FRET-based Sensor Research Articles

FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d.

FRET-Based Biosensor for Oleic Acid in Nanomolar Range with Quantum Dots As an Energy Donor

A self-assembling sensor for oleic acid has been developed. The sensor consists of a self-assembling fluorescent dye labeled BSA and quantum dots CdSe/ZnS capped with 3-mercaptopropionic acid. The detection limit of the new sensor is 10-1000 nM. The influence of the quantum dot size on the FRET efficiency in the course of the interaction of the sensor system with the analyte has been studied. The pH dependence, aggregation stability. and electrophoretic properties of the sensor have been examined. The data suggest a new approach for the development of nanoscale FRET-based sensors operating effectively due to unique fluorescent properties of quantum dots as well as due to selective protein-ligand interactions.

Calcium signal regulates temperature-dependent transformation of sporozoites in malaria parasite development

The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle.

Depletion of PIP2, Rather than Calcium Rise or PKC Activation, is Responsible for Inhibition of KCNQ2/3 Channels by Gq-Coupled Receptors in TSA201 Cells

Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyses the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) into the second messengers diacylgycerol and inositoltrisphosphate, leading to (i) protein kinase C (PKC) activation, (ii) calcium release, and in some cases (iii) PIP2 depletion. Which of these three events is responsible for the inhibition of KCNQ2/3 (Kv7.2/7.3) potassium channels? We measure in tsA201 cells PKC activity by a FRET-based sensor (CKAR), calcium by Fura4F, PIP2 by PH domains (PH-PLCδ1), and find that activating endogenous purinergic receptors by UTP leads to PKC activation and calcium release, but not PIP2 depletion. With activation of overexpressed M1 muscarinic receptors by 10 µM oxotremorine-M (oxo-M), the amplitude and time course of the CKAR and Fura responses are much like with UTP, but PIP2 is depleted. KCNQ2/3 channels are inhibited only by oxo-M (85%) and not by UTP (<1%), indicating that it is the PIP2 depletion that inhibits KCNQ2/3 in response to oxo-M. PLC activation by UTP can be boosted by overexpressing G-proteins or P2Y2 receptors. Then UTP depletes PIP2 and does inhibit KCNQ2/3 (92%). Overexpression of AKAP79 or CaM-YFP does not increase KCNQ2/3 inhibition by UTP, but with AKAP79, KCNQ2/3 can be partly inhibited (15%) by adding OAG to maximally activate PKC.We conclude that: (1) PLC activation can elicit full-size calcium and PKC responses without depleting PIP2. (2) G-protein abundance regulates downstream effects of purinergic receptors in tsA201 cells. (3) In response to UTP and oxo-M the PIP2 depletion inhibits KCNQ2/3 in tsA201 cells, but PIP2 depletion is not necessary for KCNQ2/3 inhibition under all circumstances.Supported by NIH grant R01 NS08174 and the Human Frontier Science Program Organization

Targeted in Vivo O-GlcNAc Sensors Reveal Discrete Compartment-specific Dynamics during Signal Transduction

β-O-N-acetyl-D-glucosamine (O-GlcNAc) is a post-translational modification involved in a plethora of biological systems ranging from cellular stress to insulin signaling. This modification shares many hallmarks with phosphorylation, including its dynamic cycling onto a host of proteins such as transcription factors, kinases, and phosphatases, and regulation of cellular functions, including cell signaling. Herein, we report the development of an improved genetically based O-GlcNAc FRET sensor and compartmentalized targeted variants for the characterization of the spatiotemporal dynamics of O-GlcNAc. During serum-stimulated signal transduction, rapid increases in O-GlcNAc activity were observed at both the plasma membrane and the nucleus, with a concomitant decrease detected in the cytoplasm. These findings suggest the existence of compartment specific dynamics for O-GlcNAc in response to signal-inducing stimuli, pointing to complex regulation of this modification. In addition, inhibition of the PI3K pathway by wortmannin abolished the O-GlcNAc response, suggesting that the activity observed is modulated downstream of the PI3K pathway. Taken together, our data argues that O-GlcNAc is a rapidly induced component of signaling and that the interplay between O-GlcNAc and kinase signaling may be more akin to the complex relationship between kinase pathways.

Bidirectional Fluorescence Resonance Energy Transfer (FRET) in Mutated and Chemically Modified Yellow Fluorescent Protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Förster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.

FRET-Based Ratiometric Detection System for Mercury Ions in Water with Polymeric Particles as Scaffolds

Mercury pollution is a global problem, and the development of stable and sensitive fluorescent probes for mercury ions in the water phase has long been sought. In this work, a novel fluorescence resonance energy transfer (FRET)-based ratiometric sensor for detecting Hg(2+) in pure water was demonstrated. Polymeric nanoparticles prepared by miniemulsion polymerization of methyl methacrylate and acrylic acid were used as the scaffold for the FRET-based sensor. A hydrophobic fluorescent dye nitrobenzoxadiazolyl derivative (NBD) was embedded in the nanoparticles during the polymerization and used as the donor. A spirolactam rhodamine derivative SRHB-NH(2) was synthesized and then covalently linked onto the particle surface and used as an ion recognition element. The presence of Hg(2+) in the water dispersion of nanoparticles induced the ring-opening reaction of the spirolactam rhodamine moieties and led to the occurrence of the FRET process, affording the nanoparticle system a ratiometric sensor for Hg(2+). The nanoparticle sensor can selectively detect the Hg(2+) in water with the detection limit of 100 nM (ca. 20 ppb). It has been found that the FRET-based system with smaller nanoparticles as the scaffold exhibited higher energy transfer efficiency and was more preferred for the accurate ratiometric detection. Moreover, the FRET-based sensor was applicable in a relatively wide pH range (pH 4-8) in water; thus, this approach may provide a new strategy for ratiometric detection of analytes in environmental and biological applications.

A FRET-based ratiometric sensor for mercury ions in water with multi-layered silica nanoparticles as the scaffold

A FRET system was built for ratiometric sensing of Hg(2+) in water with multilayered silica nanoparticles as the scaffold. This architecture ensures the control over the location of both donor and acceptor and their separation distance within nanoparticles, affording higher energy transfer efficiency and higher signal-to-background ratio for particle-based sensing.

Temporally resolved cAMP monitoring in endothelial cells uncovers a thrombin‐induced [cAMP] elevation mediated via the Ca2+‐dependent production of prostacyclin

The barrier function of the endothelium is controlled by the second messengers Ca2+ and cAMP that differentially regulate the permeability of endothelial cells. The Ca2+-elevating agent thrombin has been demonstrated to increase endothelial permeability and to decrease cAMP levels as detected via enzyme immunoassays. To study the effects of thrombin on cAMP with high temporal resolution, we utilised the FRET-based cAMP sensor Epac1-camps in single intact human umbilical vein endothelial cells (HUVECs). In these cells, thrombin induced a delayed increase in [cAMP], initiating after about 40 s, with maximum cAMP levels after 130 s of thrombin application. This increase of cAMP levels was Ca2+-dependent, but did not require calmodulin (CaM). Pharmacological approaches revealed that phospholipase A2 (PLA2) activity and cyclooxygenase (COX)-mediated synthesis of prostaglandins was required for the thrombin-induced elevation of [cAMP]. Furthermore, preincubation of HUVECs with a prostacyclin-receptor antagonist significantly reduced the thrombin-induced increase in [cAMP]. We conclude that thrombin causes the synthesis of prostacyclin in endothelial cells and that the subsequent stimulation of Gs-coupled prostacyclin receptors then results in an increase in [cAMP].

