Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP/sfGFP and mVenus/mKOκ fluorescent protein pairs

Su, Ting; Pan, Shaotao; Luo, Qingming

doi:10.1016/j.bios.2013.02.024

Cited by 23 publications

(15 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Commonly, FRET is measured by the fluorescence intensity ratio of the acceptor to the donor. In that case, whatever the two fluorescent protein FRET pairs chosen, CFP/YFP and mOrange/mCherry21, mTFP1/mCitrine and mAmetrine/tdTomato2223, mTagBFP/sfGFP and mVenus/mKok24, the multiplexed approach suffers from two limitations: (i) a spectral bleed-through of the first acceptor in the second donor emission band that depends directly on the respective quantities of the two biosensors and (ii) the multiple excitation wavelength which requires sequential acquisition, and thus is not adequate to follow fast signal dynamics or signal changes in highly motile sample.…”

Section: Discussionmentioning

confidence: 99%

“…Multi-parameter biosensing experiments have become essential to correlate biochemical activities during a dedicated cellular process. A very exciting challenge has thus been to follow several FRET biosensors in the same sample at the same time and location2021222324. Although simultaneous recording of multiple cellular events was managed, the multiplex FRET biosensors approaches suffer from two limitations: (i) a spectral bleed-through of the first acceptor in the second donor emission band dependent on biosensors concentration, and (ii) the multiple excitation wavelengths necessitate sequential acquisitions that are not optimal for simultaneous observation of several biosensors.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Déméautis

Sipieter

Roul

et al. 2017

Sci Rep

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Déméautis

Sipieter

Roul

et al. 2017

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Commonly, FRET is measured by the fluorescence intensity ratio of the acceptor to the donor. In that case, whatever the two fluorescent protein FRET pairs chosen, CFP/YFP and mOrange/mCherry [42], mTFP1/mCitrine and mAmetrine/tdTomato [43,44], mTagBFP/sfGFP and mVenus/ mKok [45], the multiplex approach suffers from two limitations: (i) a spectral bleed-through of the first acceptor in the second donor emission band that depends directly on the respective quantities of the two biosensors and (ii) the multiple excitation wavelength which requires sequential acquisition that does not adequately follow fast signal dynamics or signal changes in highly motile samples.…”

Section: New Methodological Insights For Multiplexing Kinase Biosensorsmentioning

confidence: 99%

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

Sizaire¹

2017

Protein Phosphorylation

View full text Add to dashboard Cite

Fluorescence microscopy is widely used in biology to localize, to track, or to quantify proteins in single cells. However, following particular events in living cells with good spatio-temporal resolution is much more complex. In this context, Forster resonance energy transfer (FRET) biosensors are tools that have been developed to monitor various events such as dimerization, cleavage, elasticity, or the activation state of a protein. In particular, genetically encoded FRET biosensors are strong tools to study mechanisms of activation and activity of a large panel of kinases in living cells. Their principles are based on a conformational change of a genetically encoded probe that modulates the distance between a pair of fluorescent proteins leading to FRET variations. Recent advances in fluorescence microscopy such as fluorescence lifetime imaging microscopy (FLIM) have made the quantification of FRET efficiency easier. This review aims to address the different kinase biosensors that have been developed, how they allow specific tracking of the activity or activation of a kinase, and to give an overview of the future challenging methods to simultaneously track several biosensors in the same system.

show abstract

“…More recently, FRET biosensors to probe kinase activities have been engineered with different AFP couples such as Venus, mTurquoise, Clover and mRuby, For example, Su, et.al developed a Src sensor based on mVenus/mKO FRET pair [125]. Moreover, since many kinases often exhibit cross-talk within a given signaling pathway and perform their functions in a concerted fashion or in interactive feedback loops, in response to activation of another kinase or enzymatic regulator, there is a growing interest to monitor several kinase activities simultaneously.…”

Section: Probing Protein Kinase Activities In Living Cells With Gementioning

confidence: 99%

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

González‐Vera

Morris

2015

Proteomes

View full text Add to dashboard Cite

Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.

show abstract

Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP/sfGFP and mVenus/mKOκ fluorescent protein pairs

Cited by 23 publications

References 21 publications

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

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

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

Contact Info

Product

Resources

About