Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution.

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

[Make the invisible visible-innovation in the single fluorescent protein-based indicators].

Biological phenomena are generated by the cooperative and hierarchical relationships between a variety of biomolecules, such as proteins, metabolites, signaling molecules, and ions. In many cases, however, these biomolecules do not have color, and it is difficult to observe them as they are. Therefore, it is necessary to "visualize" each molecule with color or fluorescence, and to analyze the functional relationships between them. The live cell imaging technology using single fluorescent protein (FP)-based indicators has contributed to the visualization of biomolecules. Single FP-based indicators, which change their fluorescence intensity upon binding to the target molecule, have been revolutionized into multicolor indicators by a series of innovative screening methods. On the other hand, we have established an original screening method using semi-rational molecular design and molecular evolution, and have developed many single FP-based indicators for various molecules such as cAMP and glucose. In this article, we focus on single FP-based indicators and introduce their development strategy and the history of screening method.

Mitochondrial ATP concentration decreases immediately after glucose administration to glucose-deprived hepatocytes.

Hepatocytes can switch their metabolic processes in response to nutrient availability. However, the dynamics of metabolites (such as lactate, pyruvate, and ATP) in hepatocytes during the metabolic switch remain unknown. In this study, we visualized metabolite dynamics in primary cultured hepatocytes during recovery from glucose-deprivation. We observed a decrease in the mitochondrial ATP concentration when glucose was administered to hepatocytes under glucose-deprivation conditions. In contrast, there was slight change in the cytoplasmic ATP concentration. A decrease in mitochondrial ATP concentration was associated with increased protein synthesis rather than glycogen synthesis, activation of urea cycle, and production of reactive oxygen species. These results suggest that mitochondrial ATP is important in switching metabolic processes in the hepatocytes.

Distinct lactate metabolism between hepatocytes and myotubes revealed by live cell imaging with genetically encoded indicators.

The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP. Although the biochemical study of lactate metabolism in hepatocytes and skeletal muscle cells has been extensive, the spatial and temporal dynamics of this metabolism in live cells are still unknown. We observed the dynamics of metabolism-related molecules in primary cultured hepatocytes and a skeletal muscle cell line upon lactate overload. Our observations revealed an increase in cytoplasmic pyruvate concentration in hepatocytes, which led to glucose release. Skeletal muscle cells exhibited elevated levels of lactate and pyruvate levels in both the cytoplasm and mitochondrial matrix. However, mitochondrial ATP levels remained unaffected, indicating that the increased lactate can be converted to pyruvate but is unlikely to be utilized for ATP production. The findings suggest that excess lactate in skeletal muscle cells is taken up into mitochondria with little contribution to ATP production. Meanwhile, lactate released into the bloodstream can be converted to glucose in hepatocytes for subsequent utilization in skeletal muscle cells.

In situ FRET measurement of cellular tension using conventional confocal laser microscopy in newly established reporter mice expressing actinin tension sensor.

FRET-based sensors are utilized for real-time measurements of cellular tension. However, transfection of the sensor gene shows low efficacy and is only effective for a short period. Reporter mice expressing such sensors have been developed, but sensor fluorescence has not been measured successfully using conventional confocal microscopy. Therefore, methods for spatiotemporal measurement of cellular tension in vivo or ex vivo are still limited. We established a reporter mouse line expressing FRET-based actinin tension sensors consisting of EGFP as the donor and mCherry as the acceptor and whose FRET ratio change is observable with confocal microscopy. Tension-induced changes in FRET signals were monitored in the aorta and tail tendon fascicles, as well as aortic smooth muscle cells isolated from these mice. The pattern of FRET changes was distinctive, depending on tissue type. Indeed, aortic smooth muscle cells exhibit different sensitivity to macroscopic tensile strain in situ and in an isolated state. This mouse strain will enable novel types of biomechanical investigations of cell functions in important physiological events.

