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Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal t...
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... change improved the dynamic range of the biosensor from 38% to 58% in ratio experiments [18]. Therefore, we used the same kind of procedure on EKAR and exchanged the Venus FP with its variant Cp172 Venus, to obtain a first construction named EKAR-Cer-CpV, including the Cerulean/cpVenus FRET pair ( Figure 1A). T EPAC VV is a cAMP level FRET biosensor that exhibits a remarkable dynamic range. ...
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... donor fluorophore is characterized by a phase lifetime of 3.7 ns (instead of 2.3 for the Cerulean) [20,21], which improves FLIM measures, while the use of an acceptors dimer also improves ratiometric experiments. We thus generated a second construction based on this pair and named it EKAR-TVV ( Figure 1A). ...
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... difference between the basal ratio values corresponding to starved cells and the ratio values following EGF stimulation corresponded to the dynamic range of the biosensors. In order to compare the results among themselves, each value obtained over time for every single cell, was normalized on the average of baseline values ( Figure 1C,D). When compared to EKAR, our two constructions exhibited an increased dynamic range as shown by the normalized YFP/CFP ratios after EGF stimulation ( Figure 1C). ...
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... order to compare the results among themselves, each value obtained over time for every single cell, was normalized on the average of baseline values ( Figure 1C,D). When compared to EKAR, our two constructions exhibited an increased dynamic range as shown by the normalized YFP/CFP ratios after EGF stimulation ( Figure 1C). To highlight those changes the average of baseline values was subtracted to the average of the values upon EGF stimulation and was depicted in Figure 1E. ...
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... compared to EKAR, our two constructions exhibited an increased dynamic range as shown by the normalized YFP/CFP ratios after EGF stimulation ( Figure 1C). To highlight those changes the average of baseline values was subtracted to the average of the values upon EGF stimulation and was depicted in Figure 1E. EKAR-TVV in particular, revealed a 21.5% FRET increase upon EGF stimulation, when EKAR-Cer-CpV and EKAR reached respectively 19.7% and 14.2% ( Figure 1C,E). ...
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... highlight those changes the average of baseline values was subtracted to the average of the values upon EGF stimulation and was depicted in Figure 1E. EKAR-TVV in particular, revealed a 21.5% FRET increase upon EGF stimulation, when EKAR-Cer-CpV and EKAR reached respectively 19.7% and 14.2% ( Figure 1C,E). ...
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... previously described, cells were starved and then stimulated with EGF, and images were acquired every minute. Curves thus represented the lifetime variation to the average of baseline values ( Figure 1F,G). The difference between baseline values and EGF values was also represented for each biosensor ( Figure 1H). ...
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... thus represented the lifetime variation to the average of baseline values ( Figure 1F,G). The difference between baseline values and EGF values was also represented for each biosensor ( Figure 1H). EKAR-TVV's dynamic range was higher than EKAR's (160 ps and 60 ps respectively) whereas there was no significant improvement with EKAR-Cer-CpV. ...
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... previously reported MAPK sensor EKAREV [16] was also an improved version of EKAR notably due to the modification of a linker within the probe. We thus choose to combine this modification with the most efficient FRET pair to further increase the biosensors dynamic range ( Figure 1B). This third construction, named EKAREV-TVV, did not show improvement compared to EKAR or EKAREV using ratiometric techniques (Figure 1D,E). ...
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... thus choose to combine this modification with the most efficient FRET pair to further increase the biosensors dynamic range ( Figure 1B). This third construction, named EKAREV-TVV, did not show improvement compared to EKAR or EKAREV using ratiometric techniques (Figure 1D,E). Conversely, it strongly improved the dynamic ranges in lifetime-based experiments compared to EKAREV (192 ps and 100 ps respectively). ...
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... it strongly improved the dynamic ranges in lifetime-based experiments compared to EKAREV (192 ps and 100 ps respectively). Moreover EKAREV-TVV overrided EKAR-TVV performance (160 ps), making it our best construction for lifetime experiments ( Figure 1G,H). . Design and properties of ERK biosensors EKAR-Cer/CpV, EKAR-TVV and EKAREV-TVV. Figure 1A,B illustrates the design of new ERK biosensors. ...
