Resonance Energy Transfer Sensor Research Articles

A novel “off-on” electrochemiluminescence sensor based on highly efficient resonance energy transfer in C-g-C3N4/CuO nanocomposite

A signal “off-on” electrochemiluminescence resonance energy transfer (ECL-RET) sensor based on carboxylated graphene-like carbon nitride (C-g-C3N4) as donor and CuO nanoneedles as acceptor has been constructed. The distance between donor and acceptor is a critical factor for ECL-RET sensors. Herein, we used a new method to make CuO nanoneedles grow in situ on C-g-C3N4 to form a nanocomposite, largely reducing the distance between donor and acceptor and greatly improving ECL-RET efficiency. In this system, because the CuO could be reduced by dopamine (DA), the ECL emission was significantly enhanced. Hence, a sensitive ECL sensor was successfully fabricated for quantitative detection of DA in dopamine hydrochloride injection and human serum sample. Further, the ECL-RET sensor exhibited a wide linear range from 10 nM to 1 mM, as well as a low detection limit of 8.2 nM. With its excellent stability and selectivity, the novel strategy will enable numerous applications in biological systems.

Calcineurin Aβ-Specific Anchoring Confers Isoform-Specific Compartmentation and Function in Pathological Cardiac Myocyte Hypertrophy.

The Ca2+/calmodulin-dependent phosphatase calcineurin is a key regulator of cardiac myocyte hypertrophy in disease. An unexplained paradox is how the β isoform of the calcineurin catalytic A-subunit (CaNAβ) is required for induction of pathological myocyte hypertrophy, despite calcineurin Aα expression in the same cells. It is unclear how the pleiotropic second messenger Ca2+ drives excitation-contraction coupling while not stimulating hypertrophy by calcineurin in the normal heart. Elucidation of the mechanisms conferring this selectivity in calcineurin signaling should reveal new strategies for targeting the phosphatase in disease. Primary adult rat ventricular myocytes were studied for morphology and intracellular signaling. New Förster resonance energy transfer reporters were used to assay Ca2+ and calcineurin activity in living cells. Conditional gene deletion and adeno-associated virus-mediated gene delivery in the mouse were used to study calcineurin signaling after transverse aortic constriction in vivo. CIP4 (Cdc42-interacting protein 4)/TRIP10 (thyroid hormone receptor interactor 10) was identified as a new polyproline domain-dependent scaffold for CaNAβ2 by yeast 2-hybrid screen. Cardiac myocyte-specific CIP4 gene deletion in mice attenuated pressure overload-induced pathological cardiac remodeling and heart failure. Blockade of CaNAβ polyproline-dependent anchoring using a competing peptide inhibited concentric hypertrophy in cultured myocytes; disruption of anchoring in vivo using an adeno-associated virus gene therapy vector inhibited cardiac hypertrophy and improved systolic function after pressure overload. Live cell Förster resonance energy transfer biosensor imaging of cultured myocytes revealed that Ca2+ levels and calcineurin activity associated with the CIP4 compartment were increased by neurohormonal stimulation, but minimally by pacing. Conversely, Ca2+ levels and calcineurin activity detected by nonlocalized Förster resonance energy transfer sensors were induced by pacing and minimally by neurohormonal stimulation, providing functional evidence for differential intracellular compartmentation of Ca2+ and calcineurin signal transduction. These results support a structural model for Ca2+ and CaNAβ compartmentation in cells based on an isoform-specific mechanism for calcineurin protein-protein interaction and localization. This mechanism provides an explanation for the specific role of CaNAβ in hypertrophy and its selective activation under conditions of pathologic stress. Disruption of CaNAβ polyproline-dependent anchoring constitutes a rational strategy for therapeutic targeting of CaNAβ-specific signaling responsible for pathological cardiac remodeling in cardiovascular disease deserving of further preclinical investigation.

Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria.

