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Visualization of molecular interactions by fluorescence complementation

Nature Reviews Molecular Cell Biology volume 7, pages 449–456 (2006)Cite this article

Abstract

The visualization of protein complexes in living cells enables the examination of protein interactions in their normal environment and the determination of their subcellular localization. The bimolecular fluorescence complementation assay has been used to visualize interactions among multiple proteins in many cell types and organisms. Modified forms of this assay have been used to visualize the competition between alternative interaction partners and the covalent modification of proteins by ubiquitin-family peptides.

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Figure 1: Principle of bimolecular fluorescence complementation.
Figure 2: Principle of multicolour bimolecular fluorescence complementation.
Figure 3: Principle of ubiquitin-mediated fluorescence complementation.

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References

  1. Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Creating new fluorescent probes for cell biology. Nature Rev. Mol. Cell Biol. 3, 906?918 (2002).

    Article  CAS  Google Scholar 

  2. Jares-Erijman, E. A. & Jovin, T. M. FRET imaging. Nature Biotechnol. 21, 1387?1395 (2003).

    Article  CAS  Google Scholar 

  3. Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295?305 (2003).

    Article  CAS  Google Scholar 

  4. 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 

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

    Article  CAS  Google Scholar 

  6. Brock, R. & Jovin, T. M. Fluorescence correlation microscopy (FCM)-fluorescence correlation spectroscopy (FCS) taken into the cell. Cell. Mol. Biol. 44, 847?856 (1998).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Cabantous, S., Terwilliger, T. C. & Waldo, G. S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotechnol. 23, 102?107 (2005).

    Article  CAS  Google Scholar 

  9. Deppmann, C. D., Thornton, T. M., Utama, F. E. & Taparowsky, E. J. Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation. Biochem. J. 374, 423?431 (2003).

    Article  CAS  Google Scholar 

  10. Grinberg, A. V., Hu, C. D. & Kerppola, T. K. Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol. Cell. Biol. 24, 4294?4308 (2004).

    Article  CAS  Google Scholar 

  11. Rajaram, N. & Kerppola, T. K. Synergistic transcription activation by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract. Mol. Cell. Biol. 24, 5694?5709 (2004).

    Article  CAS  Google Scholar 

  12. Zhu, L. Q. et al. Inhibition of Mist1 homodimer formation induces pancreatic acinar-to-ductal metaplasia. Mol. Cell. Biol. 24, 2673?2681 (2004).

    Article  CAS  Google Scholar 

  13. Kanno, T. et al. Selective recognition of acetylated histones by bromodomain proteins visualized in living cells. Mol. Cell 13, 33?43 (2004).

    Article  CAS  Google Scholar 

  14. Farina, A. et al. Bromodomain protein Brd4 binds to GTPase-activating SPA-1, modulating its activity and subcellular localization. Mol. Cell. Biol. 24, 9059?9069 (2004).

    Article  CAS  Google Scholar 

  15. Diaz, I., Martinez, M., Isabel-LaMoneda, I., Rubio-Somoza, I. & Carbonero, P. The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development. Plant J. 42, 652?662 (2005).

    Article  CAS  Google Scholar 

  16. Jang, M. K. et al. The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol. Cell. 19, 523?534 (2005).

    Article  CAS  Google Scholar 

  17. Laricchia-Robbio, L. et al. Partner-regulated interaction of IFN regulatory factor 8 with chromatin visualized in live macrophages. Proc. Natl Acad. Sci. USA 102, 14368?14373 (2005).

    Article  CAS  Google Scholar 

  18. von der Lehr, N. et al. The F-Box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription. Mol. Cell. 11, 1189?1200 (2003).

    Article  CAS  Google Scholar 

  19. de Virgilio, M., Kiosses, W. B. & Shattil, S. J. Proximal, selective, and dynamic interactions between integrin αIIbβ3 and protein tyrosine kinases in living cells. J. Cell Biol. 165, 305?311 (2004).

    Article  CAS  Google Scholar 

  20. Blondel, M. et al. Degradation of Hof1 by SCFGrr1 is important for actomyosin contraction during cytokinesis in yeast. EMBO J. 24, 1440?1452 (2005).

    Article  CAS  Google Scholar 

  21. Niu, T. K., Pfeifer, A. C., Lippincott-Schwartz, J. & Jackson, C. L. Dynamics of GBF1, a brefeldin A-sensitive Arf1 exchange factor at the Golgi. Mol. Biol. Cell 16, 1213?1222 (2005).