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... and properties of ERK biosensors EKAR-Cer/CpV, EKAR-TVV and EKAREV-TVV. Figure 1A,B illustrates the design of new ERK biosensors. ERK DD corresponds to an ERK docking domain (amino acid sequence: FQFP). ...
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... and EKAR-TVV derive respectively from EKAR [14] (A) and EKAREV-TVV derives from EKAREV [16] (B). Figure 1C,D shows the YFP/CFP ratio variations upon EGF stimulation in starved HeLa cells. EKAR-Cer/CpV (n = 43 cells) and EKAR-TVV (n = 23 cells) are compared to EKAR (n = 24 cells) (C). ...
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... symbols represent a p value ≤0.01 and three symbols represent a p value ≤0.001 when compared to EKAR (circles) or EKAREV (stars). Figure 1F,G shows the fluorescence lifetime variations upon EGF stimulation in starved HeLa cells. EKAR-Cer/CpV (n = 6 cells) and EKAR-TVV (n = 8 cells) are compared to EKAR (n = 7 cells) (F). ...
Citations
... There have been multiple improvements to EKAR since its initial inception [12], including changes to the linker length [13], usage of brighter and more pH-stable CFP and YFP variants [14], and changes in FP position. Recent iterations of EKAR, EKAREN4 and EKAREN5, addressed the non-specific phosphorylation of the biosensor by CDK1, which causes non-ERK specific signals in G2/M phases of the cell cycle [15]. ...
... Accordingly, a more general form of the FRET measurement model was necessary to account for spectral cross-talk terms. Working from the imaging system model presented in the "FRET reporter measurement and correction" section of the appendix to [14] (eqns. 1-3), we model the intensity ratio of the FRET720 imaging channel to the RFP670 channel as: ...
Cell fate decisions are regulated by intricate signaling networks, with Extracellular signal-Regulated Kinase (ERK) being a central regulator. However, ERK is rarely the only signaling pathway involved, creating a need to study multiple signaling pathways simultaneously at the single-cell level. Many existing fluorescent biosensors for ERK and other pathways have significant spectral overlap, limiting their ability to be multiplexed. To address this limitation, we developed two novel red-FRET ERK biosensors, REKAR67 and REKAR76, which operate in the 670-720 nm range using miRFP670nano3 and miRFP720. REKAR67 and REKAR76 differ in fluorophore position, which impacts biosensor characteristics; REKAR67 displayed a higher dynamic range but greater signal variance than REKAR76. Mixed populations of REKAR67 or REKAR76 displayed similar Signal-to-Noise ratio (SNR), but in clonal cell populations, REKAR76 had a significantly higher SNR. Overall, our red-FRET ERK biosensors were highly consistent with existing ERK FRET biosensors and in reporting ERK activity and are spectrally compatible with CFP/YFP FRET and cpGFP -based biosensors. Both REKAR biosensors expand the available methods for measuring single-cell ERK activity.
... For example, the FRET reporter EKAR3 shows greater sensitivity than ERK-KTR to small ERK activity changes but saturates easily [51]. While the dynamic range of FRET-based reporters has increased [48,53], a head-to-head comparison between the newest FRET reporters and translocation reporters to assess their relative advantages has not yet been performed. Altogether, these differences emphasize the caveat that the amplitude of ERK reporter signals must be interpreted with caution and not as an absolute linear measurement. ...
Extracellular signal-regulated kinase (ERK) has long been studied as a key driver of both essential cellular processes and disease. A persistent question has been how this single pathway is able to direct multiple cell behaviors, including growth, proliferation, and death. Modern biosensor studies have revealed that the temporal pattern of ERK activity is highly variable and heterogeneous, and critically, that these dynamic differences modulate cell fate. This two-part review discusses the current understanding of dynamic activity in the ERK pathway, how it regulates cellular decisions, and how these cell fates lead to tissue regulation and pathology. In part 1, we cover the optogenetic and live-cell imaging technologies that first revealed the dynamic nature of ERK, as well as current challenges in biosensor data analysis. We also discuss advances in mathematical models for the mechanisms of ERK dynamics, including receptor-level regulation, negative feedback, cooperativity, and paracrine signaling. While hurdles still remain, it is clear that higher temporal and spatial resolution provide mechanistic insights into pathway circuitry. Exciting new algorithms and advanced computational tools enable quantitative measurements of single-cell ERK activation, which in turn inform better models of pathway behavior. However, the fact that current models still cannot fully recapitulate the diversity of ERK responses calls for a deeper understanding of network structure and signal transduction in general.
