title: High-Content Imaging Platform for Profiling Intracellular Signaling Network Activity in Living Cells.
abstract: Essential characteristics of cellular signaling networks include a complex interconnected architecture and temporal dynamics of protein activity. The latter can be monitored by Förster resonance energy transfer (FRET) biosensors at a single-live-cell level with high temporal resolution. However, these experiments are typically limited to the use of a couple of FRET biosensors. Here, we describe a FRET-based multi-parameter imaging platform (FMIP) that allows simultaneous high-throughput monitoring of multiple signaling pathways. We apply FMIP to monitor the crosstalk between epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor signaling, signaling perturbations caused by pathophysiologically relevant EGFR mutations, and the effects of a clinically important MEK inhibitor (selumetinib) on the EGFR network. We expect that in the future the platform will be applied to develop comprehensive models of signaling networks and will help to investigate the mechanism of action as well as side effects of therapeutic treatments.
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Kuchenov
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany.
authors.forename: Vibor
authors.initials: V
authors.lastname: Laketa
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Stein
authors.affiliation: Division of Systems Biology of Signal Transduction, Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; Translational Lung Research Center (TLRC), Member of the German Center for Lung Research (DZL), 69120 Heidelberg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Salopiata
authors.affiliation: Division of Systems Biology of Signal Transduction, Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; Translational Lung Research Center (TLRC), Member of the German Center for Lung Research (DZL), 69120 Heidelberg, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Klingmüller
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany; Translational Lung Research Center (TLRC), Member of the German Center for Lung Research (DZL), 69120 Heidelberg, Germany; Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97201, USA. Electronic address: schultz@embl.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Schultz
keywords: FRET; fluorescence microscopy; growth factors; kinases; principal component analysis; sensors; signaling cross-talk