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.

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.

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.

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.

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.

Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess.

With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O2, etc.). However, the production status of the target protein can only be known after the subsequent separation and purification process. To speed up the monitoring of the production process and screening of the higher-yield target protein variants, here we developed an antibody-based His-tag sensor Quenchbody (Q-body), which can quickly detect the C-terminally His-tagged recombinant protein produced in the culture medium. Compared with single-chain Fv-based Q-body having one dye, the Fab-based Q-body having two dyes showed a higher response. In addition, not only was fluorescence response improved but also detection sensitivity by the mutations of tyrosine to tryptophan in the heavy chain CDR region. Moreover, the effect of the mutations on antigen-binding was successfully validated by molecular docking simulation by CDOCKER. Finally, the constructed Q-body was successfully applied to monitor the amount of anti-SARS CoV-2 nanobody secreted into the Brevibacillus culture media.

Sonic hedgehog enhances calcium oscillations in hippocampal astrocytes.

Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a Gi coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of Gq/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, Gi mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.

Green Fluorescent Protein-Based Glucose Indicators Report Glucose Dynamics in Living Cells.

Glucose is the most important energy source for living animals. Here, we developed a series of single fluorescent protein (FP)-based glucose indicators, named as "Green Glifons", to understand the hierarchal and mutual relationships between molecules involved in energy metabolism. Three indicators showed a different EC50 for glucose (50, 600, and 4000 µM), producing a ∼7-fold change in fluorescence intensity in response to glucose. The indicators could visualize glucose dynamics in the cytoplasm, plasma membrane, nucleus and mitochondria of living HeLa cells and in vivo, in the pharyngeal muscle of C. elegans and could measure murine blood glucose levels. Finally, the indicators were applicable to dual-color imaging, revealing the dynamic interplay between glucose and Ca2+ in mouse pancreatic MIN6 m9 ß cells. We propose that these indicators will facilitate and contribute to in vivo and multicolor imaging of energy metabolism.

Visualization of an endogenous retinoic acid gradient across embryonic development.

In vertebrate development, the body plan is determined by primordial morphogen gradients that suffuse the embryo. Retinoic acid (RA) is an important morphogen involved in patterning the anterior-posterior axis of structures, including the hindbrain and paraxial mesoderm. RA diffuses over long distances, and its activity is spatially restricted by synthesizing and degrading enzymes. However, gradients of endogenous morphogens in live embryos have not been directly observed; indeed, their existence, distribution and requirement for correct patterning remain controversial. Here we report a family of genetically encoded indicators for RA that we have termed GEPRAs (genetically encoded probes for RA). Using the principle of fluorescence resonance energy transfer we engineered the ligand-binding domains of RA receptors to incorporate cyan-emitting and yellow-emitting fluorescent proteins as fluorescence resonance energy transfer donor and acceptor, respectively, for the reliable detection of ambient free RA. We created three GEPRAs with different affinities for RA, enabling the quantitative measurement of physiological RA concentrations. Live imaging of zebrafish embryos at the gastrula and somitogenesis stages revealed a linear concentration gradient of endogenous RA in a two-tailed source-sink arrangement across the embryo. Modelling of the observed linear RA gradient suggests that the rate of RA diffusion exceeds the spatiotemporal dynamics of embryogenesis, resulting in stability to perturbation. Furthermore, we used GEPRAs in combination with genetic and pharmacological perturbations to resolve competing hypotheses on the structure of the RA gradient during hindbrain formation and somitogenesis. Live imaging of endogenous concentration gradients across embryonic development will allow the precise assignment of molecular mechanisms to developmental dynamics and will accelerate the application of approaches based on morphogen gradients to tissue engineering and regenerative medicine.

Lysophosphatidylinositol-induced activation of the cation channel TRPV2 triggers glucagon-like peptide-1 secretion in enteroendocrine L cells.

The lysophosphatidylinositol (LPI) has crucial roles in multiple physiological processes, including insulin exocytosis from pancreatic islets. However, the role of LPI in secretion of glucagon-like peptide-1 (GLP-1), a hormone that enhances glucose-induced insulin secretion, is unclear. Here, we used the murine enteroendocrine L cell line GLUTag and primary murine small intestinal cells to elucidate the mechanism of LPI-induced GLP-1 secretion. Exogenous LPI addition increased intracellular Ca2+ concentrations ([Ca2+] i ) in GLUTag cells and induced GLP-1 secretion from both GLUTag and acutely prepared primary intestinal cells. The [Ca2+] i increase was suppressed by an antagonist for G protein-coupled receptor 55 (GPR55) and by silencing of GPR55 expression, indicating involvement of Gq and G12/13 signaling pathways in the LPI-induced increased [Ca2+] i levels and GLP-1 secretion. However, GPR55 agonists did not mimic many of the effects of LPI. We also found that phospholipase C inhibitor and Rho-associated kinase inhibitor suppressed the [Ca2+] i increase and that LPI increased the number of focal adhesions, indicating actin reorganization. Of note, blockage or silencing of transient receptor potential cation channel subfamily V member 2 (TRPV2) channels suppressed both the LPI-induced [Ca2+] i increase and GLP-1 secretion. Furthermore, LPI accelerated TRPV2 translocation to the plasma membrane, which was significantly suppressed by a GPR55 antagonist. These findings suggest that TRPV2 activation via actin reorganization induced by Gq and G12/13 signaling is involved in LPI-stimulated GLP-1 secretion in enteroendocrine L cells. Because GPR55 agonists largely failed to mimic the effects of LPI, its actions on L cells are at least partially independent of GPR55 activation.

