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Visualization of ternary complexes in living cells by using a BiFC-based FRET assay

Nature Protocols volume 3, pages 1693–1702 (2008)Cite this article

Abstract

Studies of protein interactions have increased our understanding and knowledge of biological processes. Assays that utilize fluorescent proteins, such as fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), have enabled direct visualization of protein interactions in living cells. However, these assays are primarily suitable for a pair of interacting proteins, and methods to visualize and identify multiple protein complexes in vivo are very limited. This protocol describes the recently developed BiFC–FRET assay, which allows visualization of ternary complexes in living cells. We discuss how to design the BiFC–FRET assay on the basis of the validation of BiFC and FRET assays and how to perform transfection experiments for acquisition of fluorescent images for net FRET calculation. We also provide three methods for normalization of the FRET efficiency. The assay employs a two-chromophore and three-filter FRET setup and is applicable to epifluorescence microscopes. The entire protocol takes about 2–3 weeks to complete.

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Figure 1: Principle of the BiFC–FRET assay.
Figure 2: Schematic view of cloning vectors for BiFC–FRET analysis.
Figure 3: Schematic view of BiFC–FRET analysis.
Figure 4: Calculations for CoA and CoB and net FRET (nF).

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References

  1. Hu, C.D., Chinenov, Y. & Kerppola, T.K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9, 789–798 (2002).

    Article  CAS  Google Scholar 

  2. Shyu, Y.J., Liu, H., Deng, X. & Hu, C.D. Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Biotechniques 40, 61–66 (2006).

    Article  CAS  Google Scholar 

  3. Periasamy, A. & Day, R.N. Visualizing protein interactions in living cells using digitized GFP imaging and FRET microscopy. Methods Cell Biol. 58, 293–314 (1999).

    Article  CAS  Google Scholar 

  4. Piston, D.W. & Kremers, G.J. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem. Sci. 32, 407–414 (2007).

    Article  CAS  Google Scholar 

  5. Chen, H., Puhl, H.L., Koushik, S.V., Vogel, S.S. & Ikeda, S.R. Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells. Biophys. J. 91, L39–L41 (2006).

    Article  CAS  Google Scholar 

  6. Pfleger, K.D., Seeber, R.M. & Eidne, K.A. Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein–protein interactions. Nat. Protoc. 1, 337–345 (2006).

    Article  CAS  Google Scholar 

  7. De, A., Loening, A.M. & Gambhir, S.S. An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects. Cancer Res. 67, 7175–7183 (2007).

    Article  CAS  Google Scholar 

  8. Gordon, G.W., Berry, G., Liang, X.H., Levine, B. & Herman, B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702–2713 (1998).

    Article  CAS  Google Scholar 

  9. Xia, Z. & Liu, Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81, 2395–2402 (2001).

    Article  CAS  Google Scholar 

  10. Verveer, P.J., Rocks, O., Harpur, A.G. & Bastiaens, P.I.H. Measuring FRET by sensitized emission. Cold Spring Harb. Protoc. doi:10.1101/pdb.ip15 (2006).

  11. Shyu, Y.J., Suarez, C.D. & Hu, C.D. Visualization of AP-1-NF-kappaB ternary complexes in living cells by using a BiFC-based FRET. Proc. Natl. Acad. Sci. USA 105, 151–156 (2007).

    Article  Google Scholar 

  12. Adams, P.D., Seeholzer, S. & Ohh, M. Identification of associated proteins by coimmunoprecipitation. In Protein–Protein Interactions: A Molecular Cloning Manual (ed. Golemis, E.A.) 5.59–5.74 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2002).

    Google Scholar 

  13. Ong, H.L. et al. Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store–operated and calcium release-activated calcium channel components. J. Biol. Chem. 282, 9105–9116 (2007).

    Article  CAS  Google Scholar 

  14. Canonici, A. et al. Insulin-like growth factor-I receptor, E-cadherin and alpha v integrin form a dynamic complex under the control of alpha-catenin. Int. J. Cancer 122, 572–582 (2008).

    Article  CAS  Google Scholar 

  15. Galperin, E., Verkhusha, V.V. & Sorkin, A. Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells. Nat. Methods 1, 209–217 (2004).

    Article  CAS  Google Scholar 

  16. Kinoshita, K. et al. Ternary complex formation of pVHL, elongin B and elongin C visualized in living cells by a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. FEBS J. 274, 5567–5575 (2007).

    Article  CAS  Google Scholar 

  17. Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    Article  CAS  Google Scholar 

  18. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  Google Scholar 

  19. Chen, L. et al. Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature 392, 42–48 (1998).

    Article  CAS  Google Scholar 

  20. Hogan, P.G., Chen, L., Nardone, J. & Rao, A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 17, 2205–2232 (2003).

    Article  CAS  Google Scholar 

  21. Kerppola, T.K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat. Protoc. 1, 1278–1286 (2006).

    Article  Google Scholar 

  22. Verveer, P.J., Harpur, A.G. & Bastiaens, P.I.H. Imaging protein interactions by FRET micrsocopy. In Protein–Protein Interactions. A Molecular Cloning Manual (ed. Golemis, E.A.) 10 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork, 2002).

    Google Scholar 

  23. Ghosh, I., Hamilton, A.D. & Regan, L. Antiparallel lucine zipper-directed protein reassembly: applications to the green fluorescent protein. J. Amer. Chem. Soc. 122, 5658–5659 (2000).

    Article  CAS  Google Scholar 

  24. Zhang, S., Ma, C. & Chalfie, M. Combinatorial marking of cells and organelles with reconstituted fluorescent proteins. Cell 119, 137–144 (2004).

    Article  CAS  Google Scholar 

  25. Jach, G., Pesch, M., Richter, K., Frings, S. & Uhrig, J.F. An improved mRFP1 adds red to bimolecular fluorescence complementation. Nat. Methods 3, 597–600 (2006).

    Article  CAS  Google Scholar 

  26. Fan, J.Y. et al. Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein–protein interactions in living cells. Biochem. Biophys. Res. Commun. 367, 47–53 (2008).

    Article  CAS  Google Scholar 

  27. Bastiaens, P.I.H. & Jovin, T.M. Fluorescence resonance energy transfer microscopy. In Cell Biology. A Laboratory Handbook. Vol. 3 (ed. Celis, J.E.) 136–146 (Academic Press, New York, 1998).

    Google Scholar 

  28. Cardullo, R.A., Mungavon, R.M. & Wolf, D.E. Fluorescence resonance energy transfer microscopy: theory and instrumentation. Methods Cell Biol. 72, 415–430 (2003).

    Article  Google Scholar 

  29. Sekar, R.B. & Periasamy, A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol. 160, 629–633 (2003).

    Article  CAS  Google Scholar 

  30. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    Article  CAS  Google Scholar 

  31. Tsien, R.Y. The green fluorescent protein. Ann. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  32. Koushik, S.V., Chen, H., Thaler, C., Puhl, H.L. III & Vogel, S.S. Cerulean, venus, and venusY67C FRET reference standards. Biophys. J. 91, 99–101 (2006).

    Article  Google Scholar 

  33. Hu, C.D. & Kerppola, T.K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat. Biotechnol. 21, 539–545 (2003).

    Article  CAS  Google Scholar 

  34. Hu, C.D., Grinberg, A. & Kerppola, T.K. Visualization of protein interactions in living cells using bimolecular fluorescence complementation. In Curr. Protoc. Cell Biol. (eds. Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M.) 21.3.1–21.3.21 (John Wiley & Sons, Hoboken, NJ, 2005).

    Google Scholar 

  35. Gallagher, S., Winston, S.E., Fuller, S.A. & Hurrell, G.R. Immunoblotting and immunodetection. In Curr. Protoc. Cell Biol. (eds. Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. & Yamada, K.M.) 6.2.1–6.2.20 (John Wiley & Sons, New York, 1998).

    Google Scholar 

  36. Bearer, E.L. Overview of image analysis, image importing, and image processing using freeware. In Curr. Protoc. Mol. Biol. (eds. Ausubel, F.M. et al.) 14–15 (John Wiley & Sons, Hoboken, NJ, 2003).

  37. Zal, T. & Gascoigne, N.R. Photobleaching-corrected FRET efficiency imaging of live cells. Biophys. J. 86, 3923–3939 (2004).

    Article  CAS  Google Scholar 

  38. Damelin, M. & Silver, P. Analysis of protein interactions in vivo with fluorescence resonance energy transfer (FRET). Cold Spring Harb. Protoc. doi:10.1101/pdb.prot4581 (2006).

  39. Damelin, M. & Silver, P. Experimental design for in vivo FRET analysis. Cold Spring Harb. Protoc. doi:10.1101/pdb.ip10 (2006).

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Acknowledgements

We thank Drs. David Piston and Atsushi Miyawaki for kindly providing the cDNAs for Cerulean and Venus, as well as the Hu Laboratory members for their helpful discussions. C.D.H. was supported by grants from the Purdue Cancer Center (NCI-P30CA23168), the Indiana Elks Inc., Walther Cancer Institute, National Science Foundation (0420634-MCB) and the American Heart Association (0655570Z).

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  1. Department of Medicinal Chemistry and Molecular Pharmacology and Purdue Cancer Center, Purdue University, West Lafayette, 47907, Indiana, USA

    Y John Shyu, Christopher D Suarez & Chang-Deng Hu

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Correspondence to Chang-Deng Hu.

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Shyu, Y., Suarez, C. & Hu, CD. Visualization of ternary complexes in living cells by using a BiFC-based FRET assay. Nat Protoc 3, 1693–1702 (2008). https://doi.org/10.1038/nprot.2008.157

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