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An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions

Nature Methods volume 11, pages 641–644 (2014)Cite this article

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Abstract

We report a protein-fragment complementation assay (PCA) based on the engineered Deinococcus radiodurans infrared fluorescent protein IFP1.4. Unlike previous fluorescent protein PCAs, the IFP PCA is reversible, allowing analysis of spatiotemporal dynamics of hormone-induced signaling complexes in living yeast and mammalian cells at nanometer resolution. The inherently low background of infrared fluorescence permitted detection of subcellular reorganization of a signaling complex expressed at low abundance.

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Figure 1: Design and characterization of IFP PCA.
Figure 2: IFP PCA is reversible.
Figure 3: IFP PCA reversibility in mammalian cells.
Figure 4: Spatiotemporal localization of protein complexes in living eukaryotic cells.

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Acknowledgements

We thank R.Y. Tsien (University of California) for providing the pBADb2e IFP1.4-HO1 plasmid, S. Kalynych from the M. Cygler laboratory (University of Saskatchewan) for providing us with the bacterial plasmid p15A-Kan and G. Ferbeyre (Université de Montréal) for providing the pLpC retroviral vector. We also thank C. Mazurek, P.H. Ear, M. Malleshaiah, V. Messier, V. Bourdeau, E. Querido, D. Gagné and M. Vasseur for support and discussions. The authors acknowledge support from Canadian Institutes of Health Research grants MOP-GMX-152556 and MOP-GMX-231013 and Natural Sciences and Engineering Research Council of Canada grant 194582 (to SWM).

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Authors and Affiliations

  1. Département de Biochimie, Université de Montréal, Montréal, Québec, Canada

    Emmanuelle Tchekanda, Durga Sivanesan & Stephen W Michnick

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  1. Emmanuelle Tchekanda
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  2. Durga Sivanesan
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  3. Stephen W Michnick
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Contributions

E.T. designed the experiments, engineered IFP PCA fragments, generated constructs, developed and performed assays in E. coli and S. cerevisiae, analyzed the results and wrote the manuscript. D.S. designed experiments with mammalian cells (U2OS and HeLa), generated constructs and stable cell lines, analyzed the results and wrote the manuscript. S.W.M. designed the experiments, supervised the project, analyzed the results and wrote the manuscript.

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Correspondence to Stephen W Michnick.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1–6 (PDF 825 kb)

Time-lapse movie of SHC1-GRB2 interaction in response to EGF.

HeLa cells expressing the SHC1-GRB2 IFP PCA reporter show basal level expression (time 0, frame 1). After addition of EGF at concentration of 20 ng/mL (frame 2), the SHC1-GRB2 complexes are incorporated into endosomes over time. Final frame was imaged at 49 minutes after addition of EGF. (AVI 4888 kb)

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Tchekanda, E., Sivanesan, D. & Michnick, S. An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions. Nat Methods 11, 641–644 (2014). https://doi.org/10.1038/nmeth.2934

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