov, K.A., Labas, Y.A., Semenova, T.N., Ugalde, J.A., Meyers, A., Nunez, J.M., Widder, E.A., Lukyanov, S.A., and Matz, M.V., 2004, GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol. Biol. Evol. 21:841–850.CrossRefPubMedGoogle Scholar
  25. 25.
    Zacharias, D.A., Violin, J.D., Newton, A.C., and Tsien, R.Y., 2002, Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296:913–916.CrossRefPubMedGoogle Scholar
  26. 26.
    Chen, Y., Wei, L.N., Muller, J.D., 2003, Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 100:15492–15497.CrossRefPubMedGoogle Scholar
  27. 27.
    Kenworthy, A., 2002, Peering inside lipid rafts and caveolae. Trends Biochem. Sci. 27:435–437.CrossRefPubMedGoogle Scholar
  28. 28.
    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.CrossRefPubMedGoogle Scholar
  29. 29.
    Matz, M.V., Lukyanov, K.A., and Lukyanov, S.A., 2002, Family of the green fluorescent protein: journey to the end of the rainbow. Bioessays 24:953–959.CrossRefPubMedGoogle Scholar
  30. 30.
    Baird, G.S., Zacharias, D.A., and Tsien, R.Y., 2000, Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. U S A 97:11984–11989.CrossRefPubMedGoogle Scholar
  31. 31.
    Bevis, B.J., and Glick, B.S., 2002, Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20:83–87.CrossRefPubMedGoogle Scholar
  32. 32.
    Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., and Tsien, R.Y., 2002, A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U S A 99:7877–7882.CrossRefPubMedGoogle Scholar
  33. 33.
    Voss, T.C., Demarco, I.A., Booker, C.F., and Day, R.N., 2004, A computer-assisted image analysis protocol that quantitatively measures subnuclear protein organization in cell populations. Biotechniques 36:240–247.PubMedGoogle Scholar
  34. 34.
    Wang, L., Jackson, W.C., Steinbach, P.A., and Tsien, R.Y., 2004, Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl. Acad. Sci. U S A 101:16745–16749.CrossRefPubMedGoogle Scholar
  35. 35.
    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.CrossRefPubMedGoogle Scholar
  36. 36.
    Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G.H., 2003, Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell. Biol. Suppl:S7–S14.Google Scholar
  37. 37.
    Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S., and Lukyanov, K.A., 2003a, Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21:191–194.CrossRefPubMedGoogle Scholar
  38. 38.
    Chudakov, D.M., Feofanov, A.V., Mudrik, N.N., Lukyanov, S., and Lukyanov, K.A. 2003b, Chromophore environment provides clue to “kindling fluorescent protein” riddle. J. Biol. Chem. 278:7215–7219.CrossRefPubMedGoogle Scholar
  39. 39.
    Yokoe, H. and Meyer, T., 1996, Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nat. Biotechnol. 14:1252–1256.CrossRefPubMedGoogle Scholar
  40. 40.
    Patterson, G.H. and Lippincott-Schwartz, J., 2002, A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877.CrossRefPubMedGoogle Scholar
  41. 41.
    Goldman, R.D. and Spector, D.L., 2005, Live Cell Imaging: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  42. 42.
    Misteli, T., 2001, Protein dynamics: implications for nuclear architecture and gene expression. Science 291:843–847.CrossRefPubMedGoogle Scholar
  43. 43.
    Hager, G.L., Elbi, C., and Becker, M., 2002, Protein dynamics in the nuclear compartment. Curr. Opin. Genet. Dev. 12:137–141.CrossRefPubMedGoogle Scholar
  44. 44.
    Deryusheva, S. and Gall, J.G., 2004, Dynamics of coilin in Cajal bodies of the Xenopus germinal vesicle, Proc. Natl. Acad. Sci. U S A 101:4810–4814.CrossRefPubMedGoogle Scholar
  45. 45.
    Johnson, P.F., 2005, Molecular stop signs: regulation of cell-cycle arrest by C/EBP transcription factors. J. Cell Sci. 118:2545–2555.CrossRefPubMedGoogle Scholar
  46. 46.
    Tang, Q.Q. and Lane, M.D., 1999, Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation. Genes Dev. 13: 2231–2241.CrossRefPubMedGoogle Scholar
  47. 47.