Visualizing dynamic cytoplasmic forces with a compliance-matched FRET sensor

Mechanical forces are ubiquitous modulators of cell activity but little is known about the mechanical stresses in the cell. Genetically encoded FRET-based force sensors now allow the measurement of local stress in specific host proteins in vivo in real time. For a minimally invasive probe, we designed one with a mechanical compliance matching that of many common cytoskeleton proteins. sstFRET is a cassette composed of Venus and Cerulean linked by a spectrin repeat. The stress sensitivity of the probe was measured in solution using DNA springs to push the donor and acceptor apart with 5-7 pN and this produced large changes in FRET. To measure cytoskeletal stress in vivo we inserted sstFRET into α-actinin and expressed it in HEK and BAEC cells. Time-lapse imaging showed the presence of stress gradients in time and space, often uncorrelated with obvious changes in cell shape. The gradients could be rapidly relaxed by thrombin-induced cell contraction associated with inhibition of myosin II. The tension in actinin fluctuated rapidly (scale of seconds) illustrating a cytoskeleton in dynamic equilibrium. Stress in the cytoskeleton can be driven by macroscopic stresses applied to the cell. Using sstFRET as a tool to measure internal stress, we tested the prediction that osmotic pressure increases cytoskeletal stress. As predicted, hypotonic swelling increased the tension in actinin, confirming the model derived from AFM. Anisotonic stress also produced a novel transient (~2 minutes) decrease in stress upon exposure to a hypotonic challenge, matched by a transient increase with hypertonic stress. This suggests that, at rest, the stress axis of actinin is not parallel to the stress axis of actin and that swelling can reorient actinin to lie more parallel where it can absorb a larger fraction of the total stress. Protein stress sensors are opening new perspectives in cell biology.

The ω-3 fatty acid eicosapentaenoic acid elicits cAMP generation in colonic epithelial cells via a “store-operated” mechanism

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid abundant in fish oil that exerts a wide spectrum of documented beneficial health effects in humans. Because dietary interventions are relatively inexpensive and are widely assumed to be safe, they have broad public appeal. Their endorsement can potentially have a major impact on human health, but hard mechanistic evidence that specifies how these derivatives work at the cellular level is limited. EPA (50 microM) caused a small elevation of cytoplasmic Ca(2+) concentration ([Ca(2+)]) in intact NCM460 human colonic epithelial cells as measured by fura 2 and a profound drop of [Ca(2+)] within the endoplasmic reticulum (ER) of permeabilized cells as monitored by compartmentalized mag-fura 2. Total internal reflection fluorescence microscopy showed that this loss of ER store [Ca(2+)] led to translocation of the ER-resident transmembrane Ca(2+) sensor STIM1. Using sensitive FRET-based sensors for cAMP in single cells, we further found that EPA caused a substantial increase in cellular cAMP concentration, a large fraction of which was dependent on the drop in ER [Ca(2+)], but independent of cytosolic Ca(2+). An additional component of the EPA-induced cAMP signal was sensitive to the phosphodiesterase inhibitor isobutyl methylxanthine. We conclude that EPA slowly releases ER Ca(2+) stores, resulting in the generation of cAMP. The elevated cAMP is apparently independent of classical G protein-coupled receptor activation and is likely the consequence of a newly described "store-operated" cAMP signaling pathway that is mediated by STIM1.

Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca2+. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca2+ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca2+, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks.

Endothelin and Phenylephrine Both Trigger Nuclear IP3 Elevation, but Differ in Ability to Activate Nuclear HDAC5 Export

