In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays
- Resource Type
- Authors
- Philippe I. H. Bastiaens; Jian Hou; Hernán E. Grecco; Dina C. Truxius; Thomas Frahm; Andreas Girod; Rainer Pepperkok; Pedro Roda-Navarro; Anthony Squire
- Source
- Nature methods. 7(6)
- Subject
- Fluorescence-lifetime imaging microscopy
Cell
Protein tyrosine phosphatase
Biology
Biochemistry
chemistry.chemical_compound
Cell Line, Tumor
medicine
Fluorescence Resonance Energy Transfer
Humans
Protein phosphorylation
Phosphorylation
Molecular Biology
Epidermal Growth Factor
Tyrosine phosphorylation
Cell Biology
Protein-Tyrosine Kinases
Phosphoproteins
Cell biology
ErbB Receptors
Förster resonance energy transfer
medicine.anatomical_structure
chemistry
Microscopy, Fluorescence
Tyrosine
Protein Tyrosine Phosphatases
Tyrosine kinase
Protein Processing, Post-Translational
Biotechnology
- Language
- ISSN
- 1548-7105
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.