... EKAR is composed of a YFP, WW domain, Gly linker, ERK phosphorylated peptide containing ERK docking motif, and CFP, and exhibits an increase in FRET by the phosphorylation (Fig. 2B). Following the initial report of EKAR, several researchers have reported improvements of this biosensor (Fig. 2C), either by using a long flexible linker and dimerization-prone fluorescent protein pair (EKAREV) (Komatsu et al., 2011), or optimization of fluorescent proteins and the order of domains (EKAR2G, EKAR-TVV, EKAR3, EKAR4) (Fritz et al., 2013;Vandame et al., 2014;Sparta et al., 2015;Keyes et al., 2020). These improvements have significantly enhanced the gain of the FRET signal, allowing more researchers to easily employ the FRET biosensors. ...
The extracellular signal-regulated kinase (ERK) pathway governs cell proliferation, differentiation and migration, and therefore plays key roles in various developmental and regenerative processes. Recent advances in genetically encoded fluorescent biosensors have unveiled hitherto unrecognized ERK activation dynamics in space and time and their functional importance mainly in cultured cells. However, ERK dynamics during embryonic development have still only been visualized in limited numbers of model organisms, and we are far from a sufficient understanding of the roles played by developmental ERK dynamics. In this Review, we first provide an overview of the biosensors used for visualization of ERK activity in live cells. Second, we highlight the applications of the biosensors to developmental studies of model organisms and discuss the current understanding of how ERK dynamics are encoded and decoded for cell fate decision-making.
... Testing two of the various versions of ERK1/2 FRET biosensors, EKAR-EV (Komatsu et al., 2011) and EKAR-EV-TVV (Vandame et al., 2014) in HeLa and L929 cells ( Figures 3B and S4A), we observed that the ratio decrease reported by these biosensors upon ERK1/2 inhibition was relatively slow, and therefore poorly efficient in reporting ERK1/2 activity decrease (i.e., biosensor dephosphorylation), as already mentioned in the literature (Regot et al., 2014). We reasoned that the obligated linker composition required for KAR engineering (Table S1) resulting from the recombination cloning method (MultiSite Gateway Technology) (Jones et al., 2014) might alleviate this shortcoming by adding flexibility. ...
... At the time, a comparison in HeLa cells of EKAR4.0 to others FRET biosensors for monitoring ERK1/2 activity from the same design, except EKAR3 (Sparta et al., 2015), showed EKAR4.0 to overall perform better ( Figures S6A-S6G). Of note, when compared to EKAR-EV (Komatsu et al., 2011) and t-EKAR-EV-vv (Vandame et al., 2014), EKAR4.0 also performed better in L929 cells ( Figure S4A). ...
ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.
... AC-activation sensors are available [309], as well as sensors monitoring fluctuations of intracellular second messengers like cAMP [310,311], IP1 [312] and calcium [313,314]. Additionally, there are FRET sensors available for further downstream effectors such as ERK [315,316]. ...
