Hideo Takakura

Aminoluciferins as functional bioluminogenic substrates of firefly luciferase.

Firefly luciferase is widely used as a reporter gene in assays to study gene expression, gene delivery, and so on because of its extremely high signal-to-noise ratio. The availability of a range of bioluminogenic substrates would greatly extend the applicability of the luciferin-luciferase system. Herein, we describe a design concept for functional bioluminogenic substrates based on the aminoluciferin (AL) scaffold, together with a convenient, high-yield method for synthesizing N-alkylated ALs. We confirmed the usefulness of ALs as bioluminogenic substrates by synthesizing three probes. The first was a conjugate of AL with glutamate, Glu-AL. When Glu-AL, the first membrane-impermeable bioluminogenic substrate of luciferases, was applied to cells transfected with luciferase, luminescence was not observed; that is, by using Glu-AL, we can distinguish between intracellular and extracellular events. The second was Cy5-AL, which consisted of Cy5, a near-infrared (NIR) cyanine fluorescent dye, and AL, and emitted NIR light. When Cy5-AL reacted with luciferase, luminescence derived from Cy5 was observed as a result of bioluminescence resonance energy transfer (BRET) from AL to Cy5. The NIR emission wavelength would allow a signal to be observed from deeper tissues in bioluminescence in vivo imaging. The third was biotin-DEVD-AL (DEVD = the amino acid sequence Asp-Glu-Val-Asp), which employed a caspase-3 substrate peptide as a switch to control the accessibility of the substrate to luciferase, and could detect the activity of caspase-3 in a time-dependent manner. This generalized design strategy should be applicable to other proteases. Our results indicate that the AL scaffold is appropriate for a range of functional luminophores and represents a useful alternative substrate to luciferin.

Development of Luciferin Analogues Bearing an Amino Group and Their Application as BRET Donors

We systematically synthesized bioluminogenic substrates bearing an amino group on benzothiazole, quinoline, naphthalene, and coumarin scaffolds. They emit bioluminescence in various colors: red, orange, yellow, and green. An amino-substituted coumarylluciferin derivative, coumarylaminoluciferin (CAL), showed the shortest bioluminescence wavelength among substrates reported so far. Further, the fluorescence of CAL did not exhibit solvatochromism, which suggests that its bioluminescence is not susceptible to environmental factors. We applied CAL as an energy-donor substrate for a bioluminescence resonance energy transfer (BRET) system with click beetle red luciferase (CBRluc), a mutant of firefly luciferase, as the energy-donor enzyme and yellow fluorescent protein (YFP) as the energy-acceptor fluorophore, and obtained a clearly bimodal bioluminescence spectrum. Stable bioluminescence that is not influenced by environmental factors is highly desirable for reliable measurements in biological assays.

Visualization and Quantitative Analysis of G Protein-Coupled Receptor−β-Arrestin Interaction in Single Cells and Specific Organs of Living Mice Using Split Luciferase Complementation

Methods used to assess the efficacy of potentially therapeutic reagents for G protein-coupled receptors (GPCRs) have been developed. Previously, we demonstrated sensitive detection of the interaction of GPCRs and β-arrestin2 (ARRB2) using 96-well microtiter plates and a bioluminescence microscope based on split click beetle luciferase complementation. Herein, using firefly luciferase emitting longer wavelength light, we demonstrate quantitative analysis of the interaction of β2-adrenergic receptor (ADRB2), a kind of GPCR, and ARRB2 in a 96-well plate assay with single-cell imaging. Additionally, we showed bioluminescence in vivo imaging of the ADRB2-ARRB2 interaction in two systems: cell implantation and hydrodynamic tail vein (HTV) methods. Specifically, in the HTV method, the luminescence signal from the liver upon stimulation of an agonist for ADRB2 was obtained in the intact systems of mice. The results demonstrate that this method enables noninvasive screening of the efficacy of chemicals at the specific organ in in vivo testing. This in vivo system can contribute to effective evaluation in pharmacokinetics and pharmacodynamics and expedite the development of new drugs for GPCRs.

