Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor.

Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. We previously engineered genetically encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of cocultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric one- and two-photon redox imaging in rat brain slices and Drosophila retinas. Overall, the proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms.

Dual-Mode FRET and BRET Sensors for Detecting cAMP Dynamics.

Genetically encoded fluorescent and luminescent indicators have revolutionized our ability to monitor physiology in real time, but the separate development of new sensors for each of these imaging modalities involves substantial effort and resources. Methods to rapidly engineer multimodal sensors would, therefore, significantly accelerate the diversification of sensors for simultaneous use in different systems and applications. We hypothesized that the enhanced Nano-lanterns could be incorporated into modular ratiometric sensors as an efficient approach to creating dual-mode fluorescent-luminescent sensors. As a proof-of-concept, we engineered an Epac1-based sensor that responds to cyclic adenosine monophosphate binding with a greater than 80% change in both Förster Resonance Energy Transfer and bioluminescent resonance energy transfer (BRET) modes. We also demonstrate that our new sensor reports cellular changes in G-protein-coupled signaling, and that the ratiometric BRET mode is bright enough for subcutaneous measurements in mice.

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.

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP.

Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems.

Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor.

Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first report the 1.09 Å X-ray crystal structure of mCherryTYG, exhibiting a fully matured chromophore. We next determine that it has an extraordinarily large dynamic range with a 2 ns lifetime change from pH 5.5 to 9.0. Critically, we find that the sensor maintains a p Ka of 6.8 independent of environment, whether as the purified protein in solution or expressed in live cells. Furthermore, the lifetime measurements are robustly independent of total fluorescence intensity and scatter. We demonstrate that mCherryTYG is a highly effective sensor using time-resolved fluorescence spectroscopy on live-cell suspensions, which has been previously overlooked as an easily accessible approach for quantifying intracellular pH. As a red fluorescent sensor, we also demonstrate that mCherryTYG is spectrally compatible with the ATeam sensor and EGFP for simultaneous dual-color measurements of intracellular pH, ATP, and extracellular pH. In a proof-of-concept, we quantify acute respiration-dependent pH homeostasis that exhibits a stoichiometric relationship with the ATP-generating capacity of the carbon fuel choice in E. coli. Broadly speaking, our work presents a previously unemployed methodology that will greatly facilitate continuous pH quantification.

pH- and Temperature-Dependent Peptide Binding to the Lactococcus lactis Oligopeptide-Binding Protein A Measured with a Fluorescence Anisotropy Assay.

Bacterial ATP-binding cassette transporters are a superfamily of transport systems involved in the import of various molecules including amino acids, ions, sugars, and peptides. In the lactic acid bacteria Lactococcus lactis, the oligopeptide-binding protein A (OppA) binds peptides for import to support nitrogen metabolism and cell growth. The OppA protein is of great interest because it can bind peptides over a broad variety of lengths and sequences; however, current methods to study peptide binding have employed low throughput, endpoint, or low dynamic range techniques. Therefore, in this study, we developed a fluorescence anisotropy-based peptide-binding assay that can be readily employed to quantify OppA function. To test the utility of our assay, we characterized the pH dependence of oligopeptide binding because L. lactis is commonly used in fermentation and often must survive in low pH environments caused by lactic acid export. We determined that OppA affinity increases as pH or temperature decreases, and circular dichroism spectroscopy further indicated that acidic conditions increase the thermal stability of the protein, increasing the unfolding transition temperature by 10 °C from pH 8 to pH 6. Thus, our fluorescence anisotropy assay provides an easy technique to measure peptide binding, and it can be used to understand molecular aspects of OppA function under stress conditions experienced during fermentation and other biotechnology applications.

Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry.

The regulation of pH is essential for proper organelle function, and organelle-specific changes in pH often reflect the dynamics of physiological signaling and metabolism. For example, mitochondrial energy production depends on the proton gradient maintained between the alkaline mitochondrial matrix and neutral cytosol. However, we still lack a quantitative understanding of how pH dynamics are coupled between compartments and how pH gradients are regulated at organelle boundaries. Genetically encoded pH sensors are well suited to address this problem because they can be targeted to specific subcellular locations and they facilitate live, single-cell analysis. However, most of these pH sensors are derivatives of green and yellow fluorescent proteins that are not spectrally compatible for dual-compartment imaging. Therefore, there is a need for ratiometric red fluorescent protein pH sensors that enable quantitative multicolor imaging of spatially resolved pH dynamics. In this work, we demonstrate that the I158E/Q160A mutant of the red fluorescent protein mCherry is an effective ratiometric pH sensor. It has a pKa of 7.3 and a greater than 3-fold change in ratio signal. To demonstrate its utility in cells, we measured activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells. Furthermore, we were able to image pH changes simultaneously in the cytosol and mitochondria by using the mCherryEA mutant together with the green fluorescent pH sensor, ratiometric-pHluorin. Our results demonstrate the feasibility of studying interorganelle pH dynamics in live cells over time and the broad applicability of these sensors in studying the role of pH regulation in metabolism and signaling.