Cyclic adenosine monophosphate (cAMP), the ubiquitous second messenger produced upon stimulation of GPCRs which couple to the stimulatory GS protein, orchestrates an array of physiological processes including cardiac function, neuronal plasticity, immune responses, cellular proliferation and apoptosis. By interacting with various effector proteins, among others protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), it triggers signaling cascades for the cellular response. Although the functional outcomes of GSPCR-activation are very diverse depending on the extracellular stimulus, they are all mediated exclusively by this single second messenger. Thus, the question arises how specificity in such responses may be attained. A hypothesis to explain signaling specificity is that cellular signaling architecture, and thus precise operation of cAMP in space and time would appear to be essential to achieve signaling specificity. Compartments with elevated cAMP levels would allow specific signal relay from receptors to effectors within a micro- or nanometer range, setting the molecular basis for signaling specificity. Although the paradigm of signaling compartmentation gains continuous recognition and is thoroughly being investigated, the molecular composition of such compartments and how they are maintained remains to be elucidated. In addition, such compartments would require very restricted diffusion of cAMP, but all direct measurements have indicated that it can diffuse in cells almost freely. In this work, we present the identification and characterize of a cAMP signaling compartment at a GSPCR. We created a Förster resonance energy transfer (FRET)-based receptor-sensor conjugate, allowing us to study cAMP dynamics in direct vicinity of the human glucagone-like peptide 1 receptor (hGLP1R). Additional targeting of analogous sensors to the plasma membrane and the cytosol enables assessment of cAMP dynamics in different subcellular regions. We compare both basal and stimulated cAMP levels and study cAMP crosstalk of different receptors. With the design of novel receptor nanorulers up to 60nm in length, which allow mapping cAMP levels in nanometer distance from the hGLP1R, we identify a cAMP nanodomain surrounding it. Further, we show that phosphodiesterases (PDEs), the only enzymes known to degrade cAMP, are decisive in constraining cAMP diffusion into the cytosol thereby maintaining a cAMP gradient. Following the discovery of this nanodomain, we sought to investigate whether downstream effectors such as PKA are present and active within the domain, additionally studying the role of A-kinase anchoring proteins (AKAPs) in targeting PKA to the receptor compartment. We demonstrate that GLP1-produced cAMP signals translate into local nanodomain-restricted PKA phosphorylation and determine that AKAP-tethering is essential for nanodomain PKA. Taken together, our results provide evidence for the existence of a dynamic, receptor associated cAMP nanodomain and give prospect for which key proteins are likely to be involved in its formation. These conditions would allow cAMP to exert its function in a spatially and temporally restricted manner, setting the basis for a cell to achieve signaling specificity. Understanding the molecular mechanism of cAMP signaling would allow modulation and thus regulation of GPCR signaling, taking advantage of it for pharmacological treatment.
... Walking down the G protein-dependent signaling cascade, numerous RET-based sensors are validated to detect the activation of adenylyl cyclases (Ritt and Sivaramakrishnan, 2016) or fluctuations of intracellular second-messenger levels like cAMP (Klarenbeek et al., 2015;Nikolaev et al., 2004), IP1 and calcium (Evanko and Haydon, 2005;Mank et al., 2006). Furthermore, RET sensors of more distal effector proteins like extracellular-signal regulated kinase (ERK) enable monitoring their regulation in realtime in living cells (Harvey et al., 2008;Vandame et al., 2014). On the side of the different GPCR-related signaling pathways, many RET systems have been developed to monitor G protein-independent events as for instance receptor oligomerization (Cottet et al., 2011) and internalization (Namkung et al., 2016). ...
G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor’s conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function.
... Une diminution de la mesure de FRET est donc un indicateur de l'activité protéase des enzymes.Plus récemment, des biosenseurs sont développés afin d'être utilisés en multiplex, qui est la possibilité de suivre simultanément ou quasi simultanément plusieurs biosenseurs dans un même système vivant(Demeautis et al., 2017). Pour cela, un biosenseur ERK a été généré à partir du biosenseur EKAR(Fritz et al., 2013;Vandame et al., 2014), en utilisant la paire de fluorphores mTFP1/shadowG. ShadowG est un bon accepteur FRET mais considéré « dark » car il n'émet quasiment pas de fluorescence(Murakoshi et al., 2015). ...