    Schaufele, F., Enwright, III J.F., Wang, X., Teoh, C., Srihari, R., Erickson, R., MacDougald, O.A., Day, R.N., 2001, CCAAT/enhancer binding protein alpha assembles essential cooperating factors in common subnuclear domains. Mol. Endocrinol. 15:1665–1676.Google Scholar
  48. 48.
    Enwright, III J.F., Kawecki-Crook, M.A., Voss, T.C., Schaufele, F., Day, R.N., 2003, A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAATT/enhancer binding protein alpha required for prolactin gene transcription. Mol. Endocrinol. 17:209–222.CrossRefPubMedGoogle Scholar
  49. 49.
    Erickson, R.L., Hemati, N., Ross, S.E., and MacDougald, O.A., 2001, p300 coactivates the adipogenic transcription factor CCAAT/Enhancer-binding Protein, J. Biol. Chem. 276:16348–16355.PubMedGoogle Scholar
  50. 50.
    Dundr, M., Hebert, M.D., Karpova, T.S., Stanek, D., Xu, H., Shpargel, K.B., Meier, U.T., Neugebauer, K.M., Matera, A.G., and Misteli, T., 2004, In vivo kinetics of Cajal body components, J. Cell Biol. 164:831–842.CrossRefPubMedGoogle Scholar
  51. 51.
    Handwerger, K.E., Murphy, C., and Gall, J.G., 2003, Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle, J. Cell Biol. 160:495–504.CrossRefPubMedGoogle Scholar
  52. 52.
    Phair, R.D. and Misteli, T., 2001, Kinetic modelling approaches to in vivo imaging. Nat. Rev. Mol. Cell. Biol. 2:898–907.CrossRefPubMedGoogle Scholar
  53. 53.
    Zimmermann, T., Rietdorf, J., and Pepperkok, R., 2003, Spectral imaging and its applications in live cell microscopy. FEBS Lett. 546:87–92.CrossRefPubMedGoogle Scholar
  54. 54.
    Zhang, J., Ma, Y., Taylor, S.S., and Tsien, R.Y., 2001, Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc. Natl Acad. Sci. USA 98:14997–15002CrossRefPubMedGoogle Scholar
  55. 55.
    Day, R.N., Voss, T.C., Enwright, J.F. 3rd, Booker, C.F., Periasamy, A., and Schaufele, F., 2003, Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha in the living pituitary cell nucleus. Mol. Endocrinol. 17:333–45.CrossRefPubMedGoogle Scholar
  56. 56.
    Wallace, W., Schaefer, L.H., and Swedlow, J.R., 2001, A workingperson's guide to deconvolution in light microscopy. Biotechniques 31:1076–1082.PubMedGoogle Scholar
  57. 57.
    Jares-Erijman, E.A. and Jovin, T.M., 2003, FRET imaging. Nat. Biotechnol. 21:1387–1395.CrossRefPubMedGoogle Scholar
  58. 58.
    Periasamy, A. and Day, R.N., 2005, Molecular Imaging: FRET Microscopy and Spectroscopy, Oxford University Press, New York, pp. 21–259.Google Scholar
  59. 59.
    Gordon, G.W., Berry, G., Liang, X.H., Levine, B., and Herman, B., 1998, Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74:2702–2713.CrossRefPubMedGoogle Scholar
  60. 60.
    Xia, Z. and Liu, Y., 2001, Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81:2395–2402.CrossRefPubMedGoogle Scholar
  61. 61.
    Elangovan, M., Wallrabe, H., Chen, Y., Day, R.N., Barroso, M., and Periasamy, A., 2003, Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods 29:58–73.CrossRefPubMedGoogle Scholar
  62. 62.
    Berney, C., and Danuser, G., 2003, FRET or no FRET: a quantitative comparison. Biophys. J. 84:3992–4010.CrossRefPubMedGoogle Scholar
  63. 63.
    Bastiaens, P.I. and Jovin, T.M., 1996, Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I. Proc. Natl. Acad. Sci. U S A 93:8407–8412.CrossRefPubMedGoogle Scholar
  64. 64.
    Kenworthy, A.K., 2001, Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24:289–296.CrossRefPubMedGoogle Scholar
  65. 65.
