Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks

GPCRs are seven transmembrane spanning receptors that regulate a wide array of intracellular signaling cascades in response to various stimuli. To do so, they couple to different heterotrimeric G proteins and adaptor proteins, including arrestins.Importantly, arrestins were shown to regulate GPCR signaling through G proteins, as well as promote G protein-independent signaling events. Several research groups have reported successful isolation of exclusively G protein-dependent and arrestindependent signaling downstream of GPCR activation using biased agonists or receptor mutants incapable of coupling to either arrestins or G proteins. In the latter category, the DRY mutant of the angiotensin II type 1 receptor was extensively used to characterize functional selectivity downstream of AT1 A R. In an attempt to understand histamine 1 receptor signaling, we characterized the signaling capacity of the H1R DRY mutant in a panel of dynamic, live cell biosensor assays, including arrestin recruitment, heterotrimeric G-protein activation, Ca 2+ signaling, protein kinase C activity, GTP binding of RhoA, and activation of ERK1/2. Here we show that both H1R DRY mutant and the AT1 A R DRY mutant (used as a reference) are capable of efficient activation of G protein-mediated signaling. Therefore, contrary to common belief, they do not constitute suitable tools for dissection of arrestin-mediated, G proteinindependent signaling downstream of these receptors.

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“…Therefore, our results once again demonstrate the strength of single cell imaging using biosensors (Greenwald et al 2018).…”

Section: Discussionsupporting

confidence: 69%

Not so dry after all – DRY mutants of the AT1_A receptor and H1 receptor can induce G protein-dependent signaling

Pietraszewska-Bogiel

Joosen

Goedhart

2019

Preprint

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“…These networks of pathways are interconnected and integrated, allowing them to coordinate signal transduction. The ability to monitor the activities of different cell signaling pathways inside living cells became possible with the development of the genetically encoded biosensor probes (1)(2)(3)(4). A variety of design strategies have been used in the development these genetically encoded probes, and many rely on the measurement of Förster resonance energy transfer (FRET) to detect the changes in biosensor conformation that accompany the targeted signaling event (4).…”

Section: Introductionmentioning

confidence: 99%

“…The sensing domain serves as a linker between the FPs and includes an element that is modified by the targeted biological event, as well as a binding motif that recognizes that modification. This allows the sensing domain to change its conformation in response to a specific cell-signaling event, altering the distance between the FP pair in the reporter (1)(2)(3)(4). The changing intramolecular FRET signal from these single chain biosensor proteins can be monitored in real time, allowing measurement of the spatiotemporal dynamics of signaling events inside living cells.…”

Section: Introductionmentioning

confidence: 99%

“…5,6). Because FRET-based biosensors rely on sensing units that contain two FPs, multiplexing more than one biosensor in the same living cells is difficult because of the limited spectral space (4). There are mathematical approaches for separating multiple fluorescence signals, but these can be challenging when applied to sensitized emission measurements of FRET (7).…”

Section: Introductionmentioning

confidence: 99%

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PIE-FLIM measurements of two different FRET-based biosensor activities in the same living cells

Day

Reissaus

Day

et al. 2020

Preprint

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We report the use of pulsed interleaved excitation-fluorescence lifetime imaging microscopy (PIE-FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells.Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using fluorescence lifetime imaging microscopy (FLIM). The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.

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“…Genetically-encoded Förster's Resonance Energy Transfer (FRET) biosensors have revolutionised our knowledge of signal transduction pathways in the cell. The capability of sensing the activation of kinases, the activity of caspases, or the transport of second messengers as Ca 2+ or cAMP opened up the possibility of following biochemical reactions in real time and with a spatiotemporal resolution (Sizaire and Tramier, 2017;Greenwald et al, 2018;Palmer et al, 2011). The novel frontier of these probes consists in combining two or more FRET biosensors at a time to unravel the interdependence of signal transduction pathways, an approach known as multiplex FRET (Piljic and Schultz, 2008;Carlson and Campbell, 2009;Ai et al, 2008;Ding et al, 2011;Su et al, 2013;Demeautis et al, 2017;Ross et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Optimised FRET pairs and quantification approaches to detect the activation of Aurora kinase A at mitosis

Bertolin

Sizaire

Déméautis

et al. 2019

Preprint

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Genetically-encoded Förster's Resonance Energy Transfer (FRET) biosensors are indispensable tools to sense the spatiotemporal dynamics of signal transduction pathways. Investigating the crosstalk between different signalling pathways is becoming increasingly important to follow cell development and fate programs. To this end, FRET biosensors must be optimised to monitor multiple biochemical activities simultaneously and in single cells. In addition, their sensitivity must be increased to follow their activation even when the abundance of the biosensor is low. We describe here the development of a second generation of Aurora kinase A/AURKA biosensors. First, we adapt the original AURKA biosensor -GFP-AURKA-mCherry-to multiplex FRET by using dark acceptors as ShadowG or ShadowY. Then, we use the novel superYFP acceptor protein to measure FRET by 2-colour Fluorescence Cross-Correlation Spectroscopy, in cytosolic regions where the abundance of AURKA is extremely low and undetectable with the original AURKA biosensor. These results pave the way to the use of FRET biosensors to follow AURKA activation in conjunction with substrate-based activity biosensors. In addition, they open up the possibility of tracking the activation of small pools of AURKA and its interaction with novel substrates, which would otherwise remain undetectable with classical biochemical approaches.

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Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks

Cited by 392 publications

References 1,025 publications

Not so dry after all – DRY mutants of the AT1_A receptor and H1 receptor can induce G protein-dependent signaling

Not so dry after all – DRY mutants of the AT1_A receptor and H1 receptor can induce G protein-dependent signaling

PIE-FLIM measurements of two different FRET-based biosensor activities in the same living cells

Optimised FRET pairs and quantification approaches to detect the activation of Aurora kinase A at mitosis

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Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks

Cited by 392 publications

References 1,025 publications

Not so dry after all – DRY mutants of the AT1A receptor and H1 receptor can induce G protein-dependent signaling

Not so dry after all – DRY mutants of the AT1A receptor and H1 receptor can induce G protein-dependent signaling

PIE-FLIM measurements of two different FRET-based biosensor activities in the same living cells

Optimised FRET pairs and quantification approaches to detect the activation of Aurora kinase A at mitosis

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Product

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Not so dry after all – DRY mutants of the AT1_A receptor and H1 receptor can induce G protein-dependent signaling

Not so dry after all – DRY mutants of the AT1_A receptor and H1 receptor can induce G protein-dependent signaling