Fluorescence fluctuations of quantum-dot sensors capture intracellular protein interaction dynamics

Abstract

We extend the in vitro principle of co-immunoprecipitation to quantify dynamic protein interactions in living cells. Using a multiresolution implementation of fluorescence correlation spectroscopy to achieve maximal temporal resolution, we monitored the interactions of endogenous bait proteins, recruited by quantum dots, with fluorescently tagged prey. With this approach, we analyzed the rapid physiological regulation of protein kinase A.

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Figure 1: Quantum dot–based visual immunoprecipitation (QD-VIP).
Figure 2: Optimizing the temporal resolution of QD-VIP by multiparametric analysis and mrFCS.
Figure 3: Monitoring PKA response to physiological stimulations.

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Acknowledgements

This work was supported by the European Molecular Biology Organization (fellowship to E.Z.) and the Federation of European Biochemical Societies (fellowship to P.H.M.L.). H.E.G. and P.H.M.L. were also supported by the Center for Systems Biology co-financed by European Regional Development Fund and the state of North-Rhine Westphalia. E.Z. was also supported by the EU integrated project grant “Interaction Proteome”. Part of the work was conducted at the Cell Biology and Biophysics Unit, European Molecular Biology Laboratory. We thank M. Zaccolo (Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, Padova) for the PKA-Cα-EYFP plasmid, S.S. Taylor and R.Y. Tsien (University of California, San Diego) for the mGFP-PKA-RIα plasmid, S. Gentz and C.F. Becker (Max Planck Institute of Molecular Physiology, Dortmund) for the synthesis of the RIAD peptide, M.A. Hink for helpful discussions and A. Krämer for help with manuscript preparation.

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Contributions

E.Z., P.H.M.L. and P.I.H.B. devised the method. E.Z. and P.H.M.L. performed the experiments and analyzed the data. E.Z., P.H.M.L., A.K. and H.E.G. developed the analysis. E.Z., P.H.M.L. and P.I.H.B. wrote the paper.

Corresponding author

Correspondence to Philippe I H Bastiaens.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4, Supplementary Tables 1–2, Supplementary Notes 1–2 (PDF 2918 kb)

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Zamir, E., Lommerse, P., Kinkhabwala, A. et al. Fluorescence fluctuations of quantum-dot sensors capture intracellular protein interaction dynamics. Nat Methods 7, 295–298 (2010). https://doi.org/10.1038/nmeth.1441

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