Efficient and rapid linker optimization with heterodimeric coiled coils improves the response of fluorescent biosensors comprising antibodies and protein M.

We developed a coiled Q-probe (CQ-probe), a fluorescent probe containing a coiled-coil peptide pair E4/K4, to convert antibodies into biosensors for homogeneous immunoassays. This probe consists of an antibody-binding protein, protein M (PM) with the E4 peptide and the K4 peptide with a fluorescent dye. Compared to PM Q-probes, which are generated by modifying the C-terminus of PM with a fluorescent dye, CQ-probe variants with various linkers are easy to prepare and therefore enable the establishment of biosensors with a significant fluorescence response by localizing the fluorescent dye at the optimal position for quenching and antigen-dependent release. The fluorescence changes of biosensors converted from anti-BGP, anti-cortisol, and anti-testosterone antibodies using the rhodamine 6G (or TAMRA)-labeled CQ-probe upon antigen addition were 13 (or 2.6), 9.7 (or 1.5), and 2.1 (or 1.2) times larger than that of the biosensors converted using the PM Q-probe. Furthermore, the CQ-probe converted anti-digoxin IgG into a functional biosensor, whereas the PM Q-probe/antibody complex showed an insufficient response. This technology exhibits a promising capacity to convert antibodies into high-response biosensors, which are expected to be applied in a wide range of fields, including clinical diagnosis, environmental surveys, food analysis, and biological research.

Nanodroplet-Based Reagent Delivery into Water-in-Fluorinated-Oil Droplets.

In vitro compartmentalization (IVC) is a technique for generating water-in-oil microdroplets to establish the genotype (DNA information)-phenotype (biomolecule function) linkage required by many biological applications. Recently, fluorinated oils have become more widely used for making microdroplets due to their better biocompatibility. However, it is difficult to perform multi-step reactions requiring the addition of reagents in water-in-fluorinated-oil microdroplets. On-chip droplet manipulation is usually used for such purposes, but it may encounter some technical issues such as low throughput or time delay of reagent delivery into different microdroplets. Hence, to overcome the above issues, we demonstrated a nanodroplet-based approach for the delivery of copper ions and middle-sized peptide molecules (human p53 peptide, 2 kDa). We confirmed the ion delivery by microscopic inspection of crystal formation inside the microdroplet, and confirmed the peptide delivery using a fluorescent immunosensor. We believe that this nanodroplet-based delivery method is a promising approach to achieving precise control for a broad range of fluorocarbon IVC-based biological applications, including molecular evolution, cell factory engineering, digital nucleic acid detection, or drug screening.

Development of green fluorescent protein-based cAMP indicators for covering a wide range of cAMP concentrations.

There is a wide range in the concentration of intracellular cyclic adenosine 3',5'-monophosphate (cAMP), which mediates specific effects as a second messenger in pathways affecting many physiological processes. Here, we developed green fluorescent cAMP indicators, named Green Falcan (Green fluorescent protein-based indicator visualizing cAMP dynamics) with various EC50 values (0.3, 1, 3, 10 µM) for covering the wide range of intracellular cAMP concentrations. The fluorescence intensity of Green Falcans increased in a cAMP dose-dependent manner, with a dynamic range of over 3-fold. Green Falcans showed a high specificity for cAMP over its structural analogues. When we expressed Green Falcans in HeLa cells, these indicators were applicable for visualization of cAMP dynamics in the low concentration range compared to the previously developed cAMP indicators, and visualized distinct kinetics of cAMP in various pathways with high spatiotemporal resolution in living cells. Furthermore, we demonstrated that Green Falcans are applicable to dual-color imaging with R-GECO, a red fluorescent Ca2+ indicator, in the cytoplasm and the nucleus. This study shows that Green Falcans open up a new avenue for understanding hierarchal and cooperative interactions with other molecules in various cAMP signaling pathways by multi-color imaging.

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions.