La surexpression d’Aurora A est un marquer majeur de certains cancers épithéliaux. Ce gène code pour la kinase multifonctionnelle Aurora A et son activation est requise pour l’entrée et la progression vers la mitose. Jusqu'à présent, aucun inhibiteur de cet oncogène n'a été approuvé par la FDA et il est donc primordial d'identifier de nouvelles molécules. Notre équipe a développé un biosenseur FRET (Forster Resonance Energy Transfer) pour l’activité kinase d’Aurora A, constitué de la kinase entière flanquée de deux fluorophores, une GFP et une mCherry. Le changement de conformation d’Aurora A lorsqu’elle est activée rapproche les fluorophores et augmente l’efficacité du FRET. Il est ainsi possible de suivre l’activation d’Aurora A dans les cellules vivantes exprimant le biosenseur à des niveaux endogènes. Nous pouvons mesurer le FRET en utilisant la technique de FLIM (Fluorescence Lifetime Imaging Microscopy) grâce à un microscope développé dans l’équipe et appelé fastFLIM. Mes travaux de thèse ont consisté à développer une stratégie de criblage robuste et automatisée en combinant les capacités du fastFLIM et le biosenseur d’activité d’Aurora A. Cette stratégie basée sur une automatisation des acquisitions et de l’analyse de données a permis de cribler une banque de molécules en plaque 96 puits afin de trouver de potentielles inhibiteurs de l’activité kinase d’Aurora A. De plus, j’ai participé à la validation du biosenseur pour un suivi de l’activité kinase dans des cellules vivantes en montrant que les variations de FRET mesurées correspondent bien à l’état de phosphorylation d’Aurora A sur le résidu Thréonine 288, marqueur de son activation. Enfin, j’ai participé à l’élaboration de nouvelles techniques de microscopie pour suivre l’activité du biosenseur. Pour cela, j’ai utilisé un biosenseur de type homoFRET avec l’enjeu de pouvoir utiliser plusieurs biosenseurs dans un contexte multiplex. J’ai aussi utilisé la technique de 2c-FCCS (2-colors Fluorescence Cross Correlation Spectroscopy) sur le biosenseur Aurora A afin de pouvoir mesurer le FRET dans des régions où celui-ci est faiblement exprimant et dont la mesure de durée de vie de fluorescence n’est pas possible par le FLIM. Ainsi, mes travaux de thèse s’inscrivent dans la tendance à développer une microscopie quantitative et autonome avec comme enjeu d’apporter un grande nombre de données phénotypiques.
... K2α: RS-K2α FR ◆ (2013) 406 RY-K2α FR ◆ (2013) 406 YC-K2α FR ◆ (2013) 406 CaN FRET ( FR ◆) CaN activation: YC-CaN FR ◆ (2013) 406 RY-CaN FR ◆ (2013) 406 CaNAR: CaNAR2 FR ◆ (2014) 459 CaNAR1 FR ◆ (2008) 460 467 RAB-EKARev I ☼ (2018) 211 EKARet FL ◇ (2017) 468 EKAR FL ◇ (2017) 469 bimEKAR FR ◆ (2015) 444 EKAR3 FR ◆ (2015) 470 EKAREV-TVV FR ◆ (2014) 471 EKAR-TVV FR ◆ (2014) 471 REV BR ◓ (2013) 472 EKAR2G FR ◆ (2013) 473 EKAREV FR ◆ (2011) 441 467 NIR AKAR FR ◆ (2018) 489 AKARet FL ◇ (2017) 468 For each signaling target, the different published biosensors are organized into families of related variants (with the family or group name shown in bold). The different read-out mechanisms utilized are shown with the different icons indicating the type of readout for a given biosensor. ...
... K2α: RS-K2α FR ◆ (2013) 406 RY-K2α FR ◆ (2013) 406 YC-K2α FR ◆ (2013) 406 CaN FRET ( FR ◆) CaN activation: YC-CaN FR ◆ (2013) 406 RY-CaN FR ◆ (2013) 406 CaNAR: CaNAR2 FR ◆ (2014) 459 CaNAR1 FR ◆ (2008) 460 467 RAB-EKARev I ☼ (2018) 211 EKARet FL ◇ (2017) 468 EKAR FL ◇ (2017) 469 bimEKAR FR ◆ (2015) 444 EKAR3 FR ◆ (2015) 470 EKAREV-TVV FR ◆ (2014) 471 EKAR-TVV FR ◆ (2014) 471 REV BR ◓ (2013) 472 EKAR2G FR ◆ (2013) 473 EKAREV FR ◆ (2011) 441 467 NIR AKAR FR ◆ (2018) 489 AKARet FL ◇ (2017) 468 For each signaling target, the different published biosensors are organized into families of related variants (with the family or group name shown in bold). The different read-out mechanisms utilized are shown with the different icons indicating the type of readout for a given biosensor. ...