Histidine kinases (HK) switch between conformational states that promote kinase and phosphatase activities to regulate diverse cellular processes. Past studies have shown that these functional states can display heterogeneity between cells in microbial communities and can vary at the subcellular level. Methods to track and correlate the kinase conformational state with the phenotypic response of living bacteria cells will offer new opportunities to interrogate bacterial signaling mechanisms. As a proof of principle, we incorporated both mClover3 (donor) and mRuby3 (acceptor) fluorescent proteins into the Caulobacter crescentus cell-cycle HK CckA as an in vivo fluorescence resonance energy transfer (FRET) sensor to detect these structural changes. Our engineered FRET sensor was responsive to CckA-specific input signals and detected subcellular changes in CckA signal integration that occurs as cells develop. We demonstrated the potential of using the CckA FRET sensor as an in vivo screening tool for HK inhibitors. In summary, we have developed a new HK FRET sensor design strategy that can be adopted to monitor in vivo changes for interrogation of a broad range of signaling mechanisms in living bacteria.

The Zn2+‐Transporter ZnT6 Plays Important Role on Regulation of Cytosolic Free Zn2+ Through Modulation of Mitochondrial Zn2+ in Hyperglycemic Cardiomyocytes

Free zinc ion (Zn2+), have physiological importance for mammals and is found to be less than 1‐nM in cytosol. However, intracellular free Zn2+ levels ([Zn2+]i) can increase dramatically under pathological conditions including hyperglycemia and cancer. Any increase in [Zn2+]i can also cause significant changes in several intracellular signaling pathways. However, that [Zn2+]i is well regulated by various ways in physiological conditions and that value can fluctuate dynamically in cardiomyocytes. It is accepted that there are two‐types of Zn2+‐transporters, one group of them is responsible Zn2+‐influx from extracellular space and/or organelles to cytosol (ZIPs) and the other group is responsible Zn2+‐efflux from cytosol (ZnTs). Since it is not known whether the Zn2+‐transporter ZnT6 has a role in the regulation of [Zn2+]i in cardiomyocytes under hyperglycemia, we used control and ZnT6 overexpressed H9c2 cells (rat embryonic ventricular cell lines) to investigate its role. In addition, H9c2 cells are incubated with normal (5.5‐mM; control) and high glucose (25‐mM; hyperglycemic) for 24‐h. Following the treatment, control and ZnT6‐overexpressed cells are transfected with Zn+2‐sensitive as well as cytosole, sarco/endoplasmic reticulum S(ER) or mitochondria targeted Förster resonance energy transfer (FRET) sensors to measure cytosolic, S(ER) and mitochondrial [Zn2+], respectively. Furthermore, we examined the mRNA, protein expression levels and intracellular localizations by using molecular, biochemical and immunofluorescence methods. Our results demonstrated that ZnT6 but not ZnT5 mRNA and protein level is increased under hyperglycemia, while ZnT6 overexpression caused increase of ZnT5 protein level. Our immunofluorescence/precipitation experiments showed that ZnT6 localized on mitochondria together with ZnT5 in H9c2 cells. Furthermore, our [Zn2+] measurements by fluorescence imaging showed that cytosolic and mitochondrial [Zn2+] are increased and S(E)R [Zn2+] is decreased in hyperglycemic H9c2 cells. We also found that, ZnT6‐overexpression caused mitochondrial [Zn2+] increase and decrease in cytosolic [Zn2+], without changing the S(E)R [Zn2+]. To understand the role of excess [Zn2+] on mitochondrial function, we used ZnT6 overexpressed or high Zn2+ incubated H9c2 cell lines to measure mitochondrial‐membrane‐potential (MMP), ROS and ATP production. We found that excess intracellular or mitochondrial [Zn2+] caused depolarization of MMP, increase in ROS and decrease in ATP production. Overall, our results showed that ZnT6 has a significant role on [Zn2+]i by modulating mitochondrial [Zn2+] and function in hyperglycemia.Support or Funding InformationTUBITAK‐SBAG‐117S386TUBITAK‐SBAG‐119S661

Axelrod Symposium 2019: Phosphoproteomic Analysis of G-Protein–Coupled Pathways

By limiting unrestricted activation of intracellular effectors, compartmentalized signaling of cyclic nucleotides confers specificity to extracellular stimuli and is critical for the development and health of cells and organisms. Dissecting the molecular mechanisms that allow local control of cyclic nucleotide signaling is essential for our understanding of physiology and pathophysiology, but mapping the dynamics and regulation of compartmentalized signaling is a challenge. In this minireview we summarize advanced imaging and proteomics techniques that have been successfully used to probe compartmentalized cAMP signaling in eukaryotic cells. Subcellularly targeted fluorescence resonance energy transfer sensors can precisely locate and measure compartmentalized cAMP, and this allows us to estimate the range of effector activation. Because cAMP effector proteins often cluster together with their targets and cAMP regulatory proteins to form discrete cAMP signalosomes, proteomics and phosphoproteomics analysis have more recently been used to identify additional players in the cAMP-signaling cascade. We propose that the synergistic use of the techniques discussed could prove fruitful in generating a detailed map of cAMP signalosomes and reveal new details of compartmentalized signaling. Compiling a dynamic map of cAMP nanodomains in defined cell types would establish a blueprint for better understanding the alteration of signaling compartments associated with disease and would provide a molecular basis for targeted therapeutic strategies. SIGNIFICANCE STATEMENT: cAMP signaling is compartmentalized. Some functionally important cellular signaling compartments operate on a nanometer scale, and their integrity is essential to maintain cellular function and appropriate responses to extracellular stimuli. Compartmentalized signaling provides an opportunity for precision medicine interventions. Our detailed understanding of the composition, function, and regulation of cAMP-signaling nanodomains in health and disease is essential and will benefit from harnessing the right combination of advanced biochemical and imaging techniques.

Observing the Myosin Super-Relaxed State (SRX) in Cardiac Thick Filaments

We aim to develop time-resolved fluorescence resonance energy transfer (FRET) sensors to detect the structural state of myosin in cardiac thick filaments. In relaxation, these filaments are hypothesized to isomerize between the disordered relaxed state (DRX), which has the potential to interact with actin and generate force in the sarcomere, and an inactive state termed super-relaxed (SRX), which has much lower ATPase activity than DRX, apparently due to inhibitory interactions between the two heads of myosin (Hooijman et al, 2011). It has been proposed that (a) regulation of the transition between these states underlies the Frank-Starling law of the heart, and (b) dysregulation of the transition (destabilization of SRX) can lead to a form of heart failure known as hypertrophic cardiomyopathy (HCM). The structural basis of the SRX remains incompletely understood, but it has been proposed that it corresponds to the interacting heads motif (IHM) that has been observed by electron microscopy. To investigate directly the structural transition between DRX and SRX, we have developed a time-resolved FRET assay that measures the intermolecular distance between regulatory light chains on the two heads of myosin. The TRFRET data is best fit to two populations of Gaussian-distributed distances, suggesting that this FRET pair observes both the shorter distance predicted for the IHM (SRX) state and the longer distance predicted for the DRX state. This assay provides a structure-based assay for quantitating these states in functional muscle fibers, and it has the potential to create a high-throughput, high-precision assay for therapeutic development.

β-Arrestin-2 BRET Biosensors Detect Different β-Arrestin-2 Conformations in Interaction with GPCRs.

β-Arrestins are critical regulators of G protein-coupled receptors (GPCRs) that desensitize G protein signaling, promote receptor internalization, and initiate signaling on their own. Recent structural findings indicate that β-arrestins adopt different conformations upon interaction with agonist-activated GPCRs. Here, we established a β-arrestin-2 conformational bioluminescence resonance energy transfer (BRET) sensor composed of the bright Nanoluc BRET donor and the red-shifted CyOFP1 BRET acceptor. The sensor monitors early intramolecular conformational changes of β-arrestin-2 in complex with a wide panel of different class A and class B GPCRs upon agonist activation and with orphan GPCRs known to spontaneously recruit β-arrestin-2. The introduction of the R170E mutant in the β-arrestin-2 sensor allowed the detection of a partially active β-arrestin-2 conformation, which is not dependent on receptor phosphorylation, while the deletion of the β-arrestin-2 finger-loop region detected the "tail-conformation" corresponding to the interaction of β-arrestin with the carboxyl-terminal domain of GPCRs. The new sensors combine the advantages of the BRET technique in terms of sensitivity, robustness, and suitability for real-time measurements with a high responsiveness toward early conformational changes to help to elucidate the different conformational states of β-arrestins associated with GPCR activation in living cells.

Effective glucose metabolism maintains low intracellular glucose in airway epithelial cells after exposure to hyperglycemia.

The airway epithelium maintains differential glucose concentrations between the airway surface liquid (ASL, ~0.4 mM) and the blood/interstitium (5–6 mM), which is important for defense against infection. Glucose primarily moves from the blood to the ASL via paracellular movement, down its concentration gradient, across the tight junctions. However, there is evidence that glucose can move transcellularly across epithelial cells. Using a Förster resonance energy transfer sensor for glucose, we investigated intracellular glucose concentrations in airway epithelial cells and the role of hexokinases in regulating intracellular glucose concentrations in normoglycemic and hyperglycemic conditions. Our findings indicated that in airway epithelial cells (H441 or primary human bronchial epithelial cells) exposed to 5 mM glucose (normoglycemia), intracellular glucose concentration is in the micromolar range. Inhibition of facilitative glucose transporters (GLUTs) with cytochalasin B reduced intracellular glucose concentration. When cells were exposed to 15 mM glucose (hyperglycemia), intracellular glucose concentration was reduced. Airway cells expressed hexokinases I, II, and III. Inhibition with 3-bromopyruvate decreased hexokinase activity by 25% and elevated intracellular glucose concentration, but levels remained in the micromolar range. Exposure to hyperglycemia increased glycolysis, glycogen, and sorbitol. Thus, glucose enters the airway cell via GLUTs and is then rapidly processed by hexokinase-dependent and hexokinase-independent metabolic pathways to maintain low intracellular glucose concentrations. We propose that this prevents transcellular transport and aids the removal of glucose from the ASL and that the main route of entry for glucose into the ASL is via the paracellular pathway.

Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor.

Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL—ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status.

Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z′ > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI’s Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody—six actually stimulated PKC activity. Cytometry, which produces higher Z′ for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol ester concentrations to be used. We conclude that CFP-YFP FRET measured in a Spectramax i3x plate reader can be used for screening under the conditions we defined. Our strategy of varying expression level and FRET ratio could be useful to others for determining conditions needed for robust cell-based intramolecular CFP-YFP FRET measurements on their instrumentation.

Nonclassical Ligand-Independent Regulation of Go Protein by an Orphan Class C G-Protein–Coupled Receptor

The orphan G-protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery, where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-β5, leading to the regulation of neighboring GPCR-induced Go protein activity. GPR158 also interacts with αo, although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain that are putatively involved in G-protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-β5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in αo binding. Using a Gαo-βγ bioluminescence resonance energy transfer (BRET) sensor, we found that GPR158 decreases the BRET signal as observed upon G-protein activation; however, no constitutive activity of GPR158 could be detected through the measurement of various G-protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of Gαo by the VCPWE motifs, possibly leading to its dissociation from βγ Such action of GPR158 is expected to prolong the βγ activity, as also observed with some activators of G-protein signaling. Taken together, our data revealed a complex functional scaffolding or signaling role for GPR158 controlling Go through an original mechanism.

