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.

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.

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.

Generation of a cGMP Indicator with an Expanded Dynamic Range by Optimization of Amino Acid Linkers between a Fluorescent Protein and PDE5α.

Here we describe the development of a single fluorescent protein (FP)-based cGMP indicator, Green cGull, based on the cGMP binding domain from mouse phosphodiesterase 5α. The dynamic range of Green cGull was enhanced to a 7.5-fold fluorescence change upon cGMP binding by optimization of the amino acid linkers between the cGMP binding domain and FP. Green cGull has excitation and emission peaks at 498 and 522 nm, respectively, and specifically responds to cGMP in a dose-dependent manner. Live cell imaging analysis revealed that addition of a nitric oxide (NO) donor induced different cGMP kinetics and was cell-type dependent. We also found that the NO donor induced an increase of intracellular cGMP, while intracellular Ca2+ exhibited a complex profile, as revealed by dual-color imaging of cGMP and Ca2+. The results suggest that Green cGull sheds new light on understanding the complex interactions between various signaling molecules by multicolor imaging and that our systematic strategy for expanding the dynamic range of single-FP-based indicators is valuable to generate indicators for molecules of interest.

Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging.

cAMP is a common second messenger that is involved in various physiological processes. To expand the colour palette of available cAMP indicators, we developed a red cAMP indicator named "Pink Flamindo" (Pink Fluorescent cAMP indicator). The fluorescence intensity of Pink Flamindo increases 4.2-fold in the presence of a saturating dose of cAMP, with excitation and emission peaks at 567 nm and 590 nm, respectively. Live-cell imaging revealed that Pink Flamindo is effective for monitoring the spatio-temporal dynamics of intracellular cAMP generated by photoactivated adenylyl cyclase in response to blue light, and in dual-colour imaging studies using a green Ca2+ indicator (G-GECO). Furthermore, we successfully monitored the elevation of cAMP levels in vivo in cerebral cortical astrocytes by two-photon imaging. We propose that Pink Flamindo will facilitate future in vivo, optogenetic studies of cell signalling and cAMP dynamics.