A bright red fluorescent genetically encoded sensor for lactate imaging.

Lactate plays a crucial role in energy metabolism and greatly impacts protein activities, exerting diverse physiological and pathological effects. Therefore, convenient lactate assays for tracking spatiotemporal dynamics in living cells are desirable. In this paper, we engineered and optimized a red fluorescent protein sensor for l-lactate named FiLa-Red. This indicator exhibited a maximal fluorescence change of 730 % and an apparent dissociation constant (Kd) of approximately 460 µM. By utilizing FiLa-Red and other sensors, we monitored energy metabolism in a multiplex manner by simultaneously tracking lactate and NAD+/NADH abundance in the cytoplasm, nucleus, and mitochondria. The FiLa-Red sensor is expected to be a useful tool for performing metabolic analysis in vitro, in living cells and in vivo.

Detection of endogenous NPY release determined by novel GRAB sensor in cultured cortical neurons.

Neuropeptide Y (NPY) is an abundantly expressed peptide in the nervous system. Its widespread distribution along with its receptors, both centrally and peripherally, indicates its broad functions in numerous biological processes. However, the low endogenous concentration and diffuse distribution of NPY make it challenging to study its actions and dynamics directly and comprehensively. Studies on the role of NPY have primarily been limited to exogenous application, transgene expression, or knock-out in biological systems, which are often combined with pharmacological probes to delineate the involvement of specific NPY receptors. Therefore, to better understand the function of NPY in time and space, direct visualization of the real-time dynamics of endogenous NPY is a valuable and desired tool. Using the first-generation and newly developed intensiometric green fluorescent G-protein-coupled NPY sensor (GRAB NPY1.0), we, for the first time, demonstrate and characterize the direct detection of endogenously released NPY in cultured cortical neurons. A dose-dependent fluorescent signal was observed upon exogenous NPY application in nearly all recorded neurons. Pharmacologically evoked neuronal activity induced a significant increase in fluorescent signal in 32% of neurons, reflecting the release of NPY, despite only 3% of all neurons containing NPY. The remaining pool of neurons expressing the sensor were either non-responsive or displayed a notable decline in the fluorescent signal. Such decline in fluorescent signal was not rescued in cortical cultures transduced with an NPY overexpression vector, where 88% of the neurons were NPY-positive. Overexpression of NPY did, however, result in sensor signals that were more readily distinguishable. This may suggest that biological factors, such as subtle changes in intracellular pH, could interfere with the fluorescent signal, and thereby underestimate the release of endogenous NPY when using this new sensor in its present configuration. However, the development of next-generation NPY GRAB sensor technology is expected soon, and will eventually enable much-wanted studies on endogenous NPY release dynamics in both cultured and intact biological systems.

Techniques for in vivo serotonin detection in the brain: State of the art.

Neuronal circuits in the brain that utilize the neurotransmitter serotonin are essential to the regulation of mood and emotional expression. Disruptions in serotonin signaling underlie neuropsychiatric conditions such as depression and anxiety. However, the cellular mechanisms that regulate serotonergic signaling in the brain in healthy and diseased states remain to be better understood. In particular, as more is learned about serotonin in the brain, we recognize an urgent need to develop techniques capable of mapping its complex spatiotemporal dynamics in awake, behaving animals. Notably, analytical methods to detect serotonin in situ, including tomography, are widely used but still recognized as limited in terms of their spatiotemporal resolution, their methodological caveats, and their technical limitations when cross-referenced with behavioral studies. To overcome such limitations, genetically encoded serotonin indicators were developed, leading to the introduction of novel imaging modalities that enable researchers to achieve remarkable spatiotemporal resolution in the study of serotonergic circuits in preclinical models of neuropsychiatric disorders. These novel approaches, while remarkably powerful, are also not without limitations. Here, we review the current techniques for detecting and quantifying serotonin in vivo within the brain and discuss how novel approaches such as genetically encoded serotonin indicators will lead to new insights into the roles of serotonergic circuits in health and disease.

Directed evolution of the genetically encoded zinc(II) FRET sensor ZapCY1.

Zinc(II) ions (Zn2+) play an essential role in living systems, with their delicate concentration balance differing among the various intracellular organelles. The spatiotemporal distribution and homeostasis of Zn2+ can be monitored through photoluminescence imaging using zinc sensors. Among such biosensors, genetically encoded fluorescent sensor proteins are attractive tools owing to their subcellular localization advantage and high biocompatibility. However, the limited fluorescent properties of these proteins, such as their insufficient quantum yield and dynamic range, restrict their practical use. In this study, we developed an expression-screening-directed evolution system and used it to improve ZapCY1, a genetically encoded fluorescence resonance energy transfer (FRET) sensor. After four rounds of directed evolution, the FRET dynamic range of the modified sensor (designated ZapTV-EH) was increased by 1.5-1.7-fold. With its enhanced signal-to-noise ratio and ability to detect a wide Zn2+ concentration range, ZapTV-EH proves to be a better visualization tool for monitoring Zn2+ at the subcellular level. Combined with the simplified subcloning and expression steps and sufficient mutant libraries, this directed evolution system may provide a more simple and efficient way to develop and optimize genetically encoded FRET sensors through high-throughput screening.