    Article  CAS  Google Scholar 

  22. Remy, I., Montmarquette, A. & Michnick, S. W. PKB/Akt modulates TGF-β signalling through a direct interaction with Smad3. Nature Cell Biol. 6, 358?365 (2004).

    Article  CAS  Google Scholar 

  23. Stolpe, T. et al. In planta analysis of protein?protein interactions related to light signaling by bimolecular fluorescence complementation. Protoplasma 226, 137?146 (2005).

    Article  CAS  Google Scholar 

  24. Hynes, T. R., Mervine, S. M., Yost, E. A., Sabo, J. L. & Berlot, C. H. Live cell imaging of Gs and the β2-adrenergic receptor demonstrates that both αs and β1γ 7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the β2-adrenergic receptor. J. Biol. Chem. 279, 44101?44112 (2004).

    Article  CAS  Google Scholar 

  25. Guo, Y. J., Rebecchi, M. & Scarlata, S. Phospholipase C β2 binds to and inhibits phospholipase C δ1 . J. Biol. Chem. 280, 1438?1447 (2005).

    Article  CAS  Google Scholar 

  26. Ozalp, C., Szczesna-Skorupa, E. & Kemper, B. Bimolecular fluorescence complementation analysis of cytochrome P450 2C2, 2E1, and NADPH?cytochrome P450 reductase molecular interactions in living cells. Drug Metab. Dispos. 33, 1382?1390 (2005).

    Article  CAS  Google Scholar 

  27. Giese, B. et al. Dimerization of the cytokine receptors gp130 and LIFR analysed in single cells. J. Cell Sci. 118, 5129?5140 (2005).

    Article  CAS  Google Scholar 

  28. Hynes, T. R. et al. Visualization of G protein βγ dimers using bimolecular fluorescence complementation demonstrates roles for both β and γ in subcellular targeting. J. Biol. Chem. 279, 30279?30286 (2004).

    Article  CAS  Google Scholar 

  29. Takahashi, Y. et al. Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis. Mol. Cell. Biol. 25, 9369?9382 (2005).

    Article  CAS  Google Scholar 

  30. Nyfeler, B., Michnick, S. W. & Hauri, H. P. Capturing protein interactions in the secretory pathway of living cells. Proc. Natl Acad. Sci. USA 102, 6350?6355 (2005).

    Article  CAS  Google Scholar 

  31. Rackham, O. & Brown, C. M. Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs. EMBO J. 23, 3346?3355 (2004).

    Article  CAS  Google Scholar 

  32. Stains, C. I., Porter, J. R., Ooi, A. T., Segal, D. J. & Ghosh, I. DNA sequence-enabled reassembly of the green fluorescent protein. J. Am. Chem. Soc. 127, 10782?10783 (2005).

    Article  CAS  Google Scholar 

  33. Remy, I. & Michnick, S. W. Regulation of apoptosis by the Ft1 protein, a new modulator of protein kinase B/Akt. Mol. Cell. Biol. 24, 1493?1504 (2004).

    Article  CAS  Google Scholar 

  34. 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 

  35. Fang, D. Y. & Kerppola, T. K. Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes. Proc. Natl Acad. Sci. USA 101, 14782?14787 (2004).

    Article  CAS  Google Scholar 

  36. Magliery, T. J. et al. Detecting protein?protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J. Am. Chem. Soc. 127, 146?157 (2005).

    Article  CAS  Google Scholar 

  37. Richards, F. M. On the enzymic activity of subtilisin-modified ribonuclease. Proc. Natl Acad. Sci. USA 44, 162?166 (1958).

    Article  CAS  Google Scholar 

  38. Ullmann, A., Perrin, D., Jacob, F. & Monod, J. Identification par complémentation in vitro et purification d'un segment peptidique de la β-galactosidase d' Escherichia coli. J. Mol. Biol. 12, 918?923. (1965).

    Article  CAS  Google Scholar 

  39. Rossi, F., Charlton, C. A. & Blau, H. M. Monitoring protein?protein interactions in intact eukaryotic cells by βgalactosidase complementation. Proc. Natl Acad.Sci. USA 94, 8405?8410 (1997).

    Article  CAS  Google Scholar 

  40. Johnsson, N. & Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl Acad. Sci. USA 91, 10340?10344 (1994).

    Article  CAS  Google Scholar 

  41. Pelletier, J. N., Campbell-Valois, F. X. & Michnick, S. W. Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl Acad. Sci. USA 95, 12141?12146 (1998).