Correction: Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation.

Correction for 'Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation' by Jiulong Su et al., Analyst, 2018, 143, 2096-2101.

Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation.

In this study, a novel noncompetitive homogeneous immunoassay for antigen detection was developed. We utilized ß-glucuronidase (GUS), a homotetrameric enzyme, the assembly of all of whose subunits is necessary to attain its activity. By using a mutant GUS (GUSm), wherein the dimerization of dimers, which is a rate-limiting step, can be effectively inhibited by a set of interface mutations, we attempted to create a biosensor for detecting various molecules. Usually, the affinity between the two variable region domains (VH and VL) of an antibody, especially for a small molecule, is relatively low. However, in the presence of an antigen, the affinity increases so that they bind tighter to each other. A pair of fusion proteins, comprising the VH and VL regions of the antibody as the detector tethered to a GUSm subunit as the reporter, was constructed to detect antigen 4-hydroxy-3-nitrophenylacetyl (NP) and bone Gla protein (BGP) through GUS activity measurement. Colorimetric and fluorescence assays could detect NP, 5-iodo-NP, and BGP within 1 h without separation steps and with a higher signal/background ratio than conventional ELISA. The instantaneous response after simple mixing of the components makes this system convenient and high-throughput. The system could be effective for the analyses of various small molecules in environmental and clinical settings.

RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells.

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

Positive Allosteric Modulation of the Calcium-sensing Receptor by Physiological Concentrations of Glucose.

The calcium-sensing receptor (CaSR) is activated by various cations, cationic compounds, and amino acids. In the present study we investigated the effect of glucose on CaSR in HEK293 cells stably expressing human CaSR (HEK-CaSR cells). When glucose concentration in the buffer was raised from 3 to 25 mm, a rapid elevation of cytoplasmic Ca2+ concentration ([Ca2+]c) was observed. This elevation was immediate and transient and was followed by a sustained decrease in [Ca2+]c The effect of glucose was detected at a concentration of 4 mm and reached its maximum at 5 mm 3-O-Methylglucose, a non-metabolizable analogue of glucose, reproduced the effect of glucose. Sucrose also induced an elevation of [Ca2+]c in HEK-CaSR cells. Similarly, sucralose was nearly as effective as glucose in inducing elevation of [Ca2+]c Glucose was not able to increase [Ca2+]c in the absence of extracellular Ca2+ The effect of glucose on [Ca2+]c was inhibited by NPS-2143, an allosteric inhibitor of CaSR. In addition, NPS-2143 also inhibited the [Ca2+]c responses to sucralose and sucrose. Glucose as well as sucralose decreased cytoplasmic cAMP concentration in HEK-CaSR cells. The reduction of cAMP induced by glucose was blocked by pertussis toxin. Likewise, sucralose reduced [cAMP]c Finally, glucose increased [Ca2+]c in PT-r parathyroid cells and in Madin-Darby canine kidney cells, both of which express endogenous CaSR. These results indicate that glucose acts as a positive allosteric modulator of CaSR.

Flashbody: A Next Generation Fluobody with Fluorescence Intensity Enhanced by Antigen Binding.

Fluorescent probes are valuable tools for visualizing the spatiotemporal dynamics of molecules in living cells. Here we developed a genetically encoded antibody probe with antigen-dependent fluorescence intensity called "Flashbody". We first created a fusion of EGFP to the single chain variable region fragment (scFv) of antibody against seven amino acids of the bone Gla protein C-terminus (BGPC7) called BGP Fluobody, which successfully showed the intracellular localization of BGPC7-tagged protein. To generate BGP Flashbody, circularly permuted GFP was inserted in between two variable region fragments, and the linkers were optimized, resulting in fluorescence intensity increase of 300% upon binding with BGPC7 in a dose-dependent manner. Live-cell imaging using BGP Flashbody showed that BGPC7 fused with cell penetrating peptide was able to enter through the plasma membrane by forming a nucleation zone, while it penetrated the nuclear membrane with different mechanism. The construction of Flashbody will be possible for a range of antibody fragments and opens up new possibilities for visualizing a myriad of molecules of interest.