    Voss, T.C., Demarco, I.A., Booker, C.F., and Day, R.N., 2005, Functional interactions with Pit-1 reorganize co-repressor complexes in the living cell nucleus. J. Cell Sci. 118:3277–3288.CrossRefPubMedGoogle Scholar
  66. 66.
    Clegg, R.M., Holub, O., and Gohlke, C., 2003, Fluorescence lifetime-resolved imaging: measuring lifetimes in an image. Methods Enzymol. 360:509–542.CrossRefPubMedGoogle Scholar
  67. 67.
    Dong, C.Y., French, T., So, P.T., Buehler, C., Berland, K.M., and Gratton, E., 2003, Fluorescence-lifetime imaging techniques for microscopy. Methods Cell. Biol. 72:431–464.CrossRefPubMedGoogle Scholar
  68. 68.
    Centonze, V.E., Sun, M., Masuda, A., Gerritsen, H., and Herman, B., 2003, Fluorescence resonance energy transfer imaging microscopy. Methods Enzymol. 360:542–560.CrossRefPubMedGoogle Scholar
  69. 69.
    Chen, Y. and Periasamy, A., 2005, Time correlated single photon counting (TCSPC) FLIM-FRET microscopy for protein localization, in: Molecular Imaging: FRET Microscopy and Spectroscopy, Periasamy, A. and Day, R.N., eds., Oxford University Press, New York, pp. 239–259.Google Scholar
  70. 70.
    Elangovan, M., Day, R.N., and Periasamy, A., 2002, Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell. J. Microsc. 205:3–14.CrossRefPubMedGoogle Scholar
  71. 71.
    Chen, Y. and Periasamy, A., 2004, Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization, Microsc. Res. Tech. 63:72–80.CrossRefPubMedGoogle Scholar
  72. 72.
    Bacskai, B.J., Hickey, G.A., Skoch, J., Kajdasz, S.T., Wang, Y., Huang, G.F., Mathis, C.A., Klunk, W.E., and Hyman, B.T., 2003, Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-beta ligand in transgenic mice. Proc. Natl Acad. Sci. USA 100(21):12462–12467.CrossRefPubMedGoogle Scholar
  73. 73.
    Striker, G., Subramaniam, V., Seidel, C.A., and Volkmer, A., 1999, Photochromicity and fluorescence lifetimes of green fluorescent protein. J. Phys. Chem. B 103:8612–8617.CrossRefGoogle Scholar
  74. 74.
    Heikal, A.A., Hess, S.T., Baird, G.S., Tsien, R.Y., and Webb, W.W., 2000, Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc. Natl. Acad. Sci. U.S.A 97:11996–12001.CrossRefPubMedGoogle Scholar
  75. 75.
    Suhling, K., Siegel, J., Phillips, D., French, P.M.W., Lévêque-Fort, S., Webb, S.E.D., and Davis, D.M., 2002, Imaging the environment of green fluorescent protein. Biophys. J. 83:3589–3595.CrossRefPubMedGoogle Scholar
  76. 76.
    Tramier, M., Kemnitz, K., Durieux, C., and Coppey-Moisan, M., 2002, Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein. J. Microsc. 213:110–118.CrossRefGoogle Scholar
  77. 77.
    Clayton, A.H., Hanley, Q.S., and Verveer, P.J., 2004, Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data. J. Microsc. 213:1–5.CrossRefPubMedGoogle Scholar
  78. 78.
    Rizzo, M.A., Springer, G.H., Granada, B., and Piston, D.W., 2004, An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22:445–449.CrossRefPubMedGoogle Scholar
  79. 79.
    Swedlow, J.R. and Platani, M., 2002, Live cell imaging using wide-field microscopy and deconvolution. Cell Struct. Funct. 27:335–342.CrossRefPubMedGoogle Scholar
  80. 80.
    Stephens, D.J. and Allan, V.J., 2003, Light microscopy techniques for live cell imaging. Science 300:82–86.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Richard N. Day
    • 1
  • Ammasi Periasamy
    • 2
  • Ignacio Demarco
    • 3
  1. 1.Departments of Medicine and Cell BiologyUniversity of Virginia Health ServicesCharlottesvilleUSA
  2. 2.Keck Center for Cellular Imaging, Department of BiologyUniversity of VirginiaCharlottesvilleUSA
  3. 3.Department of Cell BiologyUniversity of Virginia Health ServicesCharlottesvilleUSA

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