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor.

Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens.

Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells.

Reactive oxygen species (ROS) mediate both intercellular and intraorganellar signaling, and ROS propagate oxidative stress between cellular compartments such as mitochondria and the cytosol. Each cellular compartment contains its own sources of ROS as well as antioxidant mechanisms, which contribute to dynamic fluctuations in ROS levels that occur during signaling, metabolism, and stress. However, the coupling of redox dynamics between cellular compartments has not been well studied because of the lack of available sensors to simultaneously measure more than one subcellular compartment in the same cell. Currently, the redox-sensitive green fluorescent protein, roGFP, has been used extensively to study compartment-specific redox dynamics because it provides a quantitative ratiometric readout and it is amenable to subcellular targeting as a genetically encoded sensor. Here, we report a new family of genetically encoded fluorescent protein sensors that extend the fluorescence emission of roGFP via Förster-type resonance energy transfer to an acceptor red fluorescent protein for dual-color live-cell microscopy. We characterize the redox and optical properties of the sensor proteins, and we demonstrate that they can be used to simultaneously measure cytosolic and mitochondrial ROS in living cells. Furthermore, we use these sensors to reveal cell-to-cell heterogeneity in redox coupling between the cytosol and mitochondria when neuroblastoma cells are exposed to reductive and metabolic stresses.

Imaging Adenosine Triphosphate (ATP).

Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities.

Imaging changes in the cytosolic ATP-to-ADP ratio.

Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside the cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We, therefore, developed a genetically encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here, we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy.

Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio.

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research.

Optogenetic reporters: Fluorescent protein-based genetically encoded indicators of signaling and metabolism in the brain.

Fluorescent protein technology has evolved to include genetically encoded biosensors that can monitor levels of ions, metabolites, and enzyme activities as well as protein conformation and even membrane voltage. They are well suited to live-cell microscopy and quantitative analysis, and they can be used in multiple imaging modes, including one- or two-photon fluorescence intensity or lifetime microscopy. Although not nearly complete, there now exists a substantial set of genetically encoded reporters that can be used to monitor many aspects of neuronal and glial biology, and these biosensors can be used to visualize synaptic transmission and activity-dependent signaling in vitro and in vivo. In this review, we present an overview of design strategies for engineering biosensors, including sensor designs using circularly permuted fluorescent proteins and using fluorescence resonance energy transfer between fluorescent proteins. We also provide examples of indicators that sense small ions (e.g., pH, chloride, zinc), metabolites (e.g., glutamate, glucose, ATP, cAMP, lipid metabolites), signaling pathways (e.g., G protein-coupled receptors, Rho GTPases), enzyme activities (e.g., protein kinase A, caspases), and reactive species. We focus on examples where these genetically encoded indicators have been applied to brain-related studies and used with live-cell fluorescence microscopy.

Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor.

NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor.

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.

Functional equivalence of the nicotinic acetylcholine receptor transmitter binding sites in the open state.

The subunits of the muscle-type nicotinic acetylcholine receptor (AChR) are not uniformly oriented in the resting closed conformation: the two alpha subunits are rotated relative to its non-alpha subunits. In contrast, all the subunits overlay well with one another when agonist is bound to the AChR, suggesting that they are uniformly oriented in the open receptor. This gating-dependent increase in orientational uniformity due to rotation of the alpha subunits might affect the relative affinities of the two transmitter binding sites, making the two affinities dissimilar (functionally non-equivalent) in the initial ligand-bound closed state but similar (functionally equivalent) in the open state. To test this hypothesis, we measured single-channel activity of the alphaG153S gain-of-function mutant receptor evoked by choline, and estimated the resting closed-state and open-state affinities of the two transmitter binding sites. Both model-independent analyses and maximum-likelihood estimation of microscopic rate constants indicate that channel opening makes the binding sites' affinities more similar to each other. These results support the hypothesis that open-state affinities to the transmitter binding sites are primarily determined by the alpha subunits.

Use of calculated cation-pi binding energies to predict relative strengths of nicotinic acetylcholine receptor agonists.