Integrative function of adrenaline receptors for glucagon-like peptide-1 exocytosis in enteroendocrine L cell line GLUTag.

Adrenaline reacts with three types of adrenergic receptors, α1, α2 and ß-adrenergic receptors (ARs), inducing many physiological events including exocytosis. Although adrenaline has been shown to induce glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells, the precise molecular mechanism by which adrenaline regulates GLP-1 secretion remains unknown. Here we show by live cell imaging that all types of adrenergic receptors are stimulated by adrenaline in enteroendocrine L cell line GLUTag cells and are involved in GLP-1 exocytosis. We performed RT-PCR analysis and found that α1B-, α2A-, α2B-, and ß1-ARs were expressed in GLUTag cells. Application of adrenaline induced a significant increase of intracellular Ca(2+) and cAMP concentration ([Ca(2+)]i and [cAMP]i, respectively), and GLP-1 exocytosis in GLUTag cells. Blockade of α1-AR inhibited adrenaline-induced [Ca(2+)]i increase and exocytosis but not [cAMP]i increase, while blockade of ß1-AR inhibited adrenaline-induced [cAMP]i increase and exocytosis but not [Ca(2+)]i increase. Furthermore, overexpression of α2A-AR suppressed the adrenaline-induced [cAMP]i increase and exocytosis. These results suggest that the fine-turning of GLP-1 secretion from enteroendocrine L cells is established by the balance between α1-, α2-, and ß-ARs activation.

The G protein-coupled receptor family C group 6 subtype A (GPRC6A) receptor is involved in amino acid-induced glucagon-like peptide-1 secretion from GLUTag cells.

Although amino acids are dietary nutrients that evoke the secretion of glucagon-like peptide 1 (GLP-1) from intestinal L cells, the precise molecular mechanism(s) by which amino acids regulate GLP-1 secretion from intestinal L cells remains unknown. Here, we show that the G protein-coupled receptor (GPCR), family C group 6 subtype A (GPRC6A), is involved in amino acid-induced GLP-1 secretion from the intestinal L cell line GLUTag. Application of l-ornithine caused an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in GLUTag cells. Application of a GPRC6A receptor antagonist, a phospholipase C inhibitor, or an IP(3) receptor antagonist significantly suppressed the l-ornithine-induced [Ca(2+)](i) increase. We found that the increase in [Ca(2+)](i) stimulated by l-ornithine correlated with GLP-1 secretion and that l-ornithine stimulation increased exocytosis in a dose-dependent manner. Furthermore, depletion of endogenous GPRC6A by a specific small interfering RNA (siRNA) inhibited the l-ornithine-induced [Ca(2+)](i) increase and GLP-1 secretion. Taken together, these findings suggest that the GPRC6A receptor functions as an amino acid sensor in GLUTag cells that promotes GLP-1 secretion.

Extracellular calcium influx activates adenylate cyclase 1 and potentiates insulin secretion in MIN6 cells.

Intracellular cAMP and Ca(2+) are important second messengers that regulate insulin secretion in pancreatic ß-cells; however, the molecular mechanism underlying their mutual interaction for exocytosis is not fully understood. In the present study, we investigated the interplay between intracellular cAMP and Ca(2+) concentrations ([cAMP](i) and [Ca(2+)](i) respectively) in the pancreatic ß-cell line MIN6 using total internal reflection fluorescence microscopy. For measuring [cAMP](i), we developed a genetically encoded yellow fluorescent biosensor for cAMP [Flamindo (fluorescent cAMP indicator)], which changes fluorescence intensity with cAMP binding. Application of high-KCl or glucose to MIN6 cells induced the elevation of [cAMP](i) and exocytosis. Furthermore, application of an L-type Ca(2+) channel agonist or ionomycin to induce extracellular Ca(2+) influx evoked the elevation of [cAMP](i), whereas application of carbachol or thapsigargin, which mobilize Ca(2+) from internal stores, did not evoke the elevation of [cAMP](i). We performed RT (reverse transcription)-PCR analysis and found that Ca(2+)-sensitive Adcy1 (adenylate cyclase 1) was expressed in MIN6 cells. Knockdown of endogenous ADCY1 by small interference RNA significantly suppressed glucose-induced exocytosis and the elevation of both [cAMP](i) and [Ca(2+)](i). Taken together, the findings of the present study demonstrate that ADCY1 plays an important role in the control of pancreatic ß-cell cAMP homoeostasis and insulin secretion.

[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.