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed. Different genotype variants induced by mutagens are encapsulated in microemulsions and cultured to secrete proteins inside the emulsions. The secreted protein of interest is detected as a fluorescence signal by the fluorescent immunosensor quenchbody (Q-body), and a cell sorter is used to select emulsions containing improved protein-secreting strains based on the fluorescence intensity. The concept of the screening method is demonstrated by culturing Corynebacterium glutamicum in emulsions and detecting the secreted proteins. Finally, productive strains of fibroblast growth factor 9 (FGF9) are screened, and the FGF9 secretion increased threefold compared to that of parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag. This is a highly simple process that requires only the addition of a Q-body to the medium and does not require the addition of any substrates or chemical treatments. Furthermore, this method shortens the development period of industrial strains for biopharmaceutical protein production.

Recent progress in homogeneous immunosensors based on fluorescence or bioluminescence using antibody engineering.

Homogeneous immunosensors integrate the advantages of both biosensors and immunoassays; they include speed, high sensitivity, and accuracy. They have been developed rapidly in the past few years and offer a cost-effective alternative technology with rapidity, sensitivity, and user-friendliness, which has been applied in a wide variety of applications. This review introduces the current directions of immunosensor development, focusing on fluorescent and bioluminescent immunosensors and highlighting the advantages, improvements, and key approaches to overcome the limitations of each.

Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells.

Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.

F-actin determines the time-dependent shift in docking dynamics of glucagon-like peptide-1 granules upon stimulation of secretion.

Although exocytosis can be categorized into several forms based on docking dynamics, temporal regulatory mechanisms of the exocytotic forms are unclear. We explored the dynamics of glucagon-like peptide-1 (GLP-1) exocytosis in murine GLUTag cells (GLP-1-secreting enteroendocrine L-cells) upon stimulation with deoxycholic acid (DCA) or high K+ to elucidate the mechanisms regulating the balance between the different types of exocytotic forms (pre-docked with the plasma membrane before stimulation; docked after stimulation and subsequently fused; or rapidly recruited and fused after stimulation, without stable docking). GLP-1 exocytosis showed a biphasic pattern, and we found that most exocytosis was from the pre-docked granules with the plasma membrane before stimulation, or granules rapidly fused to the plasma membrane without docking after stimulation. In contrast, granules docked with the plasma membrane after stimuli and eventually fused were predominant thereafter. Inhibition of actin polymerization suppressed exocytosis of the pre-docked granules. These results suggest that the docking dynamics of GLP-1 granules shows a time-dependent biphasic shift, which is determined by interaction with F-actin.

Creating a Thermostable ß-Glucuronidase Switch for Homogeneous Immunoassay by Disruption of Conserved Salt Bridges at Diagonal Interfaces.

Escherichia coli ß-glucuronidase (GUS) has been used as a reporter enzyme in molecular biology and engineered as an enzyme switch for the development of homogeneous biosensors. In this study, we developed a thermostable GUS enzyme switch based on the thermostable GUS mutant TR3337 by disrupting a conserved salt bridge (H514-E523) between the diagonal subunits of its homotetramer. A combinatorial library (240 variants) was screened using a novel high-throughput strategy, which led to the identification of mutant DLW (H514D/M516L/Y517W) as a functional enzyme switch in a caffeine-recognizing immunosensor. Molecular dynamics simulations were performed to predict the topology change around position 514, and a side-chain flip of D514 (repulsion with E523) was observed in the DLW mutant. Up to 1.8-fold of signal-to-background ratio was confirmed when measured at up to 45 °C, thereby highlighting the DLW mutant as a versatile tool for developing thermostable immunosensors for in vitro and in cellulo applications.

The Patrol Yeast: A new biosensor armed with antibody-receptor chimera detecting a range of toxic substances associated with food poisoning.

