論文 - 花岡　健二郎

Design of spontaneously blinking fluorophores for live-cell super-resolution imaging based on quantum-chemical calculations

Tachibana R., Kamiya M., Morozumi A., Miyazaki Y., Fujioka H., Nanjo A., Kojima R., Komatsu T., Ueno T., Hanaoka K., Yoshihara T., Tobita S., Urano Y.

Chemical Communications （Chemical Communications）  56 （ 86 ） 13173 - 13176 2020年11月

研究論文（学術雑誌）, 査読有り,  ISSN  13597345

　概要を見る

Spontaneously blinking fluorophores are powerful tools for live-cell super-resolution imaging under physiological conditions. Here we show that quantum-chemical calculations can predict key parameters for fluorophore design. We applied this methodology to develop a spontaneously blinking fluorophore with yellow fluorescence for super-resolution imaging of microtubules in living cells. This journal is

• Access to Document (DOI)

Antibody Clicking as a Strategy to Modify Antibody Functionalities on the Surface of Targeted Cells

Komatsu T., Kyo E., Ishii H., Tsuchikama K., Yamaguchi A., Ueno T., Hanaoka K., Urano Y.

Journal of the American Chemical Society （Journal of the American Chemical Society）  142 （ 37 ） 15644 - 15648 2020年09月

研究論文（学術雑誌）, 査読有り,  ISSN  00027863

　概要を見る

We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens. Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased cargo delivery and synthetic manipulation of cellular signaling.

• Access to Document (DOI)

How Viscous Is the Solidlike Structure at the Interface of Ionic Liquids? A Study Using Total Internal Reflection Fluorescence Spectroscopy with a Fluorescent Molecular Probe Sensitive to High Viscosity

Nishi N., Yamazawa T., Sakka T., Hotta H., Ikeno T., Hanaoka K., Takahashi H.

Langmuir （Langmuir）  36 （ 35 ） 10397 - 10403 2020年09月

研究論文（学術雑誌）, 査読有り,  ISSN  07437463

　概要を見る

Aiming at the evaluation of the viscosity of the interfacial solidlike structure of ionic liquids (ILs), we performed total internal reflection fluorescence (TIRF) spectroscopy for N,N-diethyl-N′-phenyl-rhodamine (Ph-DER), a fluorescent probe that is sensitive to viscosity in a high-viscosity range. TIRF spectra at the glass interface of trioctylmethylammonium bis(nonafluorobutanesulfonyl)amide (TOMAC4C4N), a hydrophobic IL, showed that the fluorescence intensity of Ph-DER increases with the decrease of the evanescence penetration depth, suggesting that there exists a high-viscosity region at the interface. In contrast, glycerol, which is a molecular liquid with a bulk viscosity similar to that of TOMAC4C4N, did not show such a fluorescence increase, supporting that the formation of a highly viscous solidlike structure at the interface is intrinsic to ILs. A model analysis suggested that the high viscous region at the glass interface of TOMAC4C4N is at least twice thicker than the ionic multilayers at the air interface, implying that the solid substrate enhances the ordering of the interfacial structure of ILs. The viscosity at the glass interface of TOMAC4C4N was found to be at least 40 times higher than that of the liquid bulk.

• Access to Document (DOI)

Real-time in vivo imaging of extracellular atp in the brain with a hybrid-type fluorescent sensor

Kitajima N., Takikawa K., Sekiya H., Satoh K., Asanuma D., Sakamoto H., Takahashi S., Hanaoka K., Urano Y., Namiki S., Iino M., Hirose K.

eLife （eLife）  9   1 - 18 2020年07月

研究論文（学術雑誌）, 査読有り

　概要を見る

Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase e subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

• Access to Document (DOI)

A Fluorescent Probe for Rapid, High-Contrast Visualization of Folate-Receptor-Expressing Tumors In Vivo

Numasawa K., Hanaoka K., Saito N., Yamaguchi Y., Ikeno T., Echizen H., Yasunaga M., Komatsu T., Ueno T., Miura M., Nagano T., Urano Y.

Angewandte Chemie - International Edition （Angewandte Chemie - International Edition）  59 （ 15 ） 6015 - 6020 2020年04月

研究論文（学術雑誌）, 査読有り,  ISSN  14337851

　概要を見る

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near-infrared (NIR) region (650–900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1, utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30 min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.

