[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.

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.

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.

Development of a Single Fluorescent Protein-Based Green Glucose Indicator by Semirational Molecular Design and Molecular Evolution.

Advances in live-cell imaging have been accelerated by the development of various fluorescent indicators. However, indicators that are suitable for multicolor imaging remain a challenge to develop. Herein, we have developed a single fluorescent protein (FP)-based indicator using a semirational molecular design and a molecular evolution approach. We first inserted a ligand-binding domain into the vicinity of an FP chromophore to convert the conformational change induced by ligand binding into a change in fluorescence intensity. We then optimized the linker regions between the FP and the ligand-binding domain to greatly expand the dynamic range (F/F0) of the indicator. Our design and optimization methods are highly versatile and can be used to develop any single FP-based indicators, which will further advance the utility of live-cell imaging.

Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging.

Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can then produce lactate from pyruvate. Here, we developed single fluorescent protein (FP)-based lactate and pyruvate indicators with low EC50 for trace detection of metabolic molecules and live cell imaging and named them "Green Lindoblum" and "Green Pegassos," respectively. Green Lindoblum (EC50 of 30 µM for lactate) and Green Pegassos (EC50 of 70 µM for pyruvate) produced a 5.2- and 3.3-fold change in fluorescence intensity in response to lactate and pyruvate, respectively. Green Lindoblum measured lactate levels in mouse plasma, and Green Pegassos in combination with D-serine dehydratase successfully estimated D-serine levels released from mouse primary cultured neurons and astrocytes by measuring pyruvate level. Furthermore, live cell imaging analysis revealed their utility for dual-colour imaging, and the interplay between lactate, pyruvate, and Ca2+ in human induced pluripotent stem cell-derived cardiomyocytes. Therefore, Green Lindoblum and Green Pegassos will be useful tools that detect specific molecules in clinical use and monitor the interplay of metabolites and other related molecules in diverse cell types.

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.