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In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays

Nature Methods volume 7, pages 467–472 (2010)Cite this article

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Abstract

Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor.

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Figure 1: Overview of CA-FLIM.
Figure 2: Global analysis of FLIM data accurately quantifies the phosphorylated fraction of proteins.
Figure 3: Identification of protein tyrosine kinase and phosphatase substrates.
Figure 4: Cell-to-cell variation in phosphorylation response reveals positive feedback motifs.
Figure 5: Cluster analysis of EGF-induced tyrosine phosphorylation response.

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Acknowledgements

P.R.-N. was supported by a Marie Curie Intra-European fellowship for career development (FP7-PEOPLE 2007-2-1-EIF). This work was supported by Interaction Proteome (Integrated Project from FP6) and the Centre for Systems Biology in Dortmund (cofinanced by the European Regional Development Fund and the State of North Rhine-Westfalia). S. Dhe-Paganon (University of Toronto) provided the pET28a-LIC-EPHA3c plasmid.

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Author notes

  1. Andreas Girod, Thomas Frahm & Anthony Squire

    Present address: Present addresses: Center for Applied Nanotechnology GmbH, Hamburg, Germany (T.F.), Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece (A.G.) and Molecular Imaging Unit, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland (A.S.).,

  2. Hernán E Grecco, Pedro Roda-Navarro and Andreas Girod: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany

    Hernán E Grecco, Pedro Roda-Navarro, Andreas Girod, Jian Hou, Thomas Frahm, Dina C Truxius & Philippe I H Bastiaens

  2. Cell Biology–Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Andreas Girod, Rainer Pepperkok & Anthony Squire

  3. Chemical Biology Department, Technical University Dortmund, Dortmund, Germany

    Philippe I H Bastiaens

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Contributions

P.I.H.B. devised the method. A.S. and R.P. contributed the initial implementation. H.E.G., P.R.-N. and A.G. designed, supervised, performed and analyzed the experiments. A.S., T.F., D.C.T. and J.H. contributed experiments. A.S. and H.E.G. built the instrument. H.E.G., P.R-N. and P.I.H.B. wrote the paper.

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Correspondence to Philippe I H Bastiaens.

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The authors declare no competing financial interests.

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Supplementary Figures 1–10, Supplementary Tables 1–2, Supplementary Protocols and Supplementary Discussion (PDF 3761 kb)

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Grecco, H., Roda-Navarro, P., Girod, A. et al. In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays. Nat Methods 7, 467–472 (2010). https://doi.org/10.1038/nmeth.1458

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