New class of bioluminogenic probe based on bioluminescent enzyme-induced electron transfer: BioLeT.

Bioluminescence imaging (BLI) has advantages for investigating biological phenomena in deep tissues of living animals, but few design strategies are available for functional bioluminescent substrates. We propose a new design strategy (designated as bioluminescent enzyme-induced electron transfer: BioLeT) for luciferin-based bioluminescence probes. Luminescence measurements of a series of aminoluciferin derivatives confirmed that bioluminescence can be controlled by means of BioLeT. Based on this concept, we developed bioluminescence probes for nitric oxide that enabled quantitative and sensitive detection even in vivo. Our design strategy should be applicable to develop a wide range of practically useful bioluminogenic probes.

Development of a Sensitive Bioluminogenic Probe for Imaging Highly Reactive Oxygen Species in Living Rats

A sensitive bioluminogenic probe for highly reactive oxygen species (hROS), SO3 H-APL, was developed based on the concept of dual control of bioluminescence emission by means of bioluminescent enzyme-induced electron transfer (BioLeT) and modulation of cell-membrane permeability. This probe enables non-invasive visualization of physiologically relevant amounts of hROS generated deep inside the body of living rats for the first time. It is expected to serve as a practical analytical tool for investigating a wide range of biological functions of hROS in vivo. The design concept should be applicable to other in vivo bioluminogenic probes.

Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein

Regulation of an intracellular acidic environment plays a pivotal role in biological processes and functions. However, spatiotemporal analysis of the acidification in complex tissues of living subjects persists as an important challenge. We developed a photo-inactivatable bioluminescent indicator, based on a combination of luciferase-fragment complementation and a photoreaction of a light, oxygen, and voltage domain from Avena sativa Phototropin1 (LOV2), to visualize temporally dynamic acidification in living tissue samples. Bioluminescence of the indicator diminished upon light irradiation and it recovered gradually in the dark state thereafter. The recovery rate was remarkably sensitive to pH changes but unsusceptible to fluctuation of luciferin or ATP concentrations. Bioluminescence imaging, taken as an index of the recovery rates, enabled long-time recording of acidification in apoptotic and autophagous processes in a cell population and an ischemic condition in living mice. This technology using the indicator is widely applicable to sense organelle-specific acidic changes in target biological tissues.

Development of 5′‐ and 7′‐Substituted Luciferin Analogues as Acid‐Tolerant Substrates of Firefly Luciferase

Firefly or beetle luciferase catalyzes the oxidation of d-luciferin (d-LH2) in the presence of adenosine triphosphate (ATP), Mg 2+ , and molecular oxygen, generating bioluminescence. This luciferin–luciferase reaction is widely used as a reporter in biochemical assays, in cell cultures, and also recently in animal models to investigate gene expression, gene delivery, tumor progression, and more due to the extremely high quantum yield of the reaction (Fbl = 0.41 ). However, a drawback of this system is its pH sensitivity ; both the luminescence wavelength and the luminescence intensity are pH-dependent, especially in the biologically relevant range from weakly acidic to neutral (pH 6.0–7.4). Although intracellular pH (pHi) is strictly regulated by various cellular homeostatic mechanisms, including Na /H exchange, vacuolar-type H-ATPase, and HCO3 transporters and exchangers, 9] abnormal pHi, especially in the acidic range, is observed in various pathological conditions including cancer and Alzheimer’s disease. 12] Indeed, pHi variations of only 0.1–0.2 units can disrupt multiple biological functions. 14] On the other hand, transient acidification is associated with electrical activity in the brain, where it influences the activities of enzymes and ion channels. Intracellular acidification also acts as a trigger for various physiological phenomena, such as apoptosis 17] and protein degradation, and is associated with various physiological states, such as hypoxia, ischemia, and muscle fatigue. Therefore, pH insensitivity, especially acid tolerance, is a highly desirable property of tools designed to investigate biological systems. In the case of the luciferin–luciferase system, various mutants and variants of luciferase and some luciferin analogues have been developed to overcome the pH sensitivity of luminescence wavelength, but little work has been done to address the problem of the pH dependence of luminescence intensity. 31] In order to extend the applicability of luciferin–luciferase-based assay systems, we have developed acid-tolerant 5’and 7’-substituted luciferin analogues, which exhibit constant luminescence wavelength and more stable luminescence intensity in response to acidic pH changes, and we have confirmed their utility for bioluminescence microscopic imaging of single cells in an acidic environment. First, we focused on the pKa value of the hydroxy group at the 6’-position of d-LH2 (pKa 8.5), which is expected to contribute to the pH sensitivity of luminescence wavelength and intensity. In order to decrease the pKa value, we introduced a halogen substituent such as fluoride or chloride at the 7’-position of d-LH2, obtaining 7F-LH2 and 7Cl-LH2 (Scheme 1). Next, since almost no 5’-substituted analogues of d-LH2 are available, we examined the influence of 5’-substitution on pH dependence of the luminescence properties. We initially focused on the Claisen rearrangement reaction to introduce the 5’-substituent but obtained only the 7’-substituted derivative (Scheme S1). Therefore, 2 a and 2 b were selected as starting materials with 7’-position substitutions; these compounds afforded 4 a and 4 b, respectively, in quite low yield (Scheme 1). Using these compounds, d-LH2 derivatives bearing an allyl or a hydroxymethyl (HM) group at the 5’-position: 7F5allyl-LH2, 7F5HM-LH2, 7Cl5allyl-LH2, and 7Cl5HM-LH2, were successfully synthesized (Scheme 1). The 5’or 7’-substituted substrates and aminoluciferin (AL), a typical substrate with a pH-insensitive spectrum, were compared in a bioluminescence assay with firefly luciferase (Fluc). Luminescence was observed from all substrates under various pH conditions (Figures 1 and S1). In the cases of d-LH2, 7F-LH2, and 7Cl-LH2, the luminescence emission gradually changed from yellow-green in weakly basic medium to red in weakly acid medium, whereas the luminescence emission

Long time-lapse nanoscopy with spontaneously blinking membrane probes

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.

Analysis of temporal patterns of GPCR–β-arrestin interactions using split luciferase-fragment complementation

We developed bioluminescence probes to detect quantitative interaction of GPCRs with arrestin isoforms β-arrestin1 and β-arrestin2 based on split luciferase complementation. Time-dependent GPCR-β-arrestin interactions showed two-types of remarkable variations that were consistent with a classification of GPCR classes. Positive charge residues in serine clusters located at the C-terminal region of GPCRs were necessary for binding to β-arrestin. This quantitative method enables elucidation of the mechanisms of different classes of GPCRs that regulate β-arrestin isoforms.

Cell-based assays and animal models for GPCR drug screening.

The family of G protein-coupled receptors (GPCRs) remains a central focus of basic pharmacology and drug discovery efforts. Convenient methods to assess the efficacy of potentially therapeutic reagents for GPCRs are strongly required for high-throughput screening (HTS) assay. We recently developed a rapid, sensitive, and quantitative method for detecting potential chemicals that act on GPCRs using split luciferase complementation. In principle, this is based on the detection of interactions of GPCR with β-arrestin, which translocates to the activated GPCRs. This method can facilitate the construction of HTS systems in a multi-well plate format. Particularly, the method is compatible with single-cell imaging and animal models and even deeper tissues such as organs, because of its high sensitivity, suggesting that promising candidates from HTS assay can be moved easily to the next phase for additional analysis. This system can contribute to the effective evaluation of potentially therapeutic reagents and expedite the development of new drugs for GPCRs.