We previously found that both ET-1 and phenylephrine (PE) induced similar HDAC5 phosphorylation and nuclear export in adult cardiac myocytes (which contributes to hypertrophic signaling). However, ET-1 requires IP3 receptor (IP3R) activation (at the nucleus) and CaM-CaMKII activation to mediate this full effect, while PE does not. That is, IP3R inhibition or CaMKII block do not prevent PE-induced HDAC5 nuclear export, despite the fact that both agonists can activate IP3 production. Here we test whether the apparent IP3-independence of PE signaling is due to a failure of IP3 elevation in the nucleus (i.e. IP3 produced at the plasma membrane may be degraded before reaching the nucleus). Using a nuclear targeted FRET-based IP3 sensor (Fire-1-Nuc) we assessed changes in nuclear [IP3] upon ET-1 and PE application in adult rabbit ventricular myocytes. Both ET-1 and PE induce rapid and robust elevation of nuclear [IP3] reaching an early peak in <1 min. While the ET-1-induced a slightly larger peak nuclear [IP3], the PE-induced rise is more sustained (lasting more than 10 min). These results demonstrate that a PE induces a strong rise in nuclear [IP3], and does not support the hypothesis that PE fails to induce a nuclear IP3 signal (compared to ET-1). We cannot rule out the possibility that the kinetic differences in nuclear [IP3] between these agonists contribute to different downstream signaling. Another explanation is that PE-induced nuclear IP3 is less effective than ET-1-induced IP3 in driving activation of nuclear CaM and CaMKII to phosphorylate HDAC5. That is, an IP3-independent effect of one of these agonists could prevent or promote the ability of IP3 to signal in the nucleus.

“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

BackgroundWhile intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).Methods/Principal FindingsHere we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.ConclusionsThis newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

Cyclodextrin Supramolecular Complex as a Water-Soluble Ratiometric Sensor for Ferric Ion Sensing

Heightened concern for human health and environmental protection has stimulated active research on the potential impact of transition-metal ions and their toxic effects, thus it is very demanding to design transition-metal ion detection methods that are cost-effective, rapid, facile, and applicable to the environmental and biological milieus. In this study, we demonstrated an alternative strategy for constructing a water-soluble FRET-based ratiometric sensor for ferric ion detection by forming a supramolecular beta-cyclodextrin/dye complex. This water-soluble FRET system consists of a dansyl-linked beta-cyclodextrin (beta-CD-DNS) and a spirolactam rhodamine-linked adamantane (AD-SRhB). The dansyl moiety serves as the donor, and the spirolactam-rhodamine B derivative (SRhB) was chosen as a sensitive, selective chemosensor for Fe(III) ions and a very efficient ring-opening reaction induced by Fe(III) generates the long-wavelength rhodamine B fluorophore that can act as the energy acceptor. Moreover, the adamantyl (AD) group, which is known for its capability to form stable host-guest inclusion complexes with beta-CD derivatives, was covalently linked to the spirolactam rhodamine, thus the adamantyl moiety of the ion-recognition element can be anchored inside the CD cavity. In this way, the donor-acceptor separation can be kept within the critical Forster distance; accordingly, energy transfer can take place from the donor (dansyl) to the acceptor (rhodamine derivative/Fe(III) complex), and thus ratiometric detection for Fe(III) in an aqueous medium can be fulfilled. This FRET-based supramolecular sensor can be readily formed via an inclusion process using the donor part and the acceptor part, hence this strategy could afford a robust approach for constructing a wide range of FRET-based water-soluble sensing systems simply by assembling a specifically predesigned donor-linked CD and acceptor-linked adamantane.

Real-time measurements of cAMP production in liveDictyosteliumcells

Cyclic AMP has a crucial role during the entire developmental program of the social amoebae Dictyostelium, acting both as an intracellular second messenger and, when secreted, as a directional cue that is relayed to neighboring cells during chemotaxis. Although significant knowledge about cAMP production in chemotaxing cells has been derived from studies performed on cell populations, cAMP dynamics at the single cell level have not been investigated. To examine this, we used a FRET-based cAMP sensor that possesses high cAMP sensitivity and great temporal resolution. We show the transient profile of cAMP accumulation in live Dictyostelium cells and establish that chemoattractants control intracellular cAMP dynamics by regulating synthesis via the adenylyl cyclase ACA. aca(-) cells show no significant change in FRET response following chemoattractant addition. Furthermore, cells lacking ACB, the other adenylyl cyclase expressed in chemotaxing cells, behave similarly to wild-type cells. We also establish that the RegA is the major phosphodiesterase that degrades intracellular cAMP in chemotaxis-competent cells. Interestingly, we failed to measure intracellular cAMP compartmentalization in actively chemotaxing cells. We conclude that cytosolic cAMP, which is destined to activate PKA, is regulated by ACA and RegA and does not compartmentalize during chemotaxis.

