Abstract
Information about protein interactions is crucial for the understanding of cellular processes. Current methods for the investigation of protein–protein interactions (PPIs) require either removal of the proteins from their normal cellular environment, perturbation of the cells or costly instrumentation and advanced technical expertise (Fields and Song, Nature 340:245–246, 1989; Deane et al., Mol Cell Proteomics 1:349–356, 2002; Kerppola, Nat Rev Mol Cell Biol 7:449–456, 2006; Blanchard et al., Mol Cell Proteomics 5:2175–2184, 2006; Miller et al., Mol Cell Proteomics 6:1027–1038, 2007; Miyawaki, Dev Cell 4:295–305, 2003; Parrish et al., Curr Opin Biotechnol 17:387–393, 2006; Sekar and Periasamy, J Cell Biol 160:629–633, 2003). Here, we describe a simple assay to directly visualize and analyze PPIs in single living cells. By adapting a lac operator/repressor system, we generated a stable nuclear interaction platform. A fluorescent bait protein is tethered to the interaction platform and assayed for co-localization of fluorescent prey fusion proteins. This fluorescent two-hybrid (F2H) assay allows the investigation of cell cycle dependent PPIs. With this cell based assay protein interactions even from different subcellular compartments can be visualized in real time (Zolghadr et al., Mol Cell Proteomics 7:2279–2287, 2008). The simple optical readout enables automated imaging systems to segment and analyze the acquired data for high-throughput screening of PPIs in living cells in response to external stimuli and chemical compounds.
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References
Fields, S., and Song, O. (1989) A novel genetic system to detect protein-protein interactions, Nature 340, 245–6.
Deane, C. M., Salwinski, L., Xenarios, I., and Eisenberg, D. (2002) Protein interactions: two methods for assessment of the reliability of high throughput observations, Mol Cell Proteomics 1, 349–56.
Kerppola, T. K. (2006) Visualization of molecular interactions by fluorescence complementation, Nat Rev Mol Cell Biol 7, 449–56.
Blanchard, D., Hutter, H., Fleenor, J., and Fire, A. (2006) A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm, Mol Cell Proteomics 5, 2175–84.
Miller, C. L., Arnold, M. M., Broering, T. J., Eichwald, C., Kim, J., Dinoso, J. B., and Nibert, M. L. (2007) Virus-derived platforms for visualizing protein associations inside cells, Mol Cell Proteomics 6, 1027–38.
Miyawaki, A. (2003) Visualization of the spatial and temporal dynamics of intracellular signaling, Dev Cell 4, 295–305.
Parrish, J. R., Gulyas, K. D., and Finley, R. L., Jr. (2006) Yeast two-hybrid contributions to interactome mapping, Curr Opin Biotechnol 17, 387–93.
Sekar, R. B., and Periasamy, A. (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations, J Cell Biol 160, 629–33.
Zolghadr, K., Mortusewicz, O., Rothbauer, U., Kleinhans, R., Goehler, H., Wanker, E. E., Cardoso, M. C., and Leonhardt, H. (2008) A fluorescent two-hybrid assay for direct visualization of protein interactions in living cells, Mol Cell Proteomics 7, 2279–87.
Meilinger, D., Fellinger, K., Bultmann, S., Rothbauer, U., Bonapace, I. M., Klinkert, W. E., Spada, F., and Leonhardt, H. (2009) Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells, EMBO Rep 10, 1259–64.
Fellinger, K., Rothbauer, U., Felle, M., Langst, G., and Leonhardt, H. (2009) Dimerization of DNA methyltransferase 1 is mediated by its regulatory domain, J Cell Biochem 106, 521–8.
Phair, R. D., and Misteli, T. (2000) High mobility of proteins in the mammalian cell nucleus, Nature 404, 604–9.
Tsukamoto, T., Hashiguchi, N., Janicki, S. M., Tumbar, T., Belmont, A. S., and Spector, D. L. (2000) Visualization of gene activity in living cells, Nat Cell Biol 2, 871–8.
Janicki, S. M., Tsukamoto, T., Salghetti, S. E., Tansey, W. P., Sachidanandam, R., Prasanth, K. V., Ried, T., Shav-Tal, Y., Bertrand, E., Singer, R. H., and Spector, D. L. (2004) From silencing to gene expression: real-time analysis in single cells, Cell 116, 683–98.
Robinett, C. C., Straight, A., Li, G., Willhelm, C., Sudlow, G., Murray, A., and Belmont, A. S. (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition, J Cell Biol 135, 1685–700.
Tumbar, T., Sudlow, G., and Belmont, A. S. (1999) Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain, J Cell Biol 145, 1341–54.
Dietzel, S., Zolghadr, K., Hepperger, C., and Belmont, A. S. (2004) Differential large-scale chromatin compaction and intranuclear positioning of transcribed versus non-transcribed transgene arrays containing {beta}-globin regulatory sequences, J Cell Sci 117, 4603–14.
Vazquez, J., Belmont, A. S., and Sedat, J. W. (2001) Multiple regimes of constrained chromosome motion are regulated in the interphase Drosophila nucleus, Curr Biol 11, 1227–39.
Acknowledgments
We thank Lothar Schermelleh for comments on the manuscript. We also thank D.L. Spector for providing BHK clone#2 and U2OS.2-6-3 cells containing a lac operator array. This work was supported by the Center for NanoScience (CeNS), the Nanosystems Initiative Munich (NIM), the BioImaging Network Munich (BIN), and by grants from the Deutsche Forschungsgemeinschaft (DFG).
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Zolghadr, K., Rothbauer, U., Leonhardt, H. (2012). The Fluorescent Two-Hybrid (F2H) Assay for Direct Analysis of Protein–Protein Interactions in Living Cells. In: Suter, B., Wanker, E. (eds) Two Hybrid Technologies. Methods in Molecular Biology, vol 812. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-455-1_16
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DOI: https://doi.org/10.1007/978-1-61779-455-1_16
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