Cellular signaling networks are the foundation which determines the fate and function of cells as they respond to various cues and stimuli. The discovery of fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we endeavored to assemble a comprehensive list of published engineered biosensors, and we discuss many of the molecular designs utilized in their development. Then, we review how the high temporal and spatial resolution afforded by fluorescent biosensors has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research in both biosensor design and applications that are on the forefront of biosensor development.
... The need to understand MAPK signaling activation in normal and disease states has led to the development of live reporters for visualization of kinase activity. Sensors based on Förster resonance energy transfer (FRET) provide great insights by visualizing ERK activity in cultured cells and more recent also in mice and zebrafish, but are difficult to implement and fail to accurately report the downregulation of activity (Vandame et al., 2013;Regot et al., 2014;Depry et al., 2015;Hirata et al., 2015;Hiratsuka et al., 2015;Sari et al., 2018). Regot et al. recently introduced an alternative kinase activity reporter termed kinase translocation reporter (KTR) and demonstrated its high sensitivity in vitro (Regot et al., 2014). ...
Precise regulation of signaling pathways in single cells underlies tissue development, maintenance and repair in multicellular organisms, but our ability to monitor signaling dynamics in living vertebrates is currently limited. We implemented kinase translocation reporter (KTR) technology to create DREKA (“dynamic reporter of Erk activity”) zebrafish, which allow one to observe Erk activity in vivo at single cell level with high temporal resolution. DREKA zebrafish faithfully reported Erk activity after muscle cell wounding and revealed the kinetics of small compound uptake. Our results promise that kinase translocation reporters can be adapted for further applications in developmental biology, disease modeling, and in vivo pharmacology in zebrafish.
... Furthermore, ERKTR has been used to monitor ERK activity in parallel with an another ERK activity -EKAR FRET based ERK reporter (50), and the activity of both is largely identical (51). In addition, a form of ERKTR has been generated to monitor ERK activity during in vivo in C. elegans, and genetic perturbations, that are predicted to downregulate (mpk1 null mutant) or upregulate (Lin-45/BRAF expression) ERK activity result in predicted effects on ERK activity (52). ...
Activating BRAF mutations are thought to drive melanoma tumorigenesis and metastasis by constitutively activating MEK and ERK. Small molecule inhibitors (SMIs) of BRAF or MEK have shown promise as melanoma therapeutics. However, the development of resistance to these inhibitors in both the short-and long-term is common; warranting investigation into how these SMIs influence ERK signaling dynamics. Quantitative single cell imaging of ERK activity in living cells reveals both intra-and inter-cell heterogeneity in this activity in isogenic melanoma populations harboring a BRAFV600E mutation. This heterogeneity is largely due to a cell-cycle dependent bifurcation of ERK activity. Moreover, we show there are also cell-cycle dependent responses in ERK activity following BRAF or MEK inhibition. Prior to, but not following, CDK4/6-mediated passage through the Restriction Point (RP) ERK activity is sensitive to BRAF and MEK inhibitors. In contrast, for cells that have passed the RP, ERK activity will remain elevated in the presence of BRAF or MEK inhibition until mitosis. Our results show that ERK activity-even in the presence of activating BRAF mutations-is regulated by both positive and negative feedback loops that are engaged in cell-cycle dependent fashions. CDK4/6 inhibition sensitizes ERK activity to BRAF or MEK inhibition by preventing passage the transition from a BRAF/MEK dependent to independent state. Our results have implications for the use of MEK and BRAF inhibitors as melanoma therapeutics, and offer a rational basis for the use of these inhibitors in combination with CDK4/6 inhibition during cancer therapy.