Baker's yeast is an attractive host with established safety and stability characteristics. Many yeast-based biosensors have been developed, but transmembrane signal transduction has not been used to detect membrane-impermeable substances using antigen-antibody interactions. Therefore, we created Patrol Yeast, a novel yeast-based immunosensor of various targets, particularly toxic substances in food. A membrane-based yeast two-hybrid system using split-ubiquitin was successfully used to detect practically important concentration ranges of caffeine and aflatoxins using separated variable regions of an antibody. Moreover, enterohemorrhagic Escherichia coli O157 was detected using a specific single-chain antibody, in which Zymolyase was added to partially destroy the cell wall. The incorporation of secreted Cypridina luciferase reporter further simplified the signal detection procedures without cell lysis. The methodology is more cost-effective and faster than using mammalian cells. The ability to detect various targets renders Patrol Yeast a valuable tool for ensuring food and beverage safety and addressing other environmental and technological issues.

Rapid and sensitive SARS-CoV-2 detection using a homogeneous fluorescent immunosensor Quenchbody with crowding agents.

Antigen tests for SARS-CoV-2 are widely used by the public during the ongoing COVID-19 pandemic, which demonstrates the societal impact of homogeneous immunosensor-related technologies. In this study, we used the PM Q-probe and Quenchbody technologies to develop a SARS-CoV-2 nucleocapsid protein (N protein) homogeneous immunosensor based on a human anti-N protein antibody. For the first time, we uncovered the crowding agent's role in improving the performance of the double-labeled Quenchbody, and the possible mechanisms behind this improvement are discussed. The 5% polyethylene glycol 6000 significantly improved both the response speed and sensitivity of SARS-CoV-2 Quenchbodies. The calculated limit of detection for recombinant N protein was 191 pM (9 ng mL-1) within 15 min of incubation, which was 9- to 10-fold lower than the assay without adding crowding agent. We also validated the developed immunosensor in a point-of-care test by measuring specimens from COVID-19-positive patients using a compact tube fluorometer. In brief, this work shows the feasibility of Quenchbody homogeneous immunosensors as rapid and cost-efficient tools for the diagnosis and high-throughput analysis of swab samples in large-scale monitoring and epidemiological studies of COVID-19 or other emerging infectious diseases.

Development of a red fluorescent protein-based cGMP indicator applicable for live-cell imaging.

Cyclic guanosine 3', 5'-monophosphate (cGMP) is a second messenger that regulates a variety of physiological processes. Here, we develop a red fluorescent protein-based cGMP indicator, "Red cGull". The fluorescence intensity of Red cGull increase more than sixfold in response to cGMP. The features of this indicator include an EC50 of 0.33 µM for cGMP, an excitation and emission peak at 567 nm and 591 nm, respectively. Live-cell imaging analysis reveal the utility of Red cGull for dual-colour imaging and its ability to be used in conjunction with optogenetics tools. Using enteroendocrine cell lines, Red cGull detects an increase in cGMP following the application of L-arginine. An increase in intracellular cGMP is found to be inhibited by Ca2+, and L-arginine-mediated hormone secretion is not potentiated. We propose that Red cGull will facilitate future research in cell signalling in relation to cGMP and its interplay with other signalling molecules.

Intra Q-body: an antibody-based fluorogenic probe for intracellular proteins that allows live cell imaging and sorting.

Although intracellular biomarkers can be imaged with fluorescent dye(s)-labeled antibodies, the use of such probes for precise imaging of intracellular biomarkers in living cells remains challenging due to background noise from unbound probes. Herein, we describe the development of a conditionally active Fab-type Quenchbody (Q-body) probe derived from a monoclonal antibody (DO-1) with the ability to both target and spatiotemporally visualize intracellular p53 in living cells with low background signal. p53 is a key tumor suppressor and validated biomarker for cancer diagnostics and therapeutics. The Q-body displayed up to 27-fold p53 level-dependent fluorescence enhancement in vitro with a limit of detection of 0.72 nM. In fixed and live cells, 8.3- and 8.4-fold enhancement was respectively observed. Furthermore, we demonstrate live-cell sorting based on p53 expression. This study provides the first evidence of the feasibility and applicability of Q-body probes for the live-cell imaging of intrinsically intracellular proteins and opens a novel avenue for research and diagnostic applications on intracellular target-based live-cell sorting.