A novel up-conversion fluorescence resonance energy transfer sensor for the high sensitivity detection of coumarin in cosmetics

Coumarin is widely used as spices in a variety of cosmetics. However, its concentration in cosmetics is strictly limited because it is a kind of suspected carcinogen. It is essential and highly desired to develop fast and convenient detection methods to detect trace amount of coumarin. This work presents a novel and simplified fluorescent sensor for the determination of coumarin based on the phenomenon of fluorescence resonance energy transfer (FRET). In this system, the NaYF4: Yb,Er (NaYF) behaves as energy donor, and the rhodamine 6 G (Rh6 G) behaves as energy receptor. The β-cyclodextrin (CD) was modified on NaYF and was used to ensure the selectivity towards coumarin and to bring the donor and receptor into FRET proximity through specific host-guest interaction, which is different from the previously reported FRET models that either rely on π-π stacking interaction or rely on molecular recognition. This fluorescent sensor shows high sensitivity and selectivity toward coumarin, and the linear response range is 2.5–32.5 μM with a limit of detection as low as 0.74 μM.

Intramolecular Fluorescent Protein Association in a Class of Zinc FRET Sensors Leads to Increased Dynamic Range

Genetically encoded Forster resonance energy transfer (FRET) sensors enable the visualization of ions, molecules, and processes in live cells. However, despite their widespread use, the molecular s...

Intramolecular Fluorescent Protein Association in a Class of Zinc FRET Sensors Leads to Increased Dynamic Range.

Genetically encoded Förster resonance energy transfer (FRET) sensors enable the visualization of ions, molecules, and processes in live cells. However, despite their widespread use, the molecular states that determine sensor performance are usually poorly understood, which limits efforts to improve them. We used dynamic light scattering (DLS) and time-resolved fluorescence anisotropy to uncover the sensing mechanism of ZifCV1.173, a Zn2+ FRET sensor. We found that the dynamic range (DR) of ZifCV1.173 was dominated by the high FRET efficiency of the Zn2+-free state, in which the donor and acceptor fluorescent proteins were closely associated. Mutating the donor-acceptor interface revealed that the DR of ZifCV1.173 could be increased or decreased by promoting or disrupting the donor-acceptor interaction, respectively. Adapting the same mutations to a related sensor showed the same pattern of DR tuning, supporting our sensing mechanism and suggesting that DLS and time-resolved fluorescence anisotropy might be generally useful in the biophysical characterization of other FRET sensors.

A Förster resonance energy transfer sensor for live-cell imaging of mitogen-activated protein kinase activity in Arabidopsis.

The catalytic activity of mitogen-activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using invitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live-cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss-of-function double-mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl-induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22-induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.

Rhodamine B derivative-modified up-conversion nanoparticle probes based on fluorescence resonance energy transfer (FRET) for the solid-based detection of copper ions.

Herein, a novel solid-based up-conversion fluorescence resonance energy transfer (FRET) sensor was developed using rhodamine B hydrazide, which provided a selective fluorescence response and suitable affinity towards Cu2+ ions over other biologically relevant metal ions because the Cu2+ ion could promote the hydrolysis of α-amino acid esters of rhodamine B hydrazide and yield the Cu·α-amino acid chelate. This solid-based detection system is more convenient for the detection of Cu2+ based on color change and emission spectra instead of the complicated and tedious measurements than other up-conversion sensors and up-conversion luminescent nanoparticles used as an excitation source; moreover, the proposed system shows high selectivity, minimum photo-damage to living organisms, and high chemical stability.

Sugar Partitioning between Ustilago maydis and Its Host Zea mays L during Infection.