    Article  CAS  Google Scholar 

  42. Ghosh, I., A. D. Hamilton, and L. Regan . Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc 122, 5658?5659 (2000).

    Article  CAS  Google Scholar 

  43. Hoff, B. & Kuck, U. Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum. Curr. Genet. 47, 132?138 (2005).

    Article  CAS  Google Scholar 

  44. Atmakuri, K., Ding, Z. Y. & Christie, P. J. VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens A1. Mol. Microbiol. 49, 1699?1713 (2003).

    Article  CAS  Google Scholar 

  45. Tzfira, T., Vaidya, M. & Citovsky, V. Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 431, 87?92 (2004).

    Article  CAS  Google Scholar 

  46. Loyter, A. et al. The plant VirE2 interacting protein 1. A molecular link between the Agrobacterium T-complex and the host cell chromatin? Plant Physiol. 138, 1318?1321 (2005).

    Article  CAS  Google Scholar 

  47. Lacroix, B., Vaidya, M., Tzfira, T. & Citovsky, V. The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation. EMBO J. 24, 428?437 (2005).

    Article  CAS  Google Scholar 

  48. Schmidt, C. et al. Mechanisms of proinflammatory cytokine-induced biphasic NF-κB activation. Mol. Cell. 12, 1287?1300 (2003).

    Article  CAS  Google Scholar 

  49. Blumenstein, A. et al. The Aspergillus nidulans phytochrome FphA represses sexual development in red light. Curr. Biol. 15, 1833?1838 (2005).

    Article  CAS  Google Scholar 

  50. Tsuchisaka, A. & Theologis, A. Heterodimeric interactions among the 1-amino-cyclopropane-1-carboxylate synthase polypeptides encoded by the Arabidopsis gene family. Proc. Natl Acad. Sci. USA 101, 2275?2280 (2004).

    Article  CAS  Google Scholar 

  51. Ye, H. H., Choi, H. J., Poe, J. & Smithgall, T. E. Oligomerization is required for HIV-1 nef-induced activation of the Src family protein-tyrosine kinase, Hck. Biochemistry 43, 15775?15784 (2004).

    Article  CAS  Google Scholar 

  52. Bracha-Drori, K. et al. Detection of protein?protein interactions in plants using bimolecular fluorescence complementation. Plant J. 40, 419?427 (2004).

    Article  CAS  Google Scholar 

  53. Walter, M. et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428?438 (2004).

    Article  CAS  Google Scholar 

  54. Hackbusch, J., Richter, K., Muller, J., Salamini, F. & Uhrig, J. F. A central role of Arabidopsis thaliana ovate family proteins in networking and subcellular localization of 3-aa loop extension homeodomain proteins. Proc. Natl Acad. Sci. USA 102, 4908?4912 (2005).

    Article  CAS  Google Scholar 

  55. Shimizu, H. et al. LIP19, a basic region leucine zipper protein, is a fos-like molecular switch in the cold signaling of rice plants. Plant Cell Physiol. 46, 1623?1634 (2005).

    Article  CAS  Google Scholar 

  56. Abe, M. et al. FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309, 1052?1056 (2005).

    Article  CAS  Google Scholar 

  57. Maple, J., Aldridge, C. & Moller, S. G. Plastid division is mediated by combinatorial assembly of plastid division proteins. Plant J. 43, 811?823 (2005).

    Article  CAS  Google Scholar 

  58. Demidov, V. V. et al. Fast complementation of split fluorescent protein triggered by DNA hybridization. Proc. Natl Acad. Sci. USA 103, 2052?2056 (2006).

    Article  CAS  Google Scholar 

  59. Schmidt, U., Richter, K., Berger, A. B. & Lichter, P. In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains. J. Cell Biol. 172, 373?381 (2006).

    Article  CAS  Google Scholar 

  60. Cascales, E., Atmakuri, K., Liu, Z., Binns, A. N. & Christie, P. J. Agrobacterium tumefaciens oncogenic suppressors inhibit T-DNA and VirE2 protein substrate binding to the VirD4 coupling protein. Mol. Microbiol. 58, 565?579 (2005).

    Article  CAS  Google Scholar 

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  1. Tom K. Kerppola is at the Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, 48109-0650, Michigan, USA

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Kerppola, T. Visualization of molecular interactions by fluorescence complementation. Nat Rev Mol Cell Biol 7, 449–456 (2006). https://doi.org/10.1038/nrm1929

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