Agonists and antagonists of the nicotinic acetylcholine receptor (nAChR) are used to treat nicotine addiction, neuromuscular disorders, and neurological diseases. In designing small molecule therapeutics with the nAChR as a target, it is useful to identify chemical parameters that correlate with ability to activate the receptor. Previous studies have shown that cation-pi interactions at the transmitter binding sites of the nAChR are important for receptor activation by strong agonists such as acetylcholine. We hypothesized that a calculated estimate of cation-pi binding ability could be used to predict the efficiency for channel opening (i.e., the gating efficiency) associated with activation of the acetylcholine receptor by a series of structurally related organic cations. We demonstrate that the calculated cation-pi energy is strongly correlated with gating efficiency but only weakly correlated with closed-state binding affinity. Our results suggest that cation-pi interactions contribute significantly to the open-state affinity of these cations and that the calculated cation-pi energy will be a useful parameter for designing nAChR agonists and antagonists.

An activity-based protein profiling probe for the nicotinic acetylcholine receptor.

Activity-based protein profiling (ABPP) has been used extensively to characterize the physiological functions of enzymes but has not yet been extended to ion channels. We have synthesized a state-dependent photoaffinity probe for the nicotinic acetylcholine receptor (nAChR) as a proof of concept for the development of ion channel directed ABPP probes. The candidate probe BPyneTEA comprises an nAChR binding moiety, a benzophenone moiety for photolabeling, and an alkyne moiety for biotinylation via "click chemistry". Single-molecule current measurements show that BPyneTEA blocks both the closed and open (i.e., nondesensitized) conformations of the nAChR with similar kinetics. In living cells, BPyneTEA photolabels the closed state selectively over the inactive desensitized state. BPyneTEA thus shows promise as a probe for nondesensitized nAChRs and may be useful in studying the molecular physiology of desensitization. The structure and reactivity of ion channel pores in general suggest that they will be a broadly useful target for ABPP probes.

Testing for violations of microscopic reversibility in ATP-sensitive potassium channel gating.

In pancreatic beta cells, insulin secretion is tightly controlled by the cells' metabolic state via the ATP-sensitive potassium (KATP) channel. ATP is a key mediator in this signaling process, where its role as an inhibitor of KATP channels has been extensively studied. Since the channel contains an ATPase as an accessory subunit, the possibility that ATP hydrolysis mediates KATP channel opening has also been proposed. However, a rigorous test of coupling between ATP hydrolysis and channel gating has not previously been performed. In the present work, we examine whether KATP channel gating obeys detailed balance in order to determine whether ATP hydrolysis is strongly coupled to the gating of the KATP channel. Single-channel records were obtained from inside-out patches of transiently transfected HEK-293 cells. Channel activity in membrane patches with exactly one channel shows no violations of microscopic reversibility. Although KATP channel gating shows long closed times on the time scale where ATP hydrolysis takes place, the time symmetry of channel gating indicates that it is not tightly coupled to ATP hydrolysis. This lack of coupling suggests that channel gating operates close to equilibrium; although detailed balance is not expected to hold for ATP hydrolysis, it still does so in channel gating. On the basis of these results, the function of the ATPase active site in channel gating may be to sense nucleotides by differential binding of ATP and ADP, rather than to drive a thermodynamically unfavorable conformational change.

p19(ARF) is dispensable for oncogenic stress-induced p53-mediated apoptosis and tumor suppression in vivo.

Recent studies have shown the p19(ARF) tumor suppressor to be involved in the response to oncogenic stress by regulating the activity of p53. This response is mediated by antagonizing the function of Mdm2, a negative regulator of p53, indicating a pathway for tumor suppression that involves numerous genes altered in human tumors. We previously described a transgenic mouse brain tumor model in which oncogenic stress, provided by cell-specific inactivation of the pRb pathway, triggers a p53-dependent apoptotic response. This response suppresses the growth of developing tumors and thus represents a bona fide in vivo tumor suppressor activity. We further showed that E2F1, a transcription factor known to induce p19(ARF) expression, was required for the response. Here, we use a genetic approach to test whether p19(ARF) functions to transduce the signal from E2F1 to p53 in this tumor suppression pathway. Contrary to the currently accepted hypothesis, we show that a deficiency in p19(ARF) has no impact on p53-mediated apoptosis or tumor suppression in this system. All measures of p53 function, including the level of apoptosis induced by pRb inactivation, the expression of p21 (a p53-responsive gene), and the rate of tumor growth, were comparable in mice with and without a functional p19(ARF) gene. Thus, although p19(ARF) is required in some cell types to transmit an oncogenic response signal to p53, it is dispensable for this function in an in vivo epithelial system. These results underscore the complexity of p53 tumor suppression and further indicate the existence of distinct cell-specific pathways that respond to similar stimuli.