Modulation of Local Cellular Activities using a Photothermal Dye-Based Subcellular-Sized Heat Spot.

Thermal engineering at the microscale, such as the regulation and precise evaluation of the temperature within cellular environments, is a major challenge for basic biological research and biomaterials development. We engineered a polymeric nanoparticle having a fluorescent temperature sensory dye and a photothermal dye embedded in the polymer matrix, named nanoheater-thermometer (nanoHT). When nanoHT is illuminated with a near-infrared laser at 808 nm, a subcellular-sized heat spot is generated in a live cell. Fluorescence thermometry allows the temperature increment to be read out concurrently at individual heat spots. Within a few seconds of an increase in temperature by approximately 11.4 °C from the base temperature (37 °C), we observed the death of HeLa cells. The cell death was observed to be triggered from the exact local heat spot at the subcellular level under the fluorescence microscope. Furthermore, we demonstrate the application of nanoHT for the induction of muscle contraction in C2C12 myotubes by heat release. We successfully showed heat-induced contraction to occur in a limited area of a single myotube based on the alteration of protein-protein interactions related to the contraction event. These results demonstrate that even a single heat spot provided by a photothermal material can be extremely effective in altering cellular functions.

Dopamine Negatively Regulates Insulin Secretion Through Activation of D1-D2 Receptor Heteromer.

There is increasing evidence that dopamine (DA) functions as a negative regulator of glucose-stimulated insulin secretion; however, the underlying molecular mechanism remains unknown. Using total internal reflection fluorescence microscopy, we monitored insulin granule exocytosis in primary islet cells to dissect the effect of DA. We found that D1 receptor antagonists rescued the DA-mediated inhibition of glucose-stimulated calcium (Ca2+) flux, thereby suggesting a role of D1 in the DA-mediated inhibition of insulin secretion. Overexpression of D2, but not D1, alone exerted an inhibitory and toxic effect that abolished the glucose-stimulated Ca2+ influx and insulin secretion in ß-cells. Proximity ligation and Western blot assays revealed that D1 and D2 form heteromers in ß-cells. Treatment with a D1-D2 heteromer agonist, SKF83959, transiently inhibited glucose-induced Ca2+ influx and insulin granule exocytosis. Coexpression of D1 and D2 enabled ß-cells to bypass the toxic effect of D2 overexpression. DA transiently inhibited glucose-stimulated Ca2+ flux and insulin exocytosis by activating the D1-D2 heteromer. We conclude that D1 protects ß-cells from the harmful effects of DA by modulating D2 signaling. The finding will contribute to our understanding of the DA signaling in regulating insulin secretion and improve methods for preventing and treating diabetes.

Development of red genetically encoded biosensor for visualization of intracellular glucose dynamics.

Glucose is the main source of energy for organisms, and it is important to understand the spatiotemporal dynamics of intracellular glucose. Single fluorescent protein-based glucose indicators, named "Red Glifons" have been developed that apply to live-cell and dual-color imaging. These indicators exhibited more than 3-fold increase in fluorescence intensity in the presence of 10 mM glucose. The two Red Glifons developed have different half-maximal effective concentration (EC50) values for glucose (300 µM and 3,000 µM) and are able to monitor a wide range of glucose dynamics. Red Glifon combined with green indicators allowing visualization of the interplay between glucose and ATP, lactate, or pyruvate. Glucose influx in the pharyngeal muscle of Caenorhabditis elegans, enteroendocrine cells, and human iPS cell-derived cardiac myocytes was observed using the Red Glifons. Thus these red glucose indicators serve as a multi-color imaging toolkit for investigating complex interactions in energy metabolism.