ArticleScientific reports2017
Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM.
Article in Scientific reports, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 28 papers.
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Who cites it
28 citing papers in PubMed, 51 citations in OpenAlex.
- Review
- Red-to-Near-Infrared Fluorescence Resonance Energy Transfer Reporters for Dual Imaging Applications.Small methods · 2026Article
- A Constellation of Fluorescent Biosensors to Illuminate the Galaxy of Protein Kinases.Chembiochem : a European journal of chemical biology · 2026Review
- Hedgehog pathway activity downstream of Smoothened is regulated specifically by basal ciliary PKA.Cellular & molecular biology letters · 2026Article
- Article
- Decoding Molecular Network Dynamics in Cells: Advances in Multiplexed Live Imaging of Fluorescent Biosensors.Biosensors · 2025Review
- Aurora kinase A/AURKA functionally interacts with the mitochondrial ATP synthase to regulate energy metabolism and cell death.Cell death discovery · 2023Article
- A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines.Communications biology · 2021Article
- Genetically encoded sensors towards imaging cAMP and PKA activity in vivo.Journal of neuroscience methods · 2021Review
- Article
- Fluorescent toys 'n' tools lighting the way in fungal research.FEMS microbiology reviews · 2021Article
- Simultaneous readout of multiple FRET pairs using photochromism.Nature communications · 2021Article
- Strategies for Multiplexed Biosensor Imaging to Study Intracellular Signaling Networks.Methods in molecular biology (Clifton, N.J.) · 2021Article
- A Guide to Fluorescence Lifetime Microscopy and Förster's Resonance Energy Transfer in Neuroscience.Current protocols in neuroscience · 2020Review
- Fluorescent Biosensors for Neuronal Metabolism and the Challenges of Quantitation.Current opinion in neurobiology · 2020Review
- Insights into the non-mitotic functions of Aurora kinase A: more than just cell division.Cellular and molecular life sciences : CMLS · 2020Review
- How many photons are needed for FRET imaging?Biomedical optics express · 2020Article
- Optogenetic Techniques for Manipulating and Sensing G Protein-Coupled Receptor Signaling.Methods in molecular biology (Clifton, N.J.) · 2020Review
- Live-cell FLIM-FRET using a commercially available system.Methods in cell biology · 2020Article
- ShadowR: a novel chromoprotein with reduced non-specific binding and improved expression in living cells.Scientific reports · 2019Article
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Authors and funding
7 authors at 4 institutions in 3 countries.
Funding
Abstract
Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.
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