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Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

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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.
Keywords
RESONANCE ENERGY-TRANSFER, GROWTH-FACTOR RECEPTOR, ACTIVATED, PROTEIN-KINASE, SIGNAL-REGULATED KINASE, LARGE STOKES SHIFT, FLUORESCENT, PROTEIN, MAP KINASE, A ACTIVITY, CYCLIC-AMP, ERK ACTIVITY

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MLA
Demeautis, Claire et al. “Multiplexing PKA and ERK1&2 Kinases FRET Biosensors in Living Cells Using Single Excitation Wavelength Dual Colour FLIM.” SCIENTIFIC REPORTS 7 (2017): n. pag. Print.
APA
Demeautis, C., Sipieter, F., Roul, J., Chapuis, C., Padilla-Parra, S., Riquet, F., & Tramier, M. (2017). Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM. SCIENTIFIC REPORTS, 7.
Chicago author-date
Demeautis, Claire, François Sipieter, Julien Roul, Catherine Chapuis, Sergi Padilla-Parra, Franck Riquet, and Marc Tramier. 2017. “Multiplexing PKA and ERK1&2 Kinases FRET Biosensors in Living Cells Using Single Excitation Wavelength Dual Colour FLIM.” Scientific Reports 7.
Chicago author-date (all authors)
Demeautis, Claire, François Sipieter, Julien Roul, Catherine Chapuis, Sergi Padilla-Parra, Franck Riquet, and Marc Tramier. 2017. “Multiplexing PKA and ERK1&2 Kinases FRET Biosensors in Living Cells Using Single Excitation Wavelength Dual Colour FLIM.” Scientific Reports 7.
Vancouver
1.
Demeautis C, Sipieter F, Roul J, Chapuis C, Padilla-Parra S, Riquet F, et al. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM. SCIENTIFIC REPORTS. 2017;7.
IEEE
[1]
C. Demeautis et al., “Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM,” SCIENTIFIC REPORTS, vol. 7, 2017.
@article{8543763,
  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.},
  articleno    = {41026},
  author       = {Demeautis, Claire and Sipieter, François and Roul, Julien and Chapuis, Catherine and Padilla-Parra, Sergi and Riquet, Franck and Tramier, Marc},
  issn         = {2045-2322},
  journal      = {SCIENTIFIC REPORTS},
  keywords     = {RESONANCE ENERGY-TRANSFER,GROWTH-FACTOR RECEPTOR,ACTIVATED,PROTEIN-KINASE,SIGNAL-REGULATED KINASE,LARGE STOKES SHIFT,FLUORESCENT,PROTEIN,MAP KINASE,A ACTIVITY,CYCLIC-AMP,ERK ACTIVITY},
  language     = {eng},
  pages        = {14},
  title        = {Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM},
  url          = {http://dx.doi.org/10.1038/srep41026},
  volume       = {7},
  year         = {2017},
}

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