The basidiomycete Ustilago maydis causes smut disease in maize (Zea mays) by infecting all plant aerial tissues. The infection causes leaf chlorosis and stimulates the plant to produce nutrient-rich niches (i.e. tumors), where the fungus can proliferate and complete its life cycle. Previous studies have recorded high accumulation of soluble sugars and starch within these tumors. Using interdisciplinary approaches, we found that the sugar accumulation within tumors coincided with the differential expression of plant sugars will eventually be exported transporters and the proton/sucrose symporter Sucrose Transporter1 To accumulate plant sugars, the fungus deploys its own set of sugar transporters, generating a sugar gradient within the fungal cytosol, recorded by expressing a cytosolic glucose (Glc) Förster resonance energy transfer sensor. Our measurements indicated likely elevated Glc levels in hyphal tips during infection. Growing infected plants under dark conditions led to decreased plant sugar levels and loss of the fungal tip Glc gradient, supporting a tight link between fungal sugar acquisition and host supplies. Finally, the fungal infection causes a strong imbalance in plant sugar distribution, ultimately impacting seed set and yield.

Identification of molecular determinants that govern distinct STIM2 activation dynamics.

The endoplasmic reticulum (ER) Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2, which connect ER Ca2+ depletion with extracellular Ca2+ influx, are crucial for the maintenance of Ca2+ homeostasis in mammalian cells. Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals.

Visualization of the Delivery and Release of Small RNAs Using Genetic Code Expansion and Unnatural RNA-Binding Proteins

Endogenously expressed noncoding RNAs are regulators of mRNA translation and affect diverse biological pathways spanning embryogenesis to cholesterol and fatty acid metabolism. Recently, microRNAs have become an important therapeutic target with strategies that employ oligonucleotides as both mimics and inhibitors of target microRNAs, successfully altering gene expression and cellular pathways in relevant contexts. However, delivery of these exogenous effectors remains a major challenge. Here, we present a method for evaluating noncoding RNA delivery using the viral suppressor of RNA silencing (VSRS) protein p19, optimized for cellular delivery of small RNAs. Using genetic code expansion technology, p-azidophenylalanine (AzF) was incorporated into a recombinant p19 protein and used to develop a fluorescence resonance energy transfer (FRET) sensor. AzF was used to attach FRET acceptor moieties using bioorthogonal chemistry. We show that this strategy not only gives rise to FRET signals that report on small RNA binding, but also allows for fluorescence quenching as well, convenient for measuring RNA release. We demonstrate the successful use of a modified version of the probe to track the delivery and release of small RNAs into mammalian cells. The results provide a basis for a further development of vehicles for small RNA delivery and release for intervening in noncoding RNA biology.

A Novel Multiple-Read Screen for Metabolically Active Compounds Based on a Genetically Encoded FRET Sensor for ATP

Both glycolysis and mitochondrial energetics are targets of interest for developing antiproliferative cancer therapeutics. We developed a novel multiple-read assay based on long-term expression in K562 cells of a genetically encoded intramolecular Förster resonance energy transfer sensor for adenosine triphosphate (ATP). The assay, conducted in a fluorescent plate reader, can identify compounds that inhibit oxidative phosphorylation-dependent ATP production, glycolysis, or both after short-term treatment. We screened a National Cancer Institute (NCI) compound library, identifying inhibitors of oxidative phosphorylation-dependent ATP production and glycolysis. Three glycolysis inhibitors blocked hexokinase activity, demonstrating that our assay can serve as the initial step in a workflow to identify compounds that inhibit glycolysis via a defined desired mechanism. Finally, upon reviewing the literature, we found surprisingly little evidence that inhibiting glycolysis with small molecules is antiproliferative. Using NCI data on proliferation of K562 cells, we found that inhibitors of oxidative phosphorylation-dependent ATP production were no more antiproliferative than the overall library, whereas all glycolysis inhibitors were in the top third of most effective antiproliferative compounds. Our results thus present a powerful new way to screen for compounds that affect cellular metabolism and also provide important support for the idea that blocking glycosis is antiproliferative.