Fluorescence lifetime imaging microscopy of chemotherapy‐induced apoptosis resistance in a syngenic mouse tumor model

During cancer therapy with DNA-damaging drug-agents, the development of secondary resistance to apoptosis can be observed. In the search for novel therapeutic approaches that can be used in these cases, we monitored chemotherapy-induced apoptosis resistance in a syngenic mouse tumor model. For this, syngenic murine colorectal carcinoma cells, which stably expressed a FRET-based caspase-3 activity sensor, were introduced into animals to induce peritoneal carcinomatosis or disseminated hepatic metastases. This syngenic system allowed in vitro, in vivo and ex vivo analysis of chemotherapy induced apoptosis induction by optically monitoring the caspase-3 sensor state in the tumor cells. Tumor tissue analysis of 5-FU treated mice showed the selection of 5-FU-induced apoptosis resistant tumor cells. These and chemo-naive fluorescent tumor cells could be re-isolated from treated and untreated mice and propagated in cell culture. Re-exposure to 5-FU and second line treatment modalities in this ex-vivo setting showed that 5-FU induced apoptosis resistance could be alleviated by imatinib mesylate (Gleevec). We thus show that syngenic mouse systems that stably express a FRET-based caspase-3 sensor can be employed to analyse the therapeutic efficiency of apoptosis inducing chemotherapy.

Chemical Redox Modulation of the Surface Chemistry of CdTe Quantum Dots for Probing Ascorbic Acid in Biological Fluids

Most of the fluorescence resonance energy transfer (FRET)-based sensors employing quantum dots (QDs) usually use organic fluorophores and gold nanoparticles as the quenchers. However, complex processes for the modification/immobilization of the QDs are always necessary, as the generation of FRET requires strict distance between the donor and acceptor. Herein, a simple chemical redox strategy for modulating the surface chemistry of the QDs to develop a QD-based turn-on fluorescent probe is reported. The principle of the strategy is demonstrated by employing CdTe QDs with KMnO(4) as the quencher and ascorbic acid as the target analyte. The fluorescence of CdTe QDs is quenched with a blue-shift upon addition of KMnO(4) due to the oxidation of the Te atoms on the surface of the QDs. The quenched fluorescence of the QDs is then recovered upon addition of ascorbic acid due to the reduction of CdTeO(3)/TeO(2) on the surface of the QDs to CdTe. The recovered fluorescence of the QDs increases linearly with the concentration of ascorbic acid from 0.3 to 10 microM. Thus, a novel QD-based turn-on fluorescent probe with a detection limit as low as 74 nM is developed for the sensitive and selective detection of ascorbic acid in biological fluids. The present approach avoids the complex modification/immobilization of the QDs involved in FRET-based sensors, and opens a simple pathway to developing cost-effective, sensitive, and selective QD-based fluorescence turn-on sensors/probes for biologically significant antioxidants.

Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte.

Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicle-enclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte, where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from approximately 2 microM to approximately 80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from approximately 1 microM to approximately 40 nM) relieves the inhibition of PDE3A, increasing its activity by approximately 5-fold. This causes a decrease in oocyte cAMP (from approximately 700 nM to approximately 140 nM), leading to the resumption of meiosis.

Practical and reliable FRET/FLIM pair of fluorescent proteins

BackgroundIn spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings.ResultsHere we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter).ConclusionThe combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays.