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

• Published in: Nature methods Vol. 7; no. 6; pp. 467 - 472
• Main Authors: Grecco, Hernán E, Frahm, Thomas, Hou, Jian, Truxius, Dina C, Bastiaens, Philippe I H, Roda-Navarro, Pedro, Squire, Anthony, Girod, Andreas, Pepperkok, Rainer
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.06.2010; Nature Publishing Group
• Subjects: 631/114/2391; 631/1647/245/2225; 631/1647/527/2047; 631/80/458/1733; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Cell Line, Tumor; Cellular biology; Energy transfer; Epidermal Growth Factor - pharmacology; Fluorescence; Fluorescence Resonance Energy Transfer; Humans; Life Sciences; Microscopy; Microscopy, Fluorescence - methods; Phosphoproteins - analysis; Phosphorylation; Physiological aspects; Post-translational modification; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases - metabolism; Protein-Tyrosine Kinases - metabolism; Proteins; Proteomics; Receptor, Epidermal Growth Factor - metabolism; Research methodology; Resonance; Signal transduction; Tyrosine; Tyrosine - metabolism; Greece; Germany; Finland
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth.1458

Combining reverse transfection of protein tyrosine kinase substrates on cell arrays with fluorescence resonance energy transfer (FRET) measurements by fluorescence lifetime imaging microscopy (FLIM) allows quantitative assessment of phosphorylation patterns and identification of feedback loops at single-cell resolution. 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.