• Access to Document (DOI)

Rational Design of a Near-infrared Fluorescence Probe for Ca²⁺ Based on Phosphorus-substituted Rhodamines Utilizing Photoinduced Electron Transfer

Takahashi S., Hanaoka K., Okubo Y., Echizen H., Ikeno T., Komatsu T., Ueno T., Hirose K., Iino M., Nagano T., Urano Y.

Chemistry - An Asian Journal （Chemistry - An Asian Journal）  15 （ 4 ） 524 - 530 2020年02月

研究論文（学術雑誌）, 査読有り,  ISSN  18614728

　概要を見る

Fluorescence imaging in the near-infrared (NIR) region (650–900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)-mediated fluorescence quenching of silicon- and phosphorus-substituted rhodamines (SiRs and PRs) in order to guide the development of improved far-red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca2+, CaPR-1, and its membrane-permeable acetoxymethyl derivative, CaPR-1 AM, which is distributed to the cytosol, in marked contrast to our previously reported Ca2+ far-red to NIR fluorescence probe based on the SiR scaffold, CaSiR-1 AM, which is mainly localized in lysosomes as well as cytosol in living cells. CaPR-1 showed longer-wavelength absorption and emission (up to 712 nm) than CaSiR-1. The new probe was able to image Ca2+ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.

• Access to Document (DOI)

Metabolic-Pathway-Oriented Screening Targeting S-Adenosyl- l -methionine Reveals the Epigenetic Remodeling Activities of Naturally Occurring Catechols

Ogihara S., Komatsu T., Itoh Y., Miyake Y., Suzuki T., Yanagi K., Kimura Y., Ueno T., Hanaoka K., Kojima H., Okabe T., Nagano T., Urano Y.

Journal of the American Chemical Society （Journal of the American Chemical Society）  142 （ 1 ） 21 - 26 2020年01月

研究論文（学術雑誌）, 査読有り,  ISSN  00027863

　概要を見る

Methyl transfer reactions play important roles in many biological phenomena, wherein the methylation cofactor S-adenosyl-l-methionine (SAM) serves as the important currency to orchestrate those reactions. We have developed a fluorescent-probe-based high-throughput screening (HTS) system to search for the compounds that control cellular SAM levels. HTS with a drug repositioning library revealed the importance of catechol-O-methyltransferase (COMT) and its substrates in controlling the SAM concentrations and histone methylation levels in colorectal tumor cells.

• Access to Document (DOI)

Detection of singularity in immunity and cancer by novel imaging techniques Comment

Nagai Takeharu, Chikuma Shunsuke, Hanaoka Kenjiro

BIOPHYSICS AND PHYSICOBIOLOGY 17   98 - 99 2020年

研究論文（国際会議プロシーディングス）

• Access to Document (DOI)

Design strategy for germanium-rhodamine based pH-activatable near-infrared fluorescence probes suitable for biological applications

Koide Y., Kojima R., Hanaoka K., Numasawa K., Komatsu T., Nagano T., Kobayashi H., Urano Y.

Communications Chemistry （Communications Chemistry）  2 （ 1 ）  2019年12月

研究論文（学術雑誌）, 査読有り

　概要を見る

Fluorescence probes that can detect pH are useful tools for biological research and clinical diagnosis. Here we report pH-activatable near-infrared fluorescence probes, based on hydroxymethyl germanium-rhodamine (HMGeR), that are suitable for a range of biological applications. The pKa, the ratio of the fluorescent form in an acidic environment, and the absorption/emission wavelengths can all be conveniently optimized. The most promising probe, 2-HM IGeR, offers significant advantages over currently available near-infrared pH probes, notably high quantum efficiency, appropriate pKa value for biological applications, and high photostability. Further, our molecular design strategy allows easy conjugation of the probes to biomolecules without loss of functionality. We illustrate the value of this strategy by developing probe-Herceptin® and probe-avidin conjugates to visualize pH change in cellular vesicles during endocytosis, and to visualize tumors in a mouse model, respectively. We believe 2-HM IGeR is currently among the best-in-class pH-activatable near-infrared probes for biological and medical research.

• Access to Document (DOI)

Development of ratiometric carbohydrate sensor based on boron dipyrromethene (BODIPY) scaffold

Cheruthu N.M., Komatsu T., Ueno T., Hanaoka K., Urano Y.

Bioorganic and Medicinal Chemistry Letters （Bioorganic and Medicinal Chemistry Letters）  29 （ 22 ） 126684 2019年11月

研究論文（学術雑誌）, 査読有り,  ISSN  0960894X

　概要を見る

We designed a ratiometric carbohydrate sensor consisting of the boron dipyrromethene fluorophore substituted with boronic acid at the 2-position, based upon the strong substituent dependency of the absorbance/fluorescence wavelengths of BODIPY. The substituent is in equilibrium between the boronic acid B(OH)2 and boronate (B(OH)3−) forms, which have different absorbance/fluorescence wavelengths in the visible region. Reaction of the boronic acid moiety with hydroxy groups of carbohydrate affords a cyclic ester and shifts the equilibrium in favor of the boronate (B(OR)3−) form, resulting in a carbohydrate-concentration-dependent change of the fluorescence ratio. Thus, the sensor, BA-BODIPY, can ratiometrically detect carbohydrate at a pH near the pKa of cyclic ester formation.

• Access to Document (DOI)

Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples

Ichihashi Y., Komatsu T., Kyo E., Matsuzaki H., Hata K., Watanabe T., Ueno T., Hanaoka K., Urano Y.

Analytical Chemistry （Analytical Chemistry）  91 （ 18 ） 11497 - 11501 2019年09月

研究論文（学術雑誌）, 査読有り,  ISSN  00032700

　概要を見る

We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.

• Access to Document (DOI)

Design and Synthesis of an Activatable Photoacoustic Probe for Hypochlorous Acid

Ikeno T., Hanaoka K., Iwaki S., Myochin T., Murayama Y., Ohde H., Komatsu T., Ueno T., Nagano T., Urano Y.

Analytical Chemistry （Analytical Chemistry）  91 （ 14 ） 9086 - 9092 2019年06月

研究論文（学術雑誌）, 査読有り,  ISSN  00032700

　概要を見る

Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetration of ultrasound. PA imaging contrast agents targeting various biological phenomena have been reported, but the development of activatable PA probes, which show a PA signal only in the presence of target molecules, remains challenging in spite of their potential usefulness for real-time PA imaging of specific biomolecules in vivo. To establish a simple design strategy for activatable PA probes, we first designed and synthesized a silicon-rhodamine based near-infrared nonfluorescent dye, wsSiNQ660 (water-soluble SiNQ660), as a scaffold and demonstrated that it offers a high conversion efficiency from light to ultrasound compared to typical near-infrared fluorescent dyes. Importantly, absorption off/on strategies previously established for rhodamine-based fluorescent probes are also applicable to this nonfluorescent dye scaffold. We validated this approach by synthesizing an activatable PA probe for hypochlorous acid (HOCl) and confirmed that it enables three-dimensional imaging of HOCl in mouse subcutis.

• Access to Document (DOI)

Design of Photostable, Activatable Near-Infrared Photoacoustic Probes Using Tautomeric Benziphthalocyanine as a Platform

Toriumi N., Asano N., Ikeno T., Muranaka A., Hanaoka K., Urano Y., Uchiyama M.

Angewandte Chemie - International Edition （Angewandte Chemie - International Edition）  58 （ 23 ） 7788 - 7791 2019年06月

研究論文（学術雑誌）, 査読有り,  ISSN  14337851

　概要を見る

Near-infrared (NIR) imaging techniques have attracted significant attention for biological and medicinal applications due to the ability of NIR to penetrate deeply into tissues. However, there are very few stable, activatable molecular probes that can utilize NIR light in the wavelength range beyond 800 nm. Herein, we report a new activatable NIR system for photoacoustic imaging based on tautomeric benziphthalocyanines (BPcs). We found that the existence of a free hydroxyl group is crucial for NIR absorption of BPcs. Synthesized water-soluble hydroxy BPcs exhibited high photostability and no fluorescence, which are desirable features for photoacoustic imaging. We synthesized BPcs in which the free hydroxyl group was masked by an esterase-labile or an H2O2-labile group. The photoacoustic signals of these hydroxy-masked BPcs were increased upon NIR excitation at 880 nm in the presence of esterase or H2O2, respectively. These are rare examples of activatable probes utilizing NIR light at around 900 nm.

• Access to Document (DOI)

Development of a platform for activatable fluorescent substrates of glucose transporters (GLUTs)

Takasugi T., Hanaoka K., Sasaki A., Ikeno T., Komatsu T., Ueno T., Yamada K., Urano Y.

Bioorganic and Medicinal Chemistry （Bioorganic and Medicinal Chemistry）  27 （ 10 ） 2122 - 2126 2019年05月

研究論文（学術雑誌）, 査読有り,  ISSN  09680896

　概要を見る

We have developed a platform for activatable fluorescent substrates of glucose transporters (GLUTs). We firstly conjugated fluorescein to glucosamine via an amide or methylene linker at the C-2 position of D-glucosamine, but the resulting compounds, FLG1 and FLG2, showed no uptake into MIN6 cells. So, we changed the fluorophore moiety to a fluorescein analogue, 2-Me TokyoGreen, which is less negatively charged. TokyoGreen-conjugated glucosamines TGG1 and TGG2 were successfully taken up into cells via GLUT. We further derivatized TGG1 and TGG2, and among the synthesized compounds, 2-Me-4-OMe TGG showed weak fluorescence under the acidic conditions of the extracellular environment inside tumors and in gastric cancers, and strong fluorescence at the intracellular physiological pH, under the control of a photoinduced electron transfer (PeT) process. This fluorogenic platform should be useful for developing a range of activatable fluorescent substrates targeting GLUTs, as well as derivatives that would be fluorescently activated by various intracellular enzymes, such as esterases, β-galactosidase and bioreductases.

• Access to Document (DOI)

Red Fluorescence Probe Targeted to Dipeptidylpeptidase-IV for Highly Sensitive Detection of Esophageal Cancer

Ogasawara A., Kamiya M., Sakamoto K., Kuriki Y., Fujita K., Komatsu T., Ueno T., Hanaoka K., Onoyama H., Abe H., Tsuji Y., Fujishiro M., Koike K., Fukayama M., Seto Y., Urano Y.

Bioconjugate Chemistry （Bioconjugate Chemistry）  30 （ 4 ） 1055 - 1060 2019年04月

研究論文（学術雑誌）, 査読有り,  ISSN  10431802

　概要を見る

We have developed an activatable red fluorescence probe for dipeptidylpeptidase-IV (DPP-IV) by precisely controlling the photoinduced electron transfer (PeT) process of a red fluorescent scaffold, SiR600. The developed probe exhibited an extremely low background signal and showed significant fluorescence activation upon reaction with DPP-IV, enabling sensitive detection of esophageal cancer in clinical specimens from cancer patients.

• Access to Document (DOI)

Development of novel dark quenchers and their application to imaging probes

Hanaoka K.

Yakugaku Zasshi （Yakugaku Zasshi）  139 （ 2 ） 277 - 283 2019年

研究論文（学術雑誌）, 査読有り,  ISSN  00316903

　概要を見る

Rhodamine dyes are among the most widely used fluorescent dyes for bioimaging due to their high fluorescence quantum yield and high photostability. Recently, novel far-red to near-infrared (NIR) fluorescent dyes have been developed: Si-rhodamines (SiRs), in which the O atom of conventional rhodamine dyes at the 10 position of the xanthene moiety is replaced with a Si atom. These SiRs are excellent long-wavelength fluorophores for bioimaging, as they retain the advantageous photophysical properties of conventional rhodamine dyes. Further, we focused on the QSY dark quenchers, which contain the rhodamine scaŠold bearing aromatic rings at the N atoms at the 3,6-positions of the xanthene moiety; these show no fluorescence, irrespective of solvent polarity and pH. NIR fluorescent probes based on the F orster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). We successfully developed SiR-based NIR dark quenchers (SiNQs) which show broad absorption covering this region. To demonstrate their usefulness, we designed and synthesized a NIR fluorescence probe for matrix metalloproteinase (MMP) activity using SiNQs. The developed probe was able to detect MMP activity in terms of NIR fluorescence, not only in vitro, but also in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized.

• Access to Document (DOI)

Development of fluorescent probes for detecting reactive sulfur species and their application to development of inhibitors for 3MST

Echizen H., Hanaoka K.

Folia Pharmacologica Japonica （Folia Pharmacologica Japonica）  154 （ 3 ） 121 - 127 2019年

研究論文（学術雑誌）, 査読有り,  ISSN  00155691

　概要を見る

Hydrogen sulfide (H2S) has been reported to play an important role in biological systems. More recently, sulfane sulfur (sulfur with 0 or −1 charge) molecules have been also reported to be involved in various biological phenomena such as regulation of redox signaling and antioxidant functions. Fluorescent probes are one of the important chemical tools because it is easy to use and enable the real-time detection of the target molecules in living cells and tissues. We have successfully developed a highly selective H2S-detecting fluorescent probe, HSip-1. HSip-1 has been designed on the basis of the facts that the macrocyclic polyamine ligands form a stable complex with Cu2+, and Cu2+ also reacts with H2S and make a stable CuS complex. SSip-1 is a fluorescent probe for detecting sulfane sulfur and this fluorescent probe is designed on the basis of the unique feature of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. SSip-1 is a highly selective fluorescent probe and can detect sulfane sulfur reversibly. Both HSip-1 and SSip-1 were able to be used for the live-cell fluorescence imaging. Further, we applied HSip-1 to the high-throughput screening (HTS) for the inhibitors of 3-mercaptopyruvate sulfurtransferase (3MST), one of the reactive sulfur species (RSS)-generating enzymes. We successfully found new 3MST inhibitors by screening of 174,118 compounds. We expect that these fluorescent probes and inhibitors would be useful to elucidate new functions of RSS and RSS-generating enzymes.

• Access to Document (DOI)

Rapid detection of metastatic lymph nodes of colorectal cancer with a gamma-glutamyl transpeptidase-activatable fluorescence probe

Kubo H., Hanaoka K., Kuriki Y., Komatsu T., Ueno T., Kojima R., Kamiya M., Murayama Y., Otsuji E., Urano Y.

Scientific Reports （Scientific Reports）  8 （ 1 ） 17781 2018年12月

研究論文（学術雑誌）, 査読有り

　概要を見る

Rapid diagnosis of metastatic lymph nodes (mLNs) of colorectal cancer (CRC) is desirable either intraoperatively or in resected fresh specimens. We have developed a series of activatable fluorescence probes for peptidase activities that are specifically upregulated in various tumors. We aimed to discover a target enzyme for detecting mLNs of CRC. Among our probes, we found that gGlu-HMRG, a gamma-glutamyl transpeptidase (GGT)-activatable fluorescence probe, could detect mLNs. This was unexpected, because we have previously reported that gGlu-HMRG could not detect primary CRC. We confirmed that the GGT activity of mLNs was high, whereas that of non-metastatic lymph nodes and CRC cell lines was low. We investigated the reason why GGT activity was upregulated in mLNs, and found that GGT was induced under conditions of hypoxia or low nutritional status. We utilized this feature to achieve rapid detection of mLNs with gGlu-HMRG. GGT appears to be a promising candidate enzyme for fluorescence imaging of mLNs.

• Access to Document (DOI)

Red-Shifted Fluorogenic Substrate for Detection of lacZ-Positive Cells in Living Tissue with Single-Cell Resolution

Ito H., Kawamata Y., Kamiya M., Tsuda-Sakurai K., Tanaka S., Ueno T., Komatsu T., Hanaoka K., Okabe S., Miura M., Urano Y.

Angewandte Chemie - International Edition （Angewandte Chemie - International Edition）  57 （ 48 ） 15702 - 15706 2018年11月

研究論文（学術雑誌）, 査読有り,  ISSN  14337851

　概要を見る

The Escherichia coli lacZ gene encoding β-galactosidase is a widely used reporter, but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples. Our recently reported fluorogenic substrate SPiDER-βGal is suitable for this purpose, but its hydrolysis product shows green fluorescence emission, and a red-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers. Herein, we describe the development of a red-shifted fluorogenic substrate for β-galactosidase, SPiDER-Red-βGal, based on a silicon rhodol scaffold and a carboxylic group as the intramolecular nucleophile. LacZ-positive cells were successfully labeled with SPiDER-Red-βGal at single-cell resolution in living samples, which enabled us to visualize different cell types in combination with GFP markers.

• Access to Document (DOI)

A protein-coupled fluorescent probe for organelle-specific imaging of Na⁺

Taguchi R., Terai T., Ueno T., Komatsu T., Hanaoka K., Urano Y.

Sensors and Actuators, B: Chemical （Sensors and Actuators, B: Chemical）  265   575 - 581 2018年07月

研究論文（学術雑誌）, 査読有り,  ISSN  09254005

　概要を見る

Intracellular sodium concentration plays important roles in many cellular processes, but methods to analyze dynamic change of [Na+] in organelles of interest are still lacking. To address this problem, controlled localization of fluorescent Na+ probes is a key issue. In this work, we developed a protein-coupled Na+-sensitive fluorescent probe, HaloNa-1, which covalently labelled a HaloTag protein expressed at specific organelles in living cells. Fluorescence intensity of HaloNa-1 increased by approximately three-fold when complexed with Na+, and the dissociation constant was within physiological range. Notably, by conjugating a fluorescent protein to the HaloTag protein and using it as internal standard, one can reduce unwanted variation of probe fluorescence due to different cell volume, HaloTag expression level, etc. With this technique, we could observe elevation of intracellular [Na+] after ionophore stimulation with higher reliability than conventional sensors. We believe our probe is potentially useful to understand Na+ dynamics inside cells.

• Access to Document (DOI)