Development of a novel fluorescent biosensor for dynamic monitoring of metabolic methionine redox status in cells and tissues.

Aberrant production of reactive oxygen species (ROS) leads to tissue damage accumulation, which is associated with a myriad of human pathologies. Although several sensors have been developed for ROS quantification, their applications for ROS-related human physiologies and pathologies still remain problematic due to the unstable nature of ROS. Herein, we developed Trx1-cpYFP-fRMsr (TYfR), a genetically-encoded fluorescent biosensor with the remarkable specificity and sensitivity toward fMetRO (free Methionine-R-sulfoxide), allowing for dynamic quantification of physiological levels of fMetRO, a novel indicator of ROS and methionine redox status in vitro and in vivo. Moreover, using the sensor, we observed a significant fMetRO enrichment in serum from patients with acute coronary syndrome, one of the most severe cardiovascular diseases, which becomes more evident following percutaneous coronary intervention. Collectively, this study proposes that fMetRO is a novel biomarker of tissue damage accumulation in ROS-associated human pathologies, and that TYfR is a promising tool for quantifying fMetRO with potentials in versatile applications.

Nanoscopic Visualization of Restricted Nonvolume Cholinergic and Monoaminergic Transmission with Genetically Encoded Sensors.

How neuromodulatory transmitters diffuse into the extracellular space remains an unsolved fundamental biological question, despite wide acceptance of the volume transmission model. Here, we report development of a method combining genetically encoded fluorescent sensors with high-resolution imaging and analysis algorithms which permits the first direct visualization of neuromodulatory transmitter diffusion at various neuronal and non-neuronal cells. Our analysis reveals that acetylcholine and monoamines diffuse at individual release sites with a spread length constant of ∼0.75 µm. These transmitters employ varied numbers of release sites, and when spatially close-packed release sites coactivate they can spillover into larger subcellular areas. Our data indicate spatially restricted (i.e., nonvolume) neuromodulatory transmission to be a prominent intercellular communication mode, reshaping current thinking of control and precision of neuromodulation crucial for understanding behaviors and diseases.

Illuminating NAD+ Metabolism in Live Cells and In Vivo Using a Genetically Encoded Fluorescent Sensor.

Understanding of NAD+ metabolism provides many critical insights into health and diseases, yet highly sensitive and specific detection of NAD+ metabolism in live cells and in vivo remains difficult. Here, we present ratiometric, highly responsive genetically encoded fluorescent indicators, FiNad, for monitoring NAD+ dynamics in living cells and animals. FiNad sensors cover physiologically relevant NAD+ concentrations and sensitively respond to increases and decreases in NAD+. Utilizing FiNad, we performed a head-to-head comparison study of common NAD+ precursors in various organisms and mapped their biochemical roles in enhancing NAD+ levels. Moreover, we showed that increased NAD+ synthesis controls morphofunctional changes of activated macrophages, and directly imaged NAD+ declines during aging in situ. The broad utility of the FiNad sensors will expand our mechanistic understanding of numerous NAD+-associated physiological and pathological processes and facilitate screening for drug or gene candidates that affect uptake, efflux, and metabolism of this important cofactor.

Spatiotemporal Imaging of Cellular Energy Metabolism with Genetically-Encoded Fluorescent Sensors in Brain.

The brain has very high energy requirements and consumes 20% of the oxygen and 25% of the glucose in the human body. Therefore, the molecular mechanism underlying how the brain metabolizes substances to support neural activity is a fundamental issue for neuroscience studies. A well-known model in the brain, the astrocyte-neuron lactate shuttle, postulates that glucose uptake and glycolytic activity are enhanced in astrocytes upon neuronal activation and that astrocytes transport lactate into neurons to fulfill their energy requirements. Current evidence for this hypothesis has yet to reach a clear consensus, and new concepts beyond the shuttle hypothesis are emerging. The discrepancy is largely attributed to the lack of a critical method for real-time monitoring of metabolic dynamics at cellular resolution. Recent advances in fluorescent protein-based sensors allow the generation of a sensitive, specific, real-time readout of subcellular metabolites and fill the current technological gap. Here, we summarize the development of genetically encoded metabolite sensors and their applications in assessing cell metabolism in living cells and in vivo, and we believe that these tools will help to address the issue of elucidating neural energy metabolism.

Real-time and high-throughput analysis of mitochondrial metabolic states in living cells using genetically encoded NAD+/NADH sensors.

Mitochondria are central organelles that regulate cellular bioenergetics, biosynthesis, and signaling processes. NADH, a key player in cell metabolism, is often considered as a marker of mitochondrial function. However, traditional methods for NADH measurements are either destructive or unable to distinguish between NADH and NADPH. In contrast to traditional methods, genetically encoded NADH sensors can be used for the real-time tracking and quantitative measurement of subcellular NADH levels in living cells. Therefore, these sensors provide innovative tools and address the limitations of current techniques. We herein summarize the properties of different types of recently developed NADH biosensors, discuss their advantages and disadvantages, and focus on the high-throughput analysis of mitochondrial function by using highly responsive NAD+/NADH sensors.

Genetically encoded far-red fluorescent sensors for caspase-3 activity.

Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues.