MF研究者総覧

研究者データベース

詳細
永井　健治(ナガイ　タケハル)
電子科学研究所　連携研究部門
教授
researchmap個人ページ
Last Updated :2023/12/11

基本情報

所属

  • 電子科学研究所　連携研究部門

職名

  • 教授

学位

  • 博士（医学）（東京大学）

ホームページURL

J-Global ID

研究キーワード

  • BRET   FRET   蛍光タンパク質   生物（化学）発光タンパク質   トランススケールスコープ   シンギュラリティ生物学   顕微鏡   生体機能イメージング   

研究分野

  • ライフサイエンス / 生物物理学
  • ナノテク・材料 / ナノバイオサイエンス
  • ナノテク・材料 / ナノ材料科学

職歴

  • 2022年10月 - 現在 北海道大学 電子科学研究所 教授
  • 2022年01月 - 現在 奈良先端科学技術大学院大学 客員教授
  • 2020年04月 - 現在 大阪大学 産業科学研究所 副所長
  • 2018年01月 - 現在 大阪大学 先導的学際研究機構超次元ライフイメージング研究部門 部門長（兼任）
  • 2012年06月 - 現在 理化学研究所 客員主幹研究員
  • 2012年03月 - 現在 大阪大学 産業科学研究所 教授
  • 2012年03月 - 2022年09月 北海道大学 大学院医学研究科 客員教授
  • 2017年08月 - 2019年08月 大阪大学 副理事 (共創機構担当) (兼任)
  • 2017年04月 - 2019年03月 大阪大学 共創機構 産学共創本部イノベーション共創部門 部門長 (兼任)
  • 2014年04月 - 2017年09月 大阪大学 産業科学研究所 副所長
  • 2015年08月 - 2017年08月 大阪大学 副理事 (産学連携担当) (兼任)
  • 2017年04月 大阪大学 栄誉教授 称号付与
  • 2008年10月 - 2014年03月 科学技術振興機構・さきがけ 研究員 (兼任)
  • 2005年01月 - 2012年02月 北海道大学 電子科学研究所 教授
  • 2001年12月 - 2005年03月 科学技術振興機構・さきがけ 研究員
  • 2001年04月 - 2001年11月 理化学研究所脳科学総合研究センター 研究員
  • 1998年04月 - 2001年03月 理化学研究所 基礎科学特別研究員
  • 1995年04月 - 1998年03月 日本学術振興会 特別研究員 (DC1)

学歴

  •         - 1998年   東京大学大学院   医学系研究科   脳神経医学専攻
  •         - 1994年   筑波大学大学院   農学研究科 応用生物化学専攻
  •         - 1992年   筑波大学   第二学群 生物学類 基礎生物学専攻

所属学協会

  • 日本生物物理学会   日本バイオイメージング学会   日本顕微鏡学会   日本分子生物学会   日本マグネシウム学会   

研究活動情報

論文

  • Mügen Terzioglu, Kristo Veeroja, Toni Montonen, Teemu O. Ihalainen, Tiina S. Salminen, Paule Bénit, Pierre Rustin, Young-Tae Chang, Takeharu Nagai, Howard T. Jacobs
    eLife 2023年11月13日 [査読有り]
     
    Based on studies with a fluorescent reporter dye, Mito Thermo Yellow, and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell-line was estimated to be up to 15 °C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of OXPHOS complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of oxidative phosphorylation and is homeostatically maintained as a primary feature of mitochondrial metabolism. Mitochondria are up to 15 °C hotter than their external environment in living cells. In response to diverse metabolic stresses, mitochondrial temperature re-adjusts to this value whenever possible.
  • Masakazu Agetsuma, Issei Sato, Yasuhiro R. Tanaka, Luis Carrillo-Reid, Atsushi Kasai, Atsushi Noritake, Yoshiyuki Arai, Miki Yoshitomo, Takashi Inagaki, Hiroshi Yukawa, Hitoshi Hashimoto, Junichi Nabekura, Takeharu Nagai
    Nature Communications 14 1 2023年10月06日 [査読有り]
     
    Abstract Associative learning is crucial for adapting to environmental changes. Interactions among neuronal populations involving the dorso-medial prefrontal cortex (dmPFC) are proposed to regulate associative learning, but how these neuronal populations store and process information about the association remains unclear. Here we developed a pipeline for longitudinal two-photon imaging and computational dissection of neural population activities in male mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC network topology. Using regularized regression methods and graphical modeling, we found that fear conditioning drove dmPFC reorganization to generate a neuronal ensemble encoding conditioned responses (CR) characterized by enhanced internal coactivity, functional connectivity, and association with conditioned stimuli (CS). Importantly, neurons strongly responding to unconditioned stimuli during conditioning subsequently became hubs of this novel associative network for the CS-to-CR transformation. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on the activity-dependent formation of the hub network within the dmPFC.
  • Natsumi Hoshino, Takashi Kanadome, Tomomi Takasugi, Mizuho Itoh, Ryosuke Kaneko, Yukiko U. Inoue, Takayoshi Inoue, Takahiro Hirabayashi, Masahiko Watanabe, Tomoki Matsuda, Takeharu Nagai, Etsuko Tarusawa, Takeshi Yagi
    Proceedings of the National Academy of Sciences 120 38 2023年09月11日 [査読有り]
     
    Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.
  • Takashi Kanadome, Natsumi Hoshino, Takeharu Nagai, Takeshi Yagi, Tomoki Matsuda
    iScience 26 7 107238 - 107238 2023年07月 [査読有り]
  • Kenta Temma, Ryosuke Oketani, Toshiki Kubo, Kazuki Bando, Shunsuke Maeda, Kazunori Sugiura, Tomoki Matsuda, Rainer Heintzmann, Tatsuya Kaminishi, Koki Fukuda, Maho Hamasaki, Takeharu Nagai, Katsumasa Fujita
    2023年06月19日 
    Abstract Resolution enhancement in structured illumination microscopy is hindered when volumetric samples are observed owing to background signals from out-of-focus planes. In this study, we utilized selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. We demonstrated high-resolution fluorescence imaging of volumetric samples, including dense fluorescent objects in a cell spheroid.
  • Tetsuichi Wazawa, Takeharu Nagai
    Biophysics and Physicobiology 2023年06月 [査読有り]
  • Azmeree Jahan, MST Tahmina Akter, Kiwamu Takemoto, Tai Oura, Akiko Shitara, Shingo Semba, Akihiro Nezu, Satoshi Suto, Takeharu Nagai, Akihiko Tanimura
    Cell Calcium 108 102668 - 102668 2022年12月 [査読有り]
  • Kazunori Sugiura, Takeharu Nagai
    Communications Biology 5 1 2022年11月03日 [査読有り]
     
    Abstract To perform correlation analysis between different physiological parameters using fluorescent protein-based functional probes, diversification of wavelength properties of fluorescent proteins is underway. However, the shortest emission wavelength of fluorescent proteins has not been updated for more than 10 years. Here, we report the development of Sumire, a fluorescent protein emitting 414 nm violet fluorescence from a hydrated chromophore. The Sumire’s fluorescence property allows for the creation of FRET probes that can be used simultaneously with CFP-YFP based FRET probes for multi-parameter analysis.
  • Takashi Kanadome, Kanehiro Hayashi, Yusuke Seto, Mototsugu Eiraku, Kazunori Nakajima, Takeharu Nagai, Tomoki Matsuda
    Communications Biology 5 1 1065 - 1065 2022年10月07日 [査読有り]
     
    N-cadherin (NCad) is a classical cadherin that mediates cell-cell interactions in a Ca2+-dependent manner. NCad participates in various biological processes, from ontogenesis to higher brain functions, though the visualization of NCad interactions in living cells remains limited. Here, we present intensiometric NCad interaction indicators, named INCIDERs, that utilize dimerization-dependent fluorescent proteins. INCIDERs successfully visualize reversible NCad interactions across cells. Compared to FRET-based indicators, INCIDERs have a ~70-fold higher signal contrast, enabling clear identification of NCad interactions. In primary neuronal cells, NCad interactions are visualized between closely apposed processes. Furthermore, visualization of NCad interaction at cell adhesion sites in dense cell populations is achieved by two-photon microscopy. INCIDERs are useful tools in the spatiotemporal investigation of NCad interactions across cells; future research should evaluate the potential of INCIDERs in mapping complex three-dimensional architectures in multi-cellular systems.
  • Quang Tran, Kenji Osabe, Tetsuyuki Entani, Tetsuichi Wazawa, Mitsuru Hattori, Takeharu Nagai
    Plant, Cell & Environment 2022年07月21日 [査読有り]
  • Shinobu Sato, Shinsuke Ohzawa, Kojiro Sota, Naoto Sakamoto, Ayano Udo, Shinji Sueda, Tomoki Matsuda, Takeharu Nagai, Shigeori Takenaka
    Frontiers in Chemistry 10 922094  2022年07月08日 [査読有り]
     
    Potassium-sensing oligonucleotide, PSO, a conjugate of a quadruplex structure-forming oligonucleotide with a peptide incorporating a Förster Resonance Energy Transfer (FRET) chromophore pair, has been developed for fluorescent detection of potassium ion (K+) in aqueous medium. PSO 1 could be introduced into cells for real-time imaging of cytoplasmic K+ concentrations. To perform fluorescent imaging of K+ on the cell surface, we synthesized twelve PSO derivatives with different types of peptide types and lengths, and oligonucleotide sequences including thrombin-binding aptamer (TBA) sequences with FAM and TAMRA as a FRET chromophore pair, and evaluated their performance. 1 was shown to respond selectively to K+, not to most ions present in vivo, and to show reciprocal fluorescence changes in response to K+ concentration. For the peptide chains and oligonucleotide sequences examined in this study, the PSO derivatives had K d values for K+ in the range of 5-30 mM. All PSO derivatives showed high K+ selectivity even in the presence of excess Na+. The PSO derivatives were successfully localized to the cell surface by biotinylated concanavalin A (ConA) or sulfo-NHS-biotin via streptavidin (StAv). Fluorescence imaging of extracellular K+ upon addition of apoptosis inducers was successfully achieved by 1 localized to the cell surface.
  • Kai Lu, Tetsuichi Wazawa, Joe Sakamoto, Cong Quang Vu, Masahiro Nakano, Yasuhiro Kamei, Takeharu Nagai
    Nano Letters 22 14 5698 - 5707 2022年07月06日 [査読有り]
  • Le Zhai, Ryosuke Nakashima, Hajime Shinoda, Yoshimasa Ike, Tomoki Matsuda, Takeharu Nagai
    Protein Science 31 5 e4285  2022年04月04日 [査読有り]
     
    GFP-like chromoproteins (CPs) with non-fluorescence ability have been used as bioimaging probes. Existing CPs have voids in the optical absorption window which limits their extensibility. The development of new CP color is therefore ongoing. Here, we cloned CPs from the jellyfish, Olindias formosa, and developed a completely non-fluorescent monomeric red CP, R-Velour, with an absorption peak at 528 nm. To analyze the photophysical properties from a structural aspect, we determined the crystal structure of R-Velour at a 2.1 Å resolution. R-Velour has a trans-chromophore similar to the green fluorescence protein, Gamillus, derived from the same jellyfish. However, in contrast to the two coplanar chromophoric rings in Gamillus, R-Velour has a large torsion inducing non-fluorescence property. Through site-directed mutagenesis, we surveyed residues surrounding the chromophore and found a key residue, Ser155, which contributes to the generation of four-color variants with the bathochromic and hypsochromic shift of the absorption peak, ranging from 506 to 554 nm. The recently proposed spectrum shift theory, based on the Marcus-Hush model, supports the spectrum shift of these mutants. These findings may support further development of R-Velour variants with useful absorption characteristics for bioimaging, including fluorescence lifetime imaging and photoacoustic imaging.
  • Takashi Kanadome, Natsumi Hoshino, Takeharu Nagai, Tomoki Matsuda, Takeshi Yagi
    Scientific Reports 11 1 22237  2021年11月15日 [査読有り]
     
    Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Forster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.
  • Akihiro Goto, Ayaka Bota, Ken Miya, Jingbo Wang, Suzune Tsukamoto, Xinzhi Jiang, Daichi Hirai, Masanori Murayama, Tomoki Matsuda, Thomas J. McHugh, Takeharu Nagai, Yasunori Hayashi
    Science 374 6569 857 - 863 2021年11月12日 [査読有り]
     
    Where and when of memory consolidation Episodic memory is initially encoded in the hippocampus and later transferred to other brain regions for long-term storage. Synaptic plasticity underlies learning and plays a critical role in memory consolidation. However, it remains largely unknown where and when synaptic plasticity occurs and how it shapes the neuronal representation. Goto et al . developed a new tool for controlling early structural long-term potentiation (sLTP). By selectively manipulating sLTP, the authors showed that the local circuitry in hippocampal area CA1 is required for memory formation shortly after the encoding event. The local circuitry is also important for offline memory consolidation within 24 hours. The anterior cingulate cortex, another brain region directly connected with area CA1, is crucial for memory consolidation during sleep on the second night. —PRS
  • Mitsuru Hattori, Nae Sugiura, Tetsuichi Wazawa, Tomoki Matsuda, Takeharu Nagai
    Analytical Chemistry 93 40 13520 - 13526 2021年09月27日 [査読有り]
     
    Hemostasis is an essential function that repairs tissues and maintains the survival of living organisms. To prevent diseases caused by the abnormality of the blood coagulation mechanism, it is important to carry out a blood test periodically by a method that is convenient and less burdensome for examiners. Thrombin is a protease that catalyzes the conversion of fibrinogen, and its cleavage activity can be an index of coagulation activity. Here, we developed a ratiometric bioluminescent indicator, Thrombastor (thrombin activity sensing indicator), which reflects the thrombin cleavage activity in blood by changing the emission color from green to blue. Compared to the current thrombin activity indicator, the rapid color change of the emission indicated a 2.5-fold decrease in the Km for thrombin, and the cleavage rate was more than two times faster. By improving the absolute bioluminescence intensity, detection from a small amount of plasma could be achieved with a smartphone camera. Using Thrombastor and a versatile device, an effective diagnosis for preventing coagulation disorders can be provided.
  • Toshiki Kubo, Kenta Temma, Kazunori Sugiura, Hajime Shinoda, Kai Lu, Nicholas Isaac Smith, Tomoki Matsuda, Takeharu Nagai, Katsumasa Fujita
    ACS Photonics 8 9 2666 - 2673 2021年08月30日 [査読有り]
  • Cong Quang Vu, Shun-Ichi Fukushima, Tetsuichi Wazawa, Takeharu Nagai
    Scientific Reports 11 1 16519 - 16519 2021年08月13日 [査読有り]
     
    Genetically encoded temperature indicators (GETIs) allow for real-time measurement of subcellular temperature dynamics in live cells. However, GETIs have suffered from poor temperature sensitivity, which may not be sufficient to resolve small heat production from a biological process. Here, we develop a highly-sensitive GETI, denoted as ELP-TEMP, comprised of a temperature-responsive elastin-like polypeptide (ELP) fused with a cyan fluorescent protein (FP), mTurquoise2 (mT), and a yellow FP, mVenus (mV), as the donor and acceptor, respectively, of Förster resonance energy transfer (FRET). At elevated temperatures, the ELP moiety in ELP-TEMP undergoes a phase transition leading to an increase in the FRET efficiency. In HeLa cells, ELP-TEMP responded to the temperature from 33 to 40 °C with a maximum temperature sensitivity of 45.1 ± 8.1%/°C, which was the highest ever temperature sensitivity among hitherto-developed fluorescent nanothermometers. Although ELP-TEMP showed sensitivity not only to temperature but also to macromolecular crowding and self-concentration, we were able to correct the output of ELP-TEMP to achieve accurate temperature measurements at a subcellular resolution. We successfully applied ELP-TEMP to accurately measure temperature changes in cells induced by a local heat spot, even if the temperature difference was as small as < 1 °C, and to visualize heat production from stimulated Ca2+ influx in live HeLa cells induced by a chemical stimulation. Furthermore, we investigated temperatures in the nucleus and cytoplasm of live HeLa cells and found that their temperatures were almost the same within the temperature resolution of our measurement. Our study would contribute to better understanding of cellular temperature dynamics, and ELP-TEMP would be a useful GETI for the investigation of cell thermobiology.
  • T. Ichimura, T. Kakizuka, K. Horikawa, K. Seiriki, A. Kasai, H. Hashimoto, K. Fujita, T. M. Watanabe, T. Nagai
    Scientific Reports 11 1 16539  2021年08月08日 [査読有り]
     
    In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.
  • Tomomi Kaku, Kazunori Sugiura, Tetsuyuki Entani, Kenji Osabe, Takeharu Nagai
    Scientific Reports 11 1 14994  2021年07月22日 [査読有り]
     
    Using the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.
  • Quang Tran, Kenji Osabe, Tetsuyuki Entani, Takeharu Nagai
    Plant Biotechnology 38 2 197 - 20 2021年06月25日 [査読有り]
  • Tetsuichi Wazawa, Ryohei Noma, Shusaku Uto, Kazunori Sugiura, Takashi Washio, Takeharu Nagai
    Microscopy (Oxford, England) 70 4 340 - 352 2021年01月22日 [査読有り]
     
    Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high power density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed a RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation, and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH-dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently-bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently-bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, SPoD-OnSPAN, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.
  • Yukino Itoh, Mitsuru Hattori, Tetsuichi Wazawa, Yoshiyuki Arai, Takeharu Nagai
    ACS Sensors 6 3 889 - 895 2021年01月14日 [査読有り]
  • Mitsuru Hattori, Tomoki Matsuda, Takeharu Nagai
    Methods in molecular biology (Clifton, N.J.) 2274 295 - 304 2021年 [査読有り]
     
    The application of smartphones as detectors is essential to achieve ubiquitous measurement targeting biomolecules. Because bioluminescence (BL), as a tag for a target sample, does not require an excitation light source, it can be combined with a smartphone to constitute a compact and mobile measurement system. A method was recently established to detect the spectral change of ratiometric indicators based on bioluminescence resonance energy transfer with a smartphone camera. For example, it was possible to detect changes in the BL color of the Ca2+ indicator quantitatively and easily calculate the concentration of free Ca2+ by setting appropriate image acquisition conditions in a smartphone application. In this paper, we describe techniques to obtain scientifically relevant and reliable BL data with such a convenient instrument. This protocol expands the potential of the smartphone as a personal imaging device with high mobility that can be used anywhere.
  • Toshiki Kubo, Kenta Temma, Nicholas I Smith, Kai Lu, Tomoki Matsuda, Takeharu Nagai, Katsumasa Fujita
    Optics Letters 46 1 37 - 40 2021年01月01日 [査読有り]
     
    We demonstrate hyperspectral imaging by visible-wavelength two-photon excitation microscopy using line illumination and slit-confocal detection. A femtosecond pulsed laser light at 530 nm was used for the simultaneous excitation of fluorescent proteins with different emission wavelengths. The use of line illumination enabled efficient detection of hyperspectral images and achieved simultaneous detection of three fluorescence spectra in the observation of living HeLa cells with an exposure time of 1 ms per line, which is equivalent to about 2 µs per pixel in point scanning, with 160 data points per spectrum. On combining linear spectral unmixing techniques, localization of fluorescent probes in the cells was achieved. A theoretical investigation of the imaging property revealed high-depth discrimination property attained through the combination of nonlinear excitation and slit detection.
  • Mitsuru Hattori, Sumito Shirane, Tomoki Matsuda, Kuniaki Nagayama, Takeharu Nagai
    Sensors 20 24 7166 - 7166 2020年12月14日 [査読有り]
  • Takeshi Harada, Ryota Sada, Yoshito Osugi, Shinji Matsumoto, Tomoki Matsuda, Mitsuko Hayashi-Nishino, Takeharu Nagai, Akihiro Harada, Akira Kikuchi
    Journal of Cell Science 133 21 2020年11月10日 [査読有り]
     
    Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions and in vivo xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.This article has an associated First Person interview with the first author of the paper.
  • Shuhei Nakamura, Saki Shigeyama, Satoshi Minami, Takayuki Shima, Shiori Akayama, Tomoki Matsuda, Alessandra Esposito, Gennaro Napolitano, Akiko Kuma, Tomoko Namba-Hamano, Jun Nakamura, Kenichi Yamamoto, Miwa Sasai, Ayaka Tokumura, Mika Miyamoto, Yukako Oe, Toshiharu Fujita, Seigo Terawaki, Atsushi Takahashi, Maho Hamasaki, Masahiro Yamamoto, Yukinori Okada, Masaaki Komatsu, Takeharu Nagai, Yoshitsugu Takabatake, Haoxing Xu, Yoshitaka Isaka, Andrea Ballabio, Tamotsu Yoshimori
    Nature Cell Biology 22 10 1252 - 1263 2020年09月28日 [査読有り]
     
    Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.
  • Hossain, M.N., Ishida, R., Hattori, M., Matsuda, T., Nagai, T.
    Sensors (Switzerland) 20 11 3164  2020年06月02日 [査読有り][通常論文]
     
    Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca2+/Mg2+ detection domain of human centrin 3. The binding of Ca2+/Mg2+ to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca2+/Mg2+ are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.
  • Thasaneeya Kuboki, Hiroyuki Ebata, Tomoki Matsuda, Yoshiyuki Arai, Takeharu Nagai, Satoru Kidoaki
    Cell Structure and Function 45 1 33 - 43 2020年02月22日 [査読有り][通常論文]
  • Araki, S., Nakano, M., Tsugane, M., Sunaga, F., Hattori, M., Nakano, M., Nagai, T., Suzuki, H.
    Analyst 145 2 667 - 674 2020年01月21日 [査読有り][通常論文]
     

    Capability of simple microfluidic devices having vertical sidewalls for live-cell fluorescence imaging was investigated.

  • Shigenori Inagaki, Masakazu Agetsuma, Shinya Ohara, Toshio Iijima, Hideo Yokota, Tetsuichi Wazawa, Yoshiyuki Arai, Takeharu Nagai
    Scientific Reports 9 1 2019年12月 [査読有り][通常論文]
  • Nezu, A., Morita, T., Nagai, T., Tanimura, A.
    Experimental Physiology 104 1 2019年 [査読有り][通常論文]
  • Oketani, R., Suda, H., Uegaki, K., Kubo, T., Matsuda, T., Yamanaka, M., Arai, Y., Smith, N., Nagai, T., Fujita, K.
    Journal of Biomedical Optics 25 1 1 - 5 2019年 [査読有り][通常論文]
     
    Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.
  • Farhana, I., Hossain, M.N., Suzuki, K., Matsuda, T., Nagai, T.
    ACS Sensors 4 7 1825 - 1834 2019年 [査読有り][通常論文]
  • Shinoda, H., Lu, K., Nakashima, R., Wazawa, T., Noguchi, K., Matsuda, T., Nagai, T.
    Cell Chemical Biology 26 10 1469 - 1479.e6 2019年 [査読有り][通常論文]
     
    © 2019 Elsevier Ltd Shinoda et al. developed reversibly photoswitchable variants of an acid-tolerant monomeric GFP, Gamillus, named “rsGamillus-S” and “rsGamillus-F”. Both rsGamillus proteins exhibit superior acid tolerance (pKa = 3.6) compared with other reported reversibly photoswitchable GFPs. They demonstrated the applicability of rsGamillus proteins for super-resolution imaging in acidic conditions.
  • Shiori Toba, Mingyue Jin, Masami Yamada, Kanako Kumamoto, Sakiko Matsumoto, Takuo Yasunaga, Yuko Fukunaga, Atsuo Miyazawa, Sakiko Fujita, Kyoko Itoh, Shinji Fushiki, Hiroaki Kojima, Hideki Wanibuchi, Yoshiyuki Arai, Takeharu Nagai, Shinji Hirotsune
    Scientific Reports 7 1 2018年12月01日 [査読有り][通常論文]
     
    Although α-synuclein (αSyn) has been linked to Parkinson's disease (PD), the mechanisms underlying the causative role in PD remain unclear. We previously proposed a model for a transportable microtubule (tMT), in which dynein is anchored to a short tMT by LIS1 followed by the kinesin-dependent anterograde transport however the mechanisms that produce tMTs have not been determined. Our in vitro investigations of microtubule (MT) dynamics revealed that αSyn facilitates the formation of short MTs and preferentially binds to MTs carrying 14 protofilaments (pfs). Live-cell imaging showed that αSyn co-transported with dynein and mobile βIII-tubulin fragments in the anterograde transport. Furthermore, bi-directional axonal transports are severely affected in αSyn and γSyn depleted dorsal root ganglion neurons. SR-PALM analyses further revealed the fibrous co-localization of αSyn, dynein and βIII-tubulin in axons. More importantly, 14-pfs MTs have been found in rat femoral nerve tissue, and they increased approximately 19 fold the control in quantify upon nerve ligation, indicating the unconventional MTs are mobile. Our findings indicate that αSyn facilitates to form short, mobile tMTs that play an important role in the axonal transport. This unexpected and intriguing discovery related to axonal transport provides new insight on the pathogenesis of PD.
  • Naoki Komatsu, Kenta Terai, Ayako Imanishi, Yuji Kamioka, Kenta Sumiyama, Takashi Jin, Yasushi Okada, Takeharu Nagai, Michiyuki Matsuda
    Scientific Reports 8 1 2018年12月01日 [査読有り][通常論文]
     
    Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.
  • Ryoichi Sato, Rinya Kawashima, Mai Duy Luu Trinh, Masahiro Nakano, Takeharu Nagai, Shinji Masuda
    Photosynthesis Research 139 1-3 1 - 7 2018年06月18日 [査読有り][通常論文]
     
    The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP synthesis in plants. The PMF mainly arises from the oxidation of water in photosystem II and from electron transfer within the cytochrome b6f complex. There are two electron transfer pathways related to PMF formation: linear electron flow and cyclic electron flow. Proton gradient regulation 5 (PGR5) is a major component of the cyclic electron flow pathway, and the Arabidopsis pgr5 mutant shows a substantial reduction in the PMF. How the PGR5-dependent cyclic electron flow contributes to ATP synthesis has not, however, been fully delineated. In this study, we monitored in vivo ATP levels in Arabidopsis chloroplasts in real time using a genetically encoded bioluminescence-based ATP indicator, Nano-lantern(ATP1). The increase in ATP in the chloroplast stroma of pgr5 leaves upon illumination with actinic light was significantly slower than in wild type, and the decrease in ATP levels when this illumination stopped was significantly faster in pgr5 leaves than in wild type. These results indicated that PGR5-dependent cyclic electron flow around photosystem I helps to sustain the rate of ATP synthesis, which is important for growth under fluctuating light conditions.
  • Kazushi Suzuki, Takahito Onishi, Chieko Nakada, Shunsuke Takei, Matthew J. Daniels, Masahiro Nakano, Tomoki Matsuda, Takeharu Nagai
    BMC Research Notes 11 1 2018年05月18日 [査読有り][通常論文]
     
    Objective: Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+ indicator GmNL(Ca2+), and its application in a customized microscope for high-throughput drug screening. Results: GmNL(Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.
  • Yemima Dani Riani, Tomoki Matsuda, Kiwamu Takemoto, Takeharu Nagai
    BMC Biology 16 1 50  2018年04月30日 [査読有り][通常論文]
     
    Background: Photosensitizing fluorescent proteins, which generate reactive oxygen species (ROS) upon light irradiation, are useful for spatiotemporal protein inactivation and cell ablation. They give us clues about protein function, intracellular signaling pathways and intercellular interactions. Since ROS generation of a photosensitizer is specifically controlled by certain excitation wavelengths, utilizing colour variants of photosensitizing protein would allow multi-spatiotemporal control of inactivation. To expand the colour palette of photosensitizing protein, here we developed SuperNova Green from its red predecessor, SuperNova. Results: SuperNova Green is able to produce ROS spatiotemporally upon blue light irradiation. Based on protein characterization, SuperNova Green produces insignificant amounts of singlet oxygen and predominantly produces superoxide and its derivatives. We utilized SuperNova Green to specifically inactivate the pleckstrin homology domain of phospholipase C-δ1 and to ablate cancer cells in vitro. As a proof of concept for multi-spatiotemporal control of inactivation, we demonstrate that SuperNova Green can be used with its red variant, SuperNova, to perform independent protein inactivation or cell ablation studies in a spatiotemporal manner by selective light irradiation. Conclusion: Development of SuperNova Green has expanded the photosensitizing protein toolbox to optogenetically control protein inactivation and cell ablation.
  • Tetsuichi Wazawa, Yoshiyuki Arai, Yoshinobu Kawahara, Hiroki Takauchi, Takashi Washio, Takeharu Nagai
    Microscopy 67 2 89 - 98 2018年04月01日 [査読有り][通常論文]
     
    Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102-106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70-80nm with illumination power densities as low as ~1W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of superresolution fluorescence microscopy for live-cell imaging.
  • Sayuri Hara-Kuge, Tomonobu Nishihara, Tomoki Matsuda, Tomohiro Kitazono, Takayuki Teramoto, Takeharu Nagai, Takeshi Ishihara
    PLoS ONE 13 4 2018年04月01日 [査読有り][通常論文]
     
    Sensory processing is regulated by the coordinated excitation and inhibition of neurons in neuronal circuits. The analysis of neuronal activities has greatly benefited from the recent development of genetically encoded Ca2+ indicators (GECIs). These molecules change their fluorescence intensities or colours in response to changing levels of Ca2+ and can, therefore, be used to sensitively monitor intracellular Ca2+ concentration, which enables the detection of neuronal excitation, including action potentials. These GECIs were developed to monitor increases in Ca2+ concentration therefore, neuronal inhibition cannot be sensitively detected by these GECIs. To overcome this difficulty, we hypothesised that an inverse-type of GECI, whose fluorescence intensity increases as Ca2+ levels decrease, could sensitively monitor reducing intracellular Ca2+ concentrations. We, therefore, developed a Ca2+ indicator named inverse-pericam 2.0 (IP2.0) whose fluorescent intensity decreases 25-fold upon Ca2+ binding in vitro. Using IP2.0, we successfully detected putative neuronal inhibition by monitoring the decrease in intracellular Ca2+ concentration in AWCON and ASEL neurons in Caenorhabditis elegans. Therefore, IP2.0 is a useful tool for studying neuronal inhibition and for the detailed analysis of neuronal activities in vivo.
  • Kazuhiro Maeshima, Tomoki Matsuda, Yutaka Shindo, Hiromi Imamura, Sachiko Tamura, Ryosuke Imai, Syoji Kawakami, Ryosuke Nagashima, Tomoyoshi Soga, Hiroyuki Noji, Kotaro Oka, Takeharu Nagai
    Current Biology 28 3 444 - 451.e6 2018年02月05日 [査読有り][通常論文]
     
    For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1–5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6–17], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [18–28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like “beads on a string” by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg2+, have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ increase is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell [33]. ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance. How the negatively charged long genomic DNA is organized into mitotic chromosome remains unclear. Using a newly developed Mg2+ indicator, Maeshima et al. demonstrate a transient rise in free Mg2+ released from ATP-Mg during mitosis and suggest that the rise contributes to mitotic chromosome condensation by charge neutralization.
  • Yusaku Ohta, Toshiaki Furuta, Takeharu Nagai, Kazuki Horikawa
    Scientific Reports 8 1 1866 - 1866 2018年01月30日 [査読有り][通常論文]
     
    cAMP is one of the most important second messengers in biological processes. Cellular dynamics of cAMP have been investigated using a series of fluorescent indicators; however, their sensitivity was sub-optimal for detecting cAMP dynamics at a low concentration range, due to a low ligand affinity and/or poor dynamic range. Seeking an indicator with improved detection sensitivity, we performed insertion screening of circularly permuted mApple, a red fluorescent protein, into the cAMP-binding motif of PKA regulatory subunit Iα and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (Kd = 0.3 μM) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub-μM concentrations, which could not be easily observed with existing indicators. Increased detection sensitivity also strengthened the advantages of using R-FlincA as a red fluorescent indicator, as it permits a series of applications, including multi-channel/function imaging of multiple second messengers and combinatorial imaging with photo-manipulation. These results strongly suggest that R-FlincA is a promising tool that accelerates cAMP research by revealing unobserved cAMP dynamics at a low concentration range.
  • Yuki Kushida, Yoshiyuki Arai, Ken Shimono, Takeharu Nagai
    ACS Sensors 3 1 87 - 92 2018年01月26日 [査読有り][通常論文]
     
    The chemical receptors present in living organisms are promising tools for developing biomimetic chemical sensors. However, these receptors require lipid membranes for functioning under physiological conditions, which prevents their utilization in the production of cell-free in vitro chemical sensing systems. Here, we report the development of a cell-free biomimetic sensing platform using virus-like particles (VLPs) with intact ligand-gated Ca2+ channels and genetically encoded Ca2+ indicator (GECI). We observed that targeting GECI to the plasma membrane was essential for efficient loading GECI in the VLPs. Although the physiological Ca2+ concentration [Ca2+] maintained in the cells was low (∼10 nM), the concentration in the VLPs was high. This prevented the detection of the increase in [Ca2+] caused by binding of the ligand to the receptor. To address this problem, we employed Lyn-R-CEPIA1, which had low affinity for Ca2+, and a membrane targeting sequence. Thus, we succeeded in monitoring the activation of cyclic nucleotide-gated channels (CNG) on the VLPs by measuring the increase in fluorescence of Lyn-R-CEPIA1. Our VLP-based sensing system can act as a fundamental platform for all kinds of ligand-gated channels.
  • Hossain, M.N., Suzuki, K., Iwano, M., Matsuda, T., Nagai, T.
    ACS Chemical Biology 13 7 1862 - 1871 2018年 [査読有り][通常論文]
     
    © 2018 American Chemical Society. The sarco/endoplasmic reticulum (SR/ER) is the foremost intercellular Ca2+store (at submillimolar concentrations), playing a crucial role in controlling intracellular Ca2+levels. For the investigation of SR/ER Ca2+dynamics in cells, fluorescent protein-based genetically encoded calcium indicators (GECIs) with low Ca2+affinity have been used. Recently, bioluminescent protein-based GECIs with high brightness have been reported to counter the constraints of fluorescence imaging, such as phototoxicity. However, their Ca2+affinity is high and limited for imaging in the cytosol, nucleus, or mitochondria. In this study, we developed a novel cyan color, low-affinity (Kd= 110 μM) intensiometric bioluminescent GECI, which enables monitoring of the Ca2+dynamics in the ER of HeLa cells and the SR of C2C12-derived myotubes. To facilitate the broad concentration range of Ca2+in cellular organelles, we additionally developed an intermediate affinity (Kd= 18 μM), orange color, and bioluminescent GECI, which enables monitoring of Ca2+dynamics in the mitochondria of HeLa cells. With these indicators, in conjunction with an existing high-affinity, green, bioluminescent GECI, we succeeded in multicolor bioluminescent Ca2+imaging in three distinct organelles (nuclei, mitochondria, and ER) simultaneously. The multicolor, live, bioluminescent Ca2+imaging demonstrated here can be used to stably reveal the ER Ca2+homeostasis and cooperative Ca2+regulation among organelles. This will lead to the further understanding of Ca2+-related physiological functions and pathophysiological mechanisms.
  • Arai, Y., Takauchi, H., Ogami, Y., Fujiwara, S., Nakano, M., Matsuda, T., Nagai, T.
    ACS Chemical Biology 13 8 1938 - 1943 2018年 [査読有り][通常論文]
     
    © 2018 American Chemical Society. Super-resolution imaging techniques based on single molecule localization microscopy (SMLM) broke the diffraction limit of optical microscopy in living samples with the aid of photoswitchable fluorescent probes and intricate microscopy systems. Here, we developed a fluorescent protein, SPOON, which can be switched off by excitation light illumination and switched on by thermally induced dehydration, resulting in an apparent spontaneous blinking behavior. This unique property of SPOON provides a simple SMLM-based super-resolution imaging platform which requires only a single 488 nm laser.
  • Shinoda, H., Ma, Y., Nakashima, R., Sakurai, K., Matsuda, T., Nagai, T.
    Cell Chemical Biology 25 3 330 - 338.e7 2018年 [査読有り][通常論文]
  • Shiori Toba, Mingyue Jin, Masami Yamada, Kanako Kumamoto, Sakiko Matsumoto, Takuo Yasunaga, Yuko Fukunaga, Atsuo Miyazawa, Sakiko Fujita, Kyoko Itoh, Shinji Fushiki, Hiroaki Kojima, Hideki Wanibuchi, Yoshiyuki Arai, Takeharu Nagai, Shinji Hirotsune
    Scientific Reports 8 1 2017年12月01日 [査読有り][通常論文]
     
    In this Article, Figure 5 was inadvertently published as Figures 5 and 6, leading to the incorrect publication of Figure 6 as Figure 7 and the omission of the correct Figure 7. The correct Figures 5, 6, and 7 appear below as Figures 1, 2, and 3 respectively. The Figure legends are correct. (Figure Presented).
  • Tadasu Nozaki, Ryosuke Imai, Mai Tanbo, Ryosuke Nagashima, Sachiko Tamura, Tomomi Tani, Yasumasa Joti, Masaru Tomita, Kayo Hibino, Masato T. Kanemaki, Kerstin S. Wendt, Yasushi Okada, Takeharu Nagai, Kazuhiro Maeshima
    Molecular Cell 67 2 282 - 293.e7 2017年07月20日 [査読有り][通常論文]
     
    The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy [PALM]) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ∼160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.
  • Kaoru Seiriki, Atsushi Kasai, Takeshi Hashimoto, Wiebke Schulze, Misaki Niu, Shun Yamaguchi, Takanobu Nakazawa, Ken-Ichi Inoue, Shiori Uezono, Masahiko Takada, Yuichiro Naka, Hisato Igarashi, Masato Tanuma, James A Waschek, Yukio Ago, Kenji F Tanaka, Atsuko Hayata-Takano, Kazuki Nagayasu, Norihito Shintani, Ryota Hashimoto, Yasuto Kunii, Mizuki Hino, Junya Matsumoto, Hirooki Yabe, Takeharu Nagai, Katsumasa Fujita, Toshio Matsuda, Kazuhiro Takuma, Akemichi Baba, Hitoshi Hashimoto
    Neuron 94 6 1085 - 1100 2017年06月21日 [査読有り][通常論文]
     
    Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of light-sheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases.
  • Yu-Fen Chang, Connor N. Broyles, Frances A. Brook, Mark J. Davies, Cameron W. Turtle, Takeharu Nagai, Matthew J. Daniels
    PLoS ONE 12 4 2017年04月01日 [査読有り][通常論文]
     
    Identification of drug induced electrical instability of the heart curtails development, and introduction, of potentially proarrhythmic drugs. This problem usually requires complimentary contact based approaches such as patch-clamp electrophysiology combined with field stimulation electrodes to observe and control the cell. This produces data with high signal to noise but requires direct physical contact generally preventing high-throughput, or prolonged, phenotyping of single cells or tissues. Combining genetically encoded optogenetic control and spectrally compatible calcium indicator tools into a single adenoviral vector allows the analogous capability for cell control with simultaneous cellular phenotyping without the need for contact. This combination can be applied to single rodent primary adult cardiomyocytes, and human stem cell derived cardiomyocytes, enabling contactless small molecule evaluation for inhibitors of sodium, potassium and calcium channels suggesting it may be useful for early toxicity work. In pancreatic beta-cells it reveals the effects of glucose and the KATP inhibitor gliclazide.
  • Makoto Suzuki, Masanao Sato, Hiroshi Koyama, Yusuke Hara, Kentaro Hayashi, Naoko Yasue, Hiromi Imamura, Toshihiko Fujimori, Takeharu Nagai, Robert E. Campbell, Naoto Ueno
    Development (Cambridge) 144 7 1307 - 1316 2017年04月01日 [査読有り][通常論文]
     
    Early in the development of the central nervous system, progenitor cells undergo a shape change, called apical constriction, that triggers the neural plate to form a tubular structure. How apical constriction in the neural plate is controlled and how it contributes to tissue morphogenesis are not fully understood. In this study, we show that intracellular calcium ions (Ca2+) are required for Xenopus neural tube formation and that there are two types of Ca2+- concentration changes, a single-cell and a multicellular wave-like fluctuation, in the developing neural plate. Quantitative imaging analyses revealed that transient increases in Ca2+ concentration induced cortical F-actin remodeling, apical constriction and accelerations of the closing movement of the neural plate. We also show that extracellular ATP and N-cadherin (cdh2) participate in the Ca2+-induced apical constriction. Furthermore, our mathematical model suggests that the effect of Ca2+ fluctuations on tissue morphogenesis is independent of fluctuation frequency and that fluctuations affecting individual cells are more efficient than those at the multicellular level. We propose that distinct Ca2+ signaling patterns differentially modulate apical constriction for efficient epithelial folding and that this mechanism has a broad range of physiological outcomes.
  • Shigenori Inagaki, Hidekazu Tsutsui, Kazushi Suzuki, Masakazu Agetsuma, Yoshiyuki Arai, Yuka Jinno, Guirong Bai, Matthew J. Daniels, Yasushi Okamura, Tomoki Matsuda, Takeharu Nagai
    Scientific Reports 7 42398  2017年02月13日 [査読有り][通常論文]
     
    We report development of the first genetically encoded bioluminescent indicator for membrane voltage called LOTUS-V. Since it is bioluminescent, imaging LOTUS-V does not require external light illumination. This allows bidirectional optogenetic control of cellular activity triggered by Channelrhodopsin2 and Halorhodopsin during voltage imaging. The other advantage of LOTUS-V is the robustness of a signal-to-background ratio (SBR) wherever it expressed, even in the specimens where autofluorescence from environment severely interferes fluorescence imaging. Through imaging of moving cardiomyocyte aggregates, we demonstrated the advantages of LOTUS-V in long-term imaging are attributable to the absence of phototoxicity, and photobleaching in bioluminescent imaging, combined with the ratiometric aspect of LOTUS-V design. Collectively LOTUS-V extends the scope of excitable cell control and simultaneous voltage phenotyping, which should enable applications in bioscience, medicine and pharmacology previously not possible.
  • Masahiro Nakano, Yoshiyuki Arai, Ippei Kotera, Kohki Okabe, Yasuhiro Kamei, Takeharu Nagai
    PLoS ONE 12 2 2017年02月01日 [査読有り][通常論文]
     
    Temperature is a fundamental physical parameter that plays an important role in biological reactions and events. Although thermometers developed previously have been used to investigate several important phenomena, such as heterogeneous temperature distribution in a single living cell and heat generation in mitochondria, the development of a thermometer with a sensitivity over a wide temperature range and rapid response is still desired to quantify temperature change in not only homeotherms but also poikilotherms from the cellular level to in vivo. To overcome the weaknesses of the conventional thermometers, such as a limitation of applicable species and a low temporal resolution, owing to the narrow temperature range of sensitivity and the thermometry method, respectively, we developed a genetically encoded ratiometric fluorescent temperature indicator, gTEMP, by using two fluorescent proteins with different temperature sensitivities. Our thermometric method enabled a fast tracking of the temperature change with a time resolution of 50 ms. We used this method to observe the spatiotemporal temperature change between the cytoplasm and nucleus in cells, and quantified thermogenesis from the mitochondria matrix in a single living cell after stimulation with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which was an uncoupler of oxidative phosphorylation. Moreover, exploiting the wide temperature range of sensitivity from 5°C to 50°C of gTEMP, we monitored the temperature in a living medaka embryo for 15 hours and showed the feasibility of in vivo thermometry in various living species.
  • Matsushita, J., Inagaki, S., Nishie, T., Sakasai, T., Tanaka, J., Watanabe, C., Mizutani, K.-i., Miwa, Y., Matsumoto, K., Takara, K., Naito, H., Kidoya, H., Takakura, N., Nagai, T., Takahashi, S., Ema, M.
    Scientific Reports 7 46597 - 46597 2017年 [査読有り][通常論文]
     
    Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts.
  • Takemoto, K., Iwanari, H., Tada, H., Suyama, K., Sano, A., Nagai, T., Hamakubo, T., Takahashi, T.
    Nature Biotechnology 35 1 38 - 47 2017年 [査読有り][通常論文]
     
    The synaptic delivery of neurotransmitter receptors, such as GluA1 AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, mediates important processes in cognitive function, including memory acquisition and retention. Understanding the roles of these receptors has been hampered by the lack of a method to inactivate them in vivo with high spatiotemporal precision. We developed a technique to inactivate synaptic GIuA1 AMPA receptors in vivo using chromophore-assisted light inactivation (CALI). We raised a monoclonal antibody specific for the extracellular domain of GluA1 that induced effective CALI when conjugated with a photosensitizer (eosin). Mice that had been injected in the CA1 hippocampal region with the antibody conjugate underwent a fear memory task. Exposing the hippocampus to green light using an implanted cannula erased acquired fear memory in the animals by inactivation of synaptic GluA1. Our optical technique for inactivating synaptic proteins will enable elucidation of their physiological roles in cognition.
  • Aoyama, M., Yoshioka, Y., Arai, Y., Hirai, H., Ishimoto, R., Nagano, K., Higashisaka, K., Nagai, T., Tsutsumi, Y.
    Journal of Controlled Release 260 183 - 193 2017年 [査読有り][通常論文]
     
    Little comparative information is available on the detailed intracellular dynamics (diffusion, active movement, and distribution mechanisms) of nanoparticles (<= 100 nm) and sub-micron particles (> 100 nm). Here, we quantitatively examined the intracellular movements of different-sized particles and of the endosomal vesicles containing those particles. We showed that silica nanoparticles of various sizes (30 to 100 nm) had greater motility than sub-micron particles in A549 cells. Although particles of different sizes localized in the early endosomes, late endosomes, and lysosomes in different proportions, their motilities did not vary, regardless of the vesicles in which they were localized. However, surprisingly, endosomal vesicles containing silica nanoparticles moved faster than those containing sub-micron particles. These results suggest that nanoparticles included within endosomal vesicles do not suppress the motility of the vesicles, whereas sub-micron particles perturb endosomal vesicle transport. Our data support a new hypothesis that differences in particle size influence membrane trafficking of endosomal vesicles.
  • Kim, S.-Y., Arai, Y., Tani, T., Takatsuka, H., Saito, Y., Kawashima, T., Kawakami, S., Miyawaki, A., Nagai, T.
    Microscopy 66 2 110 - 119 2017年 [査読有り][通常論文]
     
    Forster resonance energy transfer (FRET) has been widely used to design indicators for biomolecules. Conventional FRET-based indicators enable quantitative measurements of analyzes by calculating the ratio between donor and acceptor fluorophores. However, such 'hetero-FRET'-based indicators, which use multiple differently colored fluorophores, restrict the simultaneous use of other colors of fluorescent molecules. To overcome this problem, we developed a 'homo-FRET'-based Ca2+ indicator, W-Cameleon, composed of two identical yellow fluorescent proteins. The binding of Ca2+ to the indicator induces a change in FRET efficiency, which in turn transforms into changes in fluorescence anisotropy. Given that the fluorescence polarization is depolarized by light passing through a high numerical aperture lens and reflecting on a dichroic mirror, we also developed a microscopy technique that reliably detects fluorescence anisotropy with high precision. Our design is aided by photonic-crystal technology, to compensate for the fluorescence depolarization. We thereby succeeded in the simultaneous visualization of three individual intracellular events by using three different fluorescent indicators. Our system may contribute to an expansion of the number of events that can be observed, which will enable a more quantitative understanding of biological phenomena.
  • Agetsuma, M., Matsuda, T., Nagai, T.
    Cell Calcium 64 12 - 19 2017年 [査読有り][通常論文]
     
    Cells, irrespective of whether they are from multicellular or single-celled organisms, must communicate with the external environment through dynamic regulation of their internal metabolism, which are critical for their survival. Fluorescent and bioluminescent proteins, and related genetic engineering technologies, have provided new opportunities to investigate the molecular dynamics of cells and their internal compartments, with high spatio-temporal resolution. In this review article, since there is a sufficient number of previous reviews summarizing the history of their development and the techniques behind them, here we will focus on molecular features or technologies that have the potential to further open novel investigations of cellular and subcellular dynamics. (C) 2016 The Authors. Published by Elsevier Ltd.
  • Satoru Mochida, Scott Rata, Hirotsugu Hino, Takeharu Nagai, Béla Novák
    Current Biology 26 24 3361 - 3367 2016年12月19日 [査読有り][通常論文]
     
    The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex. Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15 [1, 2]. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible [2–4]. Here, we surprisingly find that Cdk1 auto-activation is dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). We show that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, we found a sharp Cdk1 threshold for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations—evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. Our results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry.
  • Kazushi Suzuki, Taichi Kimura, Hajime Shinoda, Guirong Bai, Matthew J. Daniels, Yoshiyuki Arai, Masahiro Nakano, Takeharu Nagai
    Nature Communications 7 2016年12月14日 [査読有り][通常論文]
     
    Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins. eNLs allow five-colour live-cell imaging, as well as detection of single protein complexes and even single molecules. We also develop an eNL-based Ca2+ indicator with a 500% signal change, which can image spontaneous Ca2+ dynamics in cardiomyocyte and neural cell models. These eNL probes facilitate not only multicolour imaging in living cells but also sensitive imaging of a wide repertoire of proteins, even at very low expression levels.
  • Naohiko Shimada, Minako Saito, Sayaka Shukuri, Sotaro Kuroyanagi, Thasaneeya Kuboki, Satoru Kidoaki, Takeharu Nagai, Atsushi Maruyama
    ACS Applied Materials and Interfaces 8 46 31524 - 31529 2016年11月23日 [査読有り][通常論文]
     
    Multicellular spheroids have been studied in the fields of oncology, stem cell biology, and tissue engineering. In this study, we found a new polymer material for thermo-controlled spheroid/monolayer cell culture switching. The polymers that have pendant ureido groups (ureido polymers) exhibited upper critical solution temperature-type phase separation behavior. Cells in monolayer culture were converted to spheroids by the addition of ureido polymers below phase separation temperature (Tp). Time-lapse observations indicated that cells began to migrate and aggregate to form the spheroids to avoid contact with phase-separated polymer (coacervates) on the surface of the culture dish. We supposed that the coacervates seemingly suppressed interaction between cell and the dish surface or extracellular matrices. By increasing culture temperature above Tp, the spheroids began to collapse into a monolayer of cells due to dissolution of the coacervates. These results indicated that cell morphology could be repeatedly switched by changing the culture temperature in the presence of ureido polymers.
  • Hui-Wen Lu-Walther, Wenya Hou, Martin Kielhorn, Yoshiyuki Arai, Takeharu Nagai, Michael M. Kessels, Britta Qualmann, Rainer Heintzmann
    PLoS ONE 11 10 2016年10月01日 [査読有り][通常論文]
     
    Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.
  • Yusaku Ohta, Takanori Kamagata, Asuka Mukai, Shinji Takada, Takeharu Nagai, Kazuki Horikawa
    ACS Chemical Biology 11 7 1816 - 22 2016年07月15日 [査読有り][通常論文]
     
    Genetically encoded indicators driven by the Förster resonance energy transfer (FRET) mechanism are reliable tools for live imaging. While the properties of FRET-based indicators have been improved over the years, they often suffer from a poor dynamic range due to the lack of comprehensive understanding about how to apply an appropriate strategy to optimize the FRET parameters. One of the most successful optimizations is the incorporation of circularly permuted fluorescent proteins (cpFPs). To better understand the effects of this strategy, we systematically investigated the properties of the indicators by utilizing a set of FRET backbones consisting of native or one of the most effective cp variants (cp173FPs) with considerations of their order. As a result, the ordering of donor and acceptor FPs, which has been ignored in previous studies, was found to significantly affect the dynamic range of indicators. By utilizing these backbones, we succeeded in improving a cGMP indicator with 3.6-fold increased dynamic range and in generating an ultrasensitive cAMP indicator capable of environmental imaging, demonstrating the practical importance of the ordering of donors and acceptors in the engineering of FRET-based indicators.
  • Kishor Pandey, Pedro E. Ferreira, Takeshi Ishikawa, Takeharu Nagai, Osamu Kaneko, Kazuhide Yahata
    Scientific Reports 6 2016年03月23日 [査読有り][通常論文]
     
    Calcium (Ca2+)-mediated signaling is a conserved mechanism in eukaryotes, including the human malaria parasite, Plasmodium falciparum. Due to its small size (< 10 μm) measurement of intracellular Ca2+ in Plasmodium is technically challenging, and thus Ca2+ regulation in this human pathogen is not well understood. Here we analyze Ca2+ homeostasis via a new approach using transgenic P. falciparum expressing the Ca2+ sensor yellow cameleon (YC)-Nano. We found that cytosolic Ca2+ concentration is maintained at low levels only during the intraerythrocytic trophozoite stage (30 nM), and is increased in the other blood stages (> 300 nM). We determined that the mammalian SERCA inhibitor thapsigargin and antimalarial dihydroartemisinin did not perturb SERCA activity. The change of the cytosolic Ca2+ level in P. falciparum was additionally detectable by flow cytometry. Thus, we propose that the developed YC-Nano-based system is useful to study Ca2+ signaling in P. falciparum and is applicable for drug screening.
  • Takamitsu J. Morikawa, Hideaki Fujita, Akira Kitamura, Takashi Horio, Johtaro Yamamoto, Masataka Kinjo, Akira Sasaki, Hiroaki Machiyama, Keiko Yoshizawa, Taro Ichimura, Katsumi Imada, Takeharu Nagai, Tomonobu M. Watanabe
    Scientific Reports 6 2016年03月 [査読有り][通常論文]
     
    Fluorescent proteins have been widely used in biology because of their compatibility and varied applications in living specimens. Fluorescent proteins are often undesirably sensitive to intracellular conditions such as pH and ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence of yellow fluorescent protein (YFP) depends on the protein concentration in the solution and that this dependence can be enhanced by adding a glycine residue in to the YFP; we applied this finding to construct an intracellular protein-crowding sensor. A Frster resonance energy transfer (FRET) pair, involving a cyan fluorescent protein (CFP) insensitive to protein concentration and a glycine-inserted YFP, works as a genetically encoded probe to evaluate intracellular crowding. By measuring the fluorescence of the present FRET probe, we were able to detect dynamic changes in protein crowding in living cells.
  • Lissek, T., Obenhaus, H.A., Ditzel, D.A.W., Nagai, T., Miyawaki, A., Sprengel, R., Hasan, M.T.
    Frontiers in Cellular Neuroscience 10 APR 2016年 [査読有り][通常論文]
     
    General anesthetics are commonly used in animal models to study how sensory signals are represented in the brain. Here, we used two-photon (2P) calcium activity imaging with cellular resolution to investigate how neuronal activity in layer 2/3 of the mouse barrel cortex is modified under the influence of different concentrations of chemically distinct general anesthetics. Our results show that a high isoflurane dose induces synchrony in local neuronal networks and these cortical activity patterns closely resemble those observed in EEG recordings under deep anesthesia. Moreover, ketamine and urethane also induced similar activity patterns. While investigating the effects of deep isoflurane anesthesia on whisker and auditory evoked responses in the barrel cortex, we found that dedicated spatial regions for sensory signal processing become disrupted. We propose that our isoflurane-2P imaging paradigm can serve as an attractive model system to dissect cellular and molecular mechanisms that induce the anesthetic state, and it might also provide important insight into sleep-like brain states and consciousness.
  • Sakamaki, K., Ishii, T.M., Sakata, T., Takemoto, K., Takagi, C., Takeuchi, A., Morishita, R., Takahashi, H., Nozawa, A., Shinoda, H., Chiba, K., Sugimoto, H., Saito, A., Tamate, S., Satou, Y., Jung, S.-K., Matsuoka, S., Koyamada, K., Sawasaki, T., Nagai, T., Ueno, N.
    Biochimica et Biophysica Acta - Molecular Cell Research 1863 11 2766 - 2783 2016年 [査読有り][通常論文]
     
    Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process. (C) 2016 Elsevier B.V. All rights reserved.
  • Tetsuichi Wazawa, Nobuyuki Morimoto, Takeharu Nagai, Makoto Suzuki
    Biophysics and Physicobiology 12 87 - 102 2015年12月02日 [査読有り][通常論文]
  • Itsuo Hanasaki, Satoshi Uehara, Yoshiyuki Arai, Takeharu Nagai, Satoyuki Kawano
    Japanese Journal of Applied Physics 54 12 2015年12月01日 [査読有り][通常論文]
     
    Total internal reflection fluorescence (TIRF) microscopy enables the single-molecule observation in liquid near substrate surface. However, the evaluation of the diffusion from their individually-tracked positions entails the difficulty in the treatment of molecular adsorption on the substrate. We propose a novel technique to evaluate them, and two types of near-surface Brownian motion were determined for DNA. One is the diffusion near glass surface, and the other is the adsorption-dominated motion, which is also found to be diffusive rather than anchored to the substrate. Our technique does not require the threshold values for the distinction, and even the transition between them can be captured. Objective distinction of Brownian motion with and without adsorption does not require the adsorption-free sample preparation. It is also useful for the characterization of adsorption/desorption kinetics. Our method is not limited to TIRF but applicable to many other systems involving multiple states of Brownian motion.
  • Masahito Yamanaka, Kenta Saito, Nicholas I. Smith, Yoshiyuki Arai, Kumiko Uegaki, Yasuo Yonemaru, Kentaro Mochizuki, Satoshi Kawata, Takeharu Nagai, Katsumasa Fujita
    Journal of Biomedical Optics 20 10 2015年10月01日 [査読有り][通常論文]
     
    The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.
  • Katsumasa Fujita, Takeharu Nagai, Nathan Shaner, Alexander Egner
    Journal of Biomedical Optics 20 10 2015年10月01日 [査読有り][通常論文]
  • Megumi Iwano, Kanae Ito, Sota Fujii, Mitsuru Kakita, Hiroko Asano-Shimosato, Motoko Igarashi, Pulla Kaothien-Nakayama, Tetsuyuki Entani, Asaka Kanatani, Masashi Takehisa, Masaki Tanaka, Kunihiko Komatsu, Hiroshi Shiba, Takeharu Nagai, Atsushi Miyawaki, Akira Isogai, Seiji Takayama
    Nature Plants 1 15128 - 15128 2015年09月01日 [査読有り][通常論文]
     
    Self-incompatibility in the Brassicaceae is controlled by multiple haplotypes encoding the pollen ligand (S-locus protein 11, SP11, also known as S-locus cysteine-rich protein, SCR) and its stigmatic receptor (S-receptor kinase, SRK). A haplotype-specific interaction between SP11/SCR and SRK triggers the self-incompatibility response that leads to self-pollen rejection, but the signalling pathway remains largely unknown. Here we show that Ca(2+) influx into stigma papilla cells mediates self-incompatibility signalling. Using self-incompatible Arabidopsis thaliana expressing SP11/SCR and SRK, we found that self-pollination specifically induced an increase in cytoplasmic Ca(2+) ([Ca(2+)]cyt) in papilla cells. Direct application of SP11/SCR to the papilla cell protoplasts induced Ca(2+) increase, which was inhibited by D-(-)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate receptor channel blocker. An artificial increase in [Ca(2+)]cyt in papilla cells arrested wild-type (WT) pollen hydration. Treatment of papilla cells with AP-5 interfered with self-incompatibility, and Ca(2+) increase on the self-incompatibility response was reduced in the glutamate receptor-like channel (GLR) gene mutants. These results suggest that Ca(2+) influx mediated by GLR is the essential self-incompatibility response leading to self-pollen rejection.
  • Hiroyuki Ichijo, Michito Hamada, Satoru Takahashi, Makoto Kobayashi, Takeharu Nagai, Tomoko Toyama, Masahumi Kawaguchi
    Neuroscience Research 95 27 - 37 2015年06月01日 [査読有り][通常論文]
     
    We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response.
  • Akihiro Nezu, Takao Morita, Yosuke Tojyo, Takeharu Nagai, Akihiko Tanimura
    Experimental Physiology 100 6 640 - 651 2015年06月01日 [査読有り][通常論文]
     
    New Findings: What is the central question of this study? Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the Ca< sup> 2+< /sup> -mobilizing effect of pilocarpine in salivary gland cells is extremely small. Therefore, we examined the effect of pilocarpine on Ca< sup> 2+< /sup> responses in submandibular gland cells and on secretion in vitro and in vivo. What is the main finding and its importance? Pilocarpine induces small Ca< sup> 2+< /sup> responses and reduces the effects of other mAChR agonists on Ca< sup> 2+< /sup> responses via its partial agonistic effects. These effects of pilocarpine on Ca< sup> 2+< /sup> responses in the submandibular gland were further established in vivo with a novel Ca< sup> 2+< /sup> imaging system and a genetically encoded Ca< sup> 2+< /sup> indicator. Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the effect of pilocarpine on Ca< sup> 2+< /sup> responses in dispersed salivary gland cells is extremely small. Here, we demonstrate the effect of pilocarpine on Ca< sup> 2+< /sup> responses and salivary secretion in the rat submandibular gland (SMG). In fura-2-loaded SMG cells, the maximal effect of pilocarpine on [Ca< sup> 2+< /sup> ]< inf> i< /inf> elevation was 16% of that of carbachol, and pilocarpine attenuated carbachol- and bethanechol (Bet)-induced [Ca< sup> 2+< /sup> ]< inf> i< /inf> increases, indicating that pilocarpine acts as a partial agonist for mAChR-mediated Ca< sup> 2+< /sup> responses. The partial agonistic effect of pilocarpine on Ca< sup> 2+< /sup> dynamics in the SMG was also confirmed in live animals using the genetically encoded Ca< sup> 2+< /sup> indicator, YC-Nano50. Administration of pilocarpine (3 mg kg< sup> -1< /sup> , i.p.) elicited a small increase in [Ca< sup> 2+< /sup> ]< inf> i< /inf> in the SMG. Quantitative analyses demonstrated that resting [Ca< sup> 2+< /sup> ]< inf> i< /inf> was ~37 nm, which was increased by pilocarpine (3 mg kg< sup> -1< /sup> ) and Bet (10 mg kg< sup> -1< /sup> ) to 44 and 69 nm, respectively. The inhibitory effects of pilocarpine on Bet-induced Ca< sup> 2+< /sup> responses were also elucidated in vivo. We further examined real-time changes in pilocarpine-induced SMG salivary secretion and showed that pilocarpine induced an extremely weak secretory response and reduced Bet-induced secretion. Unlike Ca< sup> 2+< /sup> responses, pilocarpine failed to reduce the effect of Bet on SMG blood flow. Our results demonstrate that pilocarpine acts as a partial agonist of mAChRs to induce weak salivary secretion that is correlated with small increases in [Ca< sup> 2+< /sup> ]< inf> i< /inf> . Furthermore, pilocarpine exhibits an antagonistic effect on mAChR-induced Ca< sup> 2+< /sup> responses and salivary secretion.
  • Yoshiyuki Arai, Takayuki Yamamoto, Takeo Minamikawa, Tetsuro Takamatsu, Takeharu Nagai
    PLoS ONE 10 5 2015年05月07日 [査読有り][通常論文]
     
    The absorption spectrum of light is known to be a "molecular fingerprint" that enables analysis of the molecular type and its amount. It would be useful to measure the absorption spectrum in single cell in order to investigate the cellular status. However, cells are too thin for their absorption spectrum to be measured. In this study, we developed an optical-cavity-enhanced absorption spectroscopic microscopy method for two-dimensional absorption imaging. The light absorption is enhanced by an optical cavity system, which allows the detection of the absorption spectrum with samples having an optical path length as small as 10 μm, at a subcellular spatial resolution. Principal component analysis of various types of cultured mammalian cells indicates absorption-based cellular diversity. Interestingly, this diversity is observed among not only different species but also identical cell types. Furthermore, this microscopy technique allows us to observe frozen sections of tissue samples without any staining and is capable of label-free biopsy. Thus, our microscopy method opens the door for imaging the absorption spectra of biological samples and thereby detecting the individuality of cells.
  • Akira Takai, Masahiro Nakano, Kenta Saito, Remi Haruno, Tomonobu M. Watanabe, Tatsuya Ohyanagi, Takashi Jin, Yasushi Okada, Takeharu Nagai
    Proceedings of the National Academy of Sciences 112 14 4352 - 4356 2015年04月07日 [査読無し][通常論文]
  • Kazunori Sugiura, Takeharu Nagai, Masahiro Nakano, Hiroshi Ichinose, Takakazu Nakabayashi, Nobuhiro Ohta, Toru Hisabori
    Biochemical and Biophysical Research Communications 457 3 242 - 248 2015年02月13日 [査読有り][通常論文]
     
    Intracellular redox state is a critical factor for fundamental cellular functions, including regulation of the activities of various metabolic enzymes as well as ROS production and elimination. Genetically-encoded fluorescent redox sensors, such as roGFP (Hanson, G. T., et al. (2004)) and Redoxfluor (Yano, T., et al. (2010)), have been developed to investigate the redox state of living cells. However, these sensors are not useful in cells that contain, for example, other colored pigments. We therefore intended to obtain simpler redox sensor proteins, and have developed oxidation-sensitive fluorescent proteins called Oba-Q (oxidation balance sensed quenching) proteins. Our sensor proteins derived from CFP and Sirius can be used to monitor the intracellular redox state as their fluorescence is drastically quenched upon oxidation. These blue-shifted spectra of the Oba-Q proteins enable us to monitor various redox states in conjunction with other sensor proteins.
  • Kim, K., Lakhanpal, G., Lu, H.E., Khan, M., Suzuki, A., Kato-Hayashi, M., Narayanan, R., Luyben, T.T., Matsuda, T., Nagai, T., Blanpied, T.A., Hayashi, Y., Okamoto, K.
    Neuron 87 4 813 - 826 2015年 [査読有り][通常論文]
     
    The structural modification of dendritic spines plays a critical role in synaptic plasticity. CaMKII is a pivotal molecule involved in this process through both kinase-dependent and independent structural functions, but the respective contributions of these two functions to the synaptic plasticity remain unclear. We demonstrate that the transient interplay between the kinase and structural functions of CaMKII during the induction of synaptic plasticity temporally gates the activity-dependent modification of the actin cytoskeleton. Inactive CaMKII binds F-actin, thereby limiting access of actin-regulating proteins to F-actin and stabilizing spine structure. CaMKII-activating stimuli trigger dissociation of CaMKII from F-actin through specific autophosphorylation reactions within the F-actin binding region and permits F-actin remodeling by regulatory proteins followed by reassociation and restabilization. Blocking the autophosphorylation impairs both functional and structural plasticity without affecting kinase activity. These results underpin the importance of the interplay between the kinase and structural functions of CaMKII in defining a time window permissive for synaptic plasticity.
  • Kim, K., Lakhanpal, G., Lu, H.E., Khan, M., Suzuki, A., Hayashi, M.K., Narayanan, R., Luyben, T.T., Matsuda, T., Nagai, T., Blanpied, T.A., Hayashi, Y., Okamoto, K.
    Neuron 88 2 433 - 433 2015年 [査読有り][通常論文]
  • Vadim Pérez Koldenkova, Tomoki Matsuda, Takeharu Nagai
    Journal of Biomedical Optics 20 10 2015年 [査読有り][通常論文]
     
    © 2015 Society of Photo-Optical Instrumentation Engineers (SPIE). Intracellular Mg2+ roles are commensurate with its abundance in the cell cytoplasm. However, little is known about Mg2+ subcellular dynamics, primarily due to the lack of suitable Mg2+-selective tools to monitor this ion in intracellular compartments. To cope with this lack, we developed a Mg2+-sensitive indicator - MagIC (indicator for Magnesium Imaging in Cell) - composed of a functionalized yellow fluorescent protein (FP) variant fused to a red-emitting FP serving as a reference, thus allowing ratiometric imaging of Mg2+. MagIC expressed in mammalian cells is homogeneously distributed between the cytosol and nucleus but its fusion with appropriate targeting sequences redirects it to mitochondria or the endoplasmic reticulum. MagIC shows little interference by intracellular Ca2+ [Kd(Mg2+)=5.1 mM; Kd(Ca2+)=4.8 mM] and its kinetic properties (koff=84 s-1) approach those of indicator dyes. With MagIC, as reported previously, we also observed a cytosolic Mg2+ increase provoked by application of 50 mM MgCl2 in the medium. This effect is, however, mimicked by 75 mM KCl or 150 mM d-sorbitol addition, indicating that it is a response to the associated hyperosmotic shock and not to Mg2+ itself. Our results confirm the functionality of MagIC as a useful tool for the long-awaited possibility of prolonged and organelle-specific monitoring of cellular Mg2+.
  • Tiwari, D.K., Arai, Y., Yamanaka, M., Matsuda, T., Agetsuma, M., Nakano, M., Fujita, K., Nagai, T.
    Nature Methods 12 6 515 - + 2015年 [査読有り][通常論文]
     
    Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.
  • Shinji Yoshiyama, Zhenyi Chen, Tsuyoshi Okagaki, Kazuhiro Kohama, Ritsuko Nasu-Kawaharada, Takashi Izumi, Noriyasu Ohshima, Takeharu Nagai, Akio Nakamura
    Atherosclerosis 237 2 464 - 470 2014年12月01日 [査読有り][通常論文]
     
    Objective: Cigarette smoking is a known risk factor for arteriosclerosis. In atheromatous plaques, vascular smooth muscle cells (VSMCs) display a phenotype that is different from the contractile type under normal conditions. Nicotine is the major pharmacological agent in cigarette smoke. However, any direct effect of nicotine on VSMCs remains uncertain. Because nicotine promotes VSMC migration, its phenotype may change due to nicotine. Approach and results: We used human aorta primary smooth muscle cells (HuAoSMCs), differentiated with transforming growth factor-β, to investigate changes in the protein levels of differentiation markers and in the activity of mitogen-activated protein kinases (MAPKs) after exposure to 0.1μM of nicotine for 48h. After nicotine exposure, the protein levels of myosin II 10 (2.93-fold) and β-actin (1.66-fold), synthetic type markers, were increased. In contrast, the levels of the contractile type markers, myosin II 11 (0.63-fold), high-molecular-weight caldesmon (0.40-fold) and SM22 (0.66-fold), which concern differentiated VSMC, were decreased. Moreover, nicotine exposure induced enhanced activation of p38 MAPK (1.30-fold) and extracellular signal-regulated kinase (1.91-fold). These results indicated that the phenotype of HuAoSMCs had changed to a synthetic-like type because of nicotine exposure. Thus, nicotine is one factor that can alter protein expression of differentiation markers in VSMCs. Besides, the increase of intracellular Ca2+ levels suggested that these effects of nicotine were mediated through nicotinic acetylcholine receptors. Conclusion: Nicotine has already been reported to promote VSMC migration from the tunica media to atheromatous plaques in the vascular intima. This phenomenon may occur because nicotine directly induces VSMC transformation from contractile type to synthetic-like type via nicotinic acetylcholine receptors and G protein-coupled receptors.
  • Yusuke Oshima, Takeshi Imamura, Atsuko Shintani, Hiroko Kajiura-Kobayashi, Terumasa Hibi, Takeharu Nagai, Shigenori Nonaka, Tomomi Nemoto
    International Journal of Molecular Sciences 15 11 19971 - 19986 2014年11月03日 [査読有り][通常論文]
     
    Yellow Cameleons are genetically encoded Ca2+ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca2+-sensor probe. To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from Rmax and Rmin values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo.
  • Kazunori Kanemaru, Hiroshi Sekiya, Ming Xu, Kaname Satoh, Nami Kitajima, Keitaro Yoshida, Yohei Okubo, Takuya Sasaki, Satoru Moritoh, Hidetoshi Hasuwa, Masaru Mimura, Kazuki Horikawa, Ko Matsui, Takeharu Nagai, Masamitsu Iino, Kenji F Tanaka
    Cell Reports 8 1 311 - 8 2014年07月10日 [査読有り][通常論文]
     
    Astrocytes generate local calcium (Ca(2+)) signals that are thought to regulate their functions. Visualization of these signals in the intact brain requires an imaging method with high spatiotemporal resolution. Here, we describe such a method using transgenic mice expressing the ultrasensitive ratiometric Ca(2+) indicator yellow Cameleon-Nano 50 (YC-Nano50) in astrocytes. In these mice, we detected a unique pattern of Ca(2+) signals. These occur spontaneously, predominantly in astrocytic fine processes, but not the cell body. Upon sensory stimulation, astrocytes initially responded with Ca(2+) signals at fine processes, which then propagated to the cell body. These observations suggest that astrocytic fine processes function as a high-sensitivity detector of neuronal activities. Thus, the method provides a useful tool for studying the activity of astrocytes in brain physiology and pathology.
  • Satoshi Uehara, Itsuo Hanasaki, Yoshiyuki Arai, Takeharu Nagai, Satoyuki Kawano
    Micro and Nano Letters 9 4 257 - 260 2014年 [査読有り][通常論文]
     
    The diffusion of single-stranded DNA (ssDNA) molecules near a glass surface by total internal reflection fluorescence microscopy has been observed. Substantially smaller diffusion coefficients compared to the bulk are caused by phenomenologically different types of surface effect that is not necessarily adsorptive. In particular, preliminary treatments of the glass substrate by low and high concentrations of KOH lead to highly and much less adsorptive effects on the ssDNA molecules, respectively. These characteristics beyond diffusion coefficients are elucidated by the difference of two ensemble-averaging methods of mean square displacement for diffusion coefficients due to the nonuniformity of the molecular motion, which is further confirmed by the distribution of displacements and tracked duration. © 2014 The Institution of Engineering and Technology.
  • Jin, M., Yamada, M., Arai, Y., Nagai, T., Hirotsune, S.
    Nature Communications 5 5295 - 5295 2014年 [査読有り][通常論文]
     
    Cytoplasmic dynein acts as a motor for the intracellular retrograde motility of vesicles and organelles along microtubules. However, the regulatory mechanism underlying release of dynactin bound cargoes from dynein motor remains largely unknown. Here we report that ADP-ribosylation factor-like 3 (Arl3) and dynein light chain LC8 induce dissociation of dynactin from dynein. Immunoprecipitation and microtubule pull-down assays revealed that Arl3(Q71L) and LC8 facilitated detachment of dynactin from dynein. We also demonstrated Arl3(Q71L) or LC8-mediated dynactin release from a dynein-dynactin complex through trace experiments using quantum dot (Qdot)-conjugated proteins. Furthermore, we disclosed interactions of Arl3 and LC8 with dynactin and dynein, respectively, by live-cell imaging. Finally, knockdown (KD) of Arl3 and LC8 by siRNA induced abnormal localizations of dynein, dynactin and related organelles. Our findings uncovered the surprising functional relevance of GTP-bound Arl3 and LC8 for the unloading regulation of dynactin-bound cargo from dynein motor.
  • Fukuda, N., Matsuda, T., Nagai, T.
    ACS Chemical Biology 9 5 1197 - 1203 2014年 [査読有り][通常論文]
     
    Calcium ion (Ca2+) is an important second messenger implicated in the control of many different cellular processes in living organisms. Ca2+ is typically studied by direct visualization using chemically or genetically encoded indicators. A complementary, and perhaps more useful, approach involves direct manipulation of Ca2+ concentration; tools for this exist but are rather poorly developed compared to the indicators at least. Here, we report a photoactivatable Ca2+-releasing protein, photoactivatable Ca2+ releaser (PACR), made by the insertion of a photosensitive protein domain (LOV2) into a Ca2+ binding protein (calmodulin fused with the M13 peptide). As the PACR is genetically encoded, and unlike conventional optical control tools (e.g., channel rhodopsin) not membrane bound, we are able to restrict expression within the cell, to allow subcellular perturbation of Ca2+ levels. In whole animals, we are able to control the behavior of Caenorhabditis elegans with light by expressing the PACR only in the touch neuron.
  • Masahito Yamanaka, Kenta Saito, Nicholas I. Smith, Satoshi Kawata, Takeharu Nagai, Katsumasa Fujita
    Interface Focus 3 5 2013年10月06日 [査読有り][通常論文]
     
    We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for sub diffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear decon-volution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy. © 2013 The Author(s) Published by the Royal Society. All rights reserved.
  • Tadasu Nozaki, Kazunari Kaizu, Chan-Gi Pack, Sachiko Tamura, Tomomi Tani, Saera Hihara, Takeharu Nagai, Koichi Takahashi, Kazuhiro Maeshima
    Nucleus 4 5 349 - 356 2013年09月 [査読有り][通常論文]
  • Dhermendra K. Tiwari, Takeharu Nagai
    Development Growth and Differentiation 55 4 491 - 507 2013年05月 [査読有り][通常論文]
     
    During the past decade, several novel fluorescence microscopy techniques have emerged that achieve incredible spatial and temporal resolution beyond the diffraction limit. These microscopy techniques depend on altered optical setups, unique fluorescent probes, or post-imaging analysis. Many of these techniques also depend strictly on the use of unique fluorescent proteins (FPs) with special photoswitching properties. These photoswitchable FPs are capable of switching between two states in response to light. All localization precision and patterned illumination techniques-such as photo-activation localization microscopy, stochastic optical reconstruction microscopy, reversible saturable optically linear transitions, and saturated structured illumination microscopy-take advantage of these inherent switching properties to achieve superior spatial resolution. This review provides extensive analysis of the positive and negative aspects of photoswitchable FPs, highlighting their application in diffraction-unlimited imaging and suggesting the most suitable fluorescent proteins for superresolution imaging. Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.
  • Kazuki Horikawa, Takeharu Nagai
    Clinical Calcium 23 4 527 - 33 2013年04月 [査読有り][通常論文]
     
    Advance in the live imaging technology revealed that cells display a variety of Ca(2 +) dynamics, such as the wave, oscillation, blip, puff and spark. Accumulating evidences suggest that not only Ca(2 +) itself, but its spatio-temporal dynamics are the important information carrier in the physiological responses. Here, we will introduce the pattern of Ca(2 +) dynamics along with the latest version of high-performance Ca(2 +) probes.
  • Takeharu Nagai, Kenta Saito, Yuriko Higuchi, Yoshiyuki Arai, Takeharu Nagai
    Protocol Exchange 2013年02月18日
  • Masami Yamada, Kanako Kumamoto, Shintaro Mikuni, Yoshiyuki Arai, Masataka Kinjo, Takeharu Nagai, Yoshikazu Tsukasaki, Tomonobu M Watanabe, Mitsuru Fukui, Mingyue Jin, Shiori Toba, Shinji Hirotsune
    Nature Communications 4 2013年 [査読有り][通常論文]
     
    Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules. We previously demonstrated that LIS1 forms an idling complex with dynein, which is transported to the plus ends of microtubules by kinesin motors. Here we report that the small GTPase Rab6a is essential for activation of idling dynein. Immunoprecipitation and microtubule pull-down assays reveal that the GTP bound mutant, Rab6a(Q72L), dissociates LIS1 from a LIS1-dynein complex, activating dynein movement in in vitro microtubule gliding assays. We monitor transient interaction between Rab6a(Q72L) and dynein in vivo using dual-colour fluorescence cross-correlation spectroscopy in dorsal root ganglion (DRG) neurons. Finally, we demonstrate that Rab6a(Q72L) mediates LIS1 release from a LIS1-dynein complex followed by dynein activation through an in vitro single-molecule assay using triple-colour quantum dots. Our findings reveal a surprising function for GTP bound Rab6a as an activator of idling dynein. © 2013 Macmillan Publishers Limited. All rights reserved.
  • Hoi, H., Matsuda, T., Nagai, T., Campbell, R.E.
    Journal of the American Chemical Society 135 1 46 - 49 2013年 [査読有り][通常論文]
     
    Two of the most powerful implementations of fluorescent protein (FP) technology are "highlighters", which can be converted from nonfluorescent to fluorescent or from one color to another by illumination, and calcium ion (Ca2+) indicators. Combining the properties of both of these FP classes into a single construct would produce a highlightable Ca2+ indicator that would enable researchers to mark a single cell spectrally in a transfected tissue and image its intracellular Ca2+ dynamics. In an effort to create such a hybrid tool, we explored three different protein design strategies. The strategy that ultimately proved successful involved the creation of a circularly permuted version of a green-to-red photoconvertible FP and its introduction into a G-CaMP-type single-FP-based Ca2+ indicator. Optimization by directed evolution led to the identification of two promising variants that exhibit excellent photoconversion properties and have an up to 4.6-fold increase in red fluorescence intensity upon binding of Ca2+. We demonstrate the utility of these variants in HeLa cells and rat hippocampal neurons.
  • Matsuda, T., Horikawa, K., Saito, K., Nagai, T.
    Scientific Reports 3 2013年 [査読有り][通常論文]
     
    Genetically encoded fluorescent indicators for bioimaging are powerful tools for visualizing biological phenomena in specified cell types or cellular compartments. However, available gene promoters or localization sequences are not applicable for visualizing all expression events. Furthermore, a visualization technique focusing on single cells or cellular compartments is required for characterizing specific cellular properties including individuality of cells in the cell population. To address these limitations, we developed a genetically encoded caged Ca2+ indicator for which expression timing and location could be controlled. This indicator, PA-TNXL, comprises a Ca2+-binding protein and troponin between a photoactivatable FRET donor (PA-GFP) and a FRET quencher (dim variant of YFP). Ultraviolet irradiation activates the FRET Ca2+ indicator. Using this indicator, we successfully imaged Ca2+ dynamics in a given set of HeLa cells and cultured hippocampal neurons. This technology can be applied for developing other photoactivatable indicators, thereby opening a new area of biological research.
  • Wu, J., Liu, L., Matsuda, T., Zhao, Y., Rebane, A., Drobizhev, M., Chang, Y.-F., Araki, S., Arai, Y., March, K., Hughes, T.E., Sagou, K., Miyata, T., Nagai, T., Li, W.-H., Campbell, R.E.
    ACS Chemical Neuroscience 4 6 963 - 972 2013年 [査読有り][通常論文]
     
    We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca2+) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca2+ indicator, R-GEC01. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca2+ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.
  • Takemoto, K., Matsuda, T., Sakai, N., Fu, D., Noda, M., Uchiyama, S., Kotera, I., Arai, Y., Horiuchi, M., Fukui, K., Ayabe, T., Inagaki, F., Suzuki, H., Nagai, T.
    Scientific Reports 3 2629  2013年 [査読有り][通常論文]
     
    Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.
  • Saera Hihara, Chan-Gi Pack, Kazunari Kaizu, Tomomi Tani, Tomo Hanafusa, Tadasu Nozaki, Satoko Takemoto, Tomohiko Yoshimi, Hideo Yokota, Naoko Imamoto, Yasushi Sako, Masataka Kinjo, Koichi Takahashi, Takeharu Nagai, Kazuhiro Maeshima
    Cell Reports 2 6 1645 - 56 2012年12月27日 [査読有り][通常論文]
     
    Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (~50 nm movement/30 ms), which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.
  • Katsuya Kominami, Takeharu Nagai, Tatsuya Sawasaki, Yuki Tsujimura, Kenta Yashima, Yasuhiro Sunaga, Masateru Tsuchimochi, Jun Nishimura, Kumiko Chiba, Jun Nakabayashi, Koji Koyamada, Yaeta Endo, Hideo Yokota, Atsushi Miyawaki, Noboru Manabe, Kazuhiro Sakamaki
    PLoS ONE 7 11 2012年11月21日 [査読有り][通常論文]
     
    Background: Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. Methodology/Principal Findings: We have established a biosensor based on fluorescence resonance energy transfer (FRET) for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET) system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3) in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. Conclusions: Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8. © 2012 Kominami et al.
  • Megumi Iwano, Quy A. Ngo, Tetsuyuki Entani, Hiroshi Shiba, Takeharu Nagai, Atsushi Miyawaki, Akira Isogai, Ueli Grossniklaus, Seiji Takayama
    Development (Cambridge) 139 22 4202 - 4209 2012年11月15日 [査読有り][通常論文]
     
    The directional growth of the pollen tube from the stigma to the embryo sac in the ovules is regulated by pollen-pistil interactions based on intercellular communication. Although pollen tube growth is regulated by the cytoplasmic Ca 2+ concentration ([Ca 2+] cyt), it is not known whether [Ca 2+] cyt is involved in pollen tube guidance and reception. Using Arabidopsis expressing the GFP-based Ca 2+ -sensor yellow cameleon 3.60 (YC3.60) in pollen tubes and synergid cells, we monitored Ca 2+ dynamics in these cells during pollen tube guidance and reception under semi-in vivo fertilization conditions. In the pollen tube growing towards the micropyle, pollen tubes initiated turning within 150 μm of the micropylar opening the [Ca 2+] cyt in these pollen tube tips was higher than in those not growing towards an ovule in assays with myb98 mutant ovules, in which pollen tube guidance is disrupted. These results suggest that attractants secreted from the ovules affect Ca 2+ dynamics in the pollen tube. [Ca 2+] cyt in synergid cells did not change when the pollen tube grew towards the micropyle or entered the ovule. Upon pollen tube arrival at the synergid cell, however, [Ca 2+] cyt oscillation began at the micropylar pole of the synergid, spreading towards the chalazal pole. Finally, [Ca 2+] cyt in the synergid cell reached a maximum at pollen tube rupture. These results suggest that signals from the pollen tube induce Ca 2+ oscillations in synergid cells, and that this Ca 2+ oscillation is involved in the interaction between the pollen tube and synergid cell. © 2012. Published by The Company of Biologists Ltd.
  • Katsuya Kominami, Jun Nakabayashi, Takeharu Nagai, Yuki Tsujimura, Kumiko Chiba, Haruna Kimura, Atsushi Miyawaki, Tatsuya Sawasaki, Hideo Yokota, Noboru Manabe, Kazuhiro Sakamaki
    Biochimica et Biophysica Acta - Molecular Cell Research 1823 10 1825 - 1840 2012年10月 [査読有り][通常論文]
     
    Caspase-8 (CASP8) is a cysteine protease that plays a pivotal role in the extrinsic apoptotic signaling pathway via death receptors. The kinetics, dynamics, and selectivity with which the pathway transmits apoptotic signals to downstream molecules upon CASP8 activation are not fully understood. We have developed a system for using high-sensitivity FRET-based biosensors to monitor the protease activity of CASP8 and its downstream effector, caspase-3, in living single cells. Using this system, we systematically investigated the caspase cascade by regulating the magnitude of extrinsic signals received by the cell. Furthermore, we determined the molar concentration of five caspases and Bid required for hierarchical transmission of apoptotic signals in a HeLa cell. Based on these quantitative experimental data, we validated a mathematical model suitable for estimation of the kinetics and dynamics of caspases, which predicts the minimal concentration of CASP8 required to act as an initiator. Consequently, we found that less than 1% of the total CASP8 proteins are sufficient to set the apoptotic program in motion if activated. Taken together, our findings demonstrate the precise cascade of CASP8-mediated apoptotic signals through the extrinsic pathway. © 2012 Elsevier B.V.
  • Noriyuki Hatsugai, Vadim Perez Koldenkova, Hiromi Imamura, Hiroyuki Noji, Takeharu Nagai
    Plant and Cell Physiology 53 10 1768 - 1775 2012年10月 [査読有り][通常論文]
     
    Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection. © The Author 2012.
  • Keiichi Ohtsuka, Shinobu Sato, Yusuke Sato, Kojiro Sota, Shinsuke Ohzawa, Tomoki Matsuda, Kiwamu Takemoto, Nobutoki Takamune, Bernard Juskowiak, Takeharu Nagai, Shigeori Takenaka
    Chemical Communications 48 39 4740 - 4742 2012年05月16日 [査読有り][通常論文]
     
    When a biotinylated FRET probe based on a peptide-thrombin binding aptamer conjugate was introduced together with streptavidin and biotinylated nuclear export signal peptide into HeLa cells, the resulting ternary complex enabled visualization of K + concentration changes in the cell. © 2012 The Royal Society of Chemistry.
  • H. J. Kwon, Y. Ohmiya, K. I. Honma, S. Honma, T. Nagai, K. Saito, K. Yasuda
    Cell Death and Disease 3 3 e278  2012年03月 [査読有り][通常論文]
     
    The skeletal elements of embryonic limb are prefigured by prechondrogenic condensation in which secreted molecules such as adhesion molecules and extracellular matrix have crucial roles. However, how the secreted molecules are controlled to organize the condensation remains unclear. In this study, we examined metabolic regulation of secretion in prechondrogenic condensation, using bioluminescent monitoring systems. We here report on ATP oscillations in the early step of chondrogenesis. The ATP oscillations depended on both glycolysis and mitochondrial respiration, and their synchronization among cells were achieved via gap junctions. In addition, the ATP oscillations were driven by Ca 2+ oscillations and led to oscillatory secretion in chondrogenesis. Blockade of the ATP oscillations prevented cellular condensation. Furthermore, the degree of cellular condensation increased with the frequency of ATP oscillations. We conclude that ATP oscillations have a critical role in prechondrogenic condensation by inducing oscillatory secretion. © 2012 Macmillan Publishers Limited All rights reserved.
  • Chang, Y.-F., Arai, Y., Nagai, T.
    Neuroscience Research 73 4 341 - 347 2012年 [査読有り][通常論文]
     
    Optogenetic tools, such as channelrhodopsin2 (ChR2), have enabled the behavior of whole organisms by light-mediated manipulation of neuronal activities. Fluorescent indicators have been used to aid in the understanding of what is happening in living cells. To date, optogenetic stimulation and imaging acquisition were sequentially performed during detector "live time." However, there is a problem with interrupting acquisition time sequences because such stimulation invades the time territory of fluorescent imaging. Here, our purpose was to show that optogenetic stimulation can be performed within the "dead time" of the charge-coupled device camera, the short interval of data transfer between frames. We show the kinetic measurement of Ca2+ dynamics in neuron-like cells upon ChR2 stimulation, by which we reveal the biphasic property of the Ca2+ increase in response to optical stimulation. (C) 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Saito, K., Chang, Y.-F., Horikawa, K., Hatsugai, N., Higuchi, Y., Hashida, M., Yoshida, Y., Matsuda, T., Arai, Y., Nagai, T.
    Nature Communications 3 1262  2012年 [査読有り][通常論文]
     
    The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca2+, cyclic adenosine monophosphate and adenosine 5'-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.
  • Yongxin Zhao, Satoko Araki, Jiahui Wu, Takayuki Teramoto, Yu-Fen Chang, Masahiro Nakano, Ahmed S. Abdelfattah, Manabi Fujiwara, Takeshi Ishihara, Takeharu Nagai, Robert E. Campbell
    Science 333 6051 1888 - 1891 2011年09月30日 [査読有り][通常論文]
     
    Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca 2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca2+ and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.
  • Masahiro Nakano, Hiromi Imamura, Takeharu Nagai, Hiroyuki Noji
    ACS Chemical Biology 6 7 709 - 715 2011年07月15日 [査読有り][通常論文]
     
    Intracellular Ca 2+ levels play a crucial role in the control of ATP synthesis. However, the spatiotemporal correlation between ATP and Ca 2+ remains unclear due to the inability to visualize these factors within same individual cells. A Förster resonance energy transfer (FRET)-based fluorescent ATP probe, named ATeam, was recently developed for ATP imaging in single living cells. However, the spectra of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) used as the FRET donor and the acceptor, respectively, significantly overlap with the ultraviolet-excitable Ca 2+ probe, fura-2. In the present work, we developed new red-shifted ATP probes, GO-ATeams, in which green fluorescent protein (GFP) and orange fluorescent protein (OFP) was used as the FRET pair to minimize spectral overlap with the fura-2 emission. The dynamics of intracellular Ca 2+ and mitochondrial ATP levels in single histamine-stimulated HeLa cells were successfully visualized by using fura-2 and GO-ATeam. The experiments showed that histamine induced increases of both intracellular Ca 2+ and mitochondrial ATP levels. The increment of mitochondrial ATP levels was proportional to that of Ca 2+. This finding suggests that cellular Ca 2+ levels might precisely control mitochondrial ATP synthesis in response to the increased ATP consumption triggered by Ca 2+. In addition, GO-ATeam has several advantages over the original ATeam. The GO-ATeam signal was more stable against acidification, which would allow ATP imaging inside acidic intracellular compartments. Also, the GO-ATeam excitation wavelength is much less phototoxic to cells, making the probe suitable for long-time observation. © 2011 American Chemical Society.
  • Mami Nomura, Takeharu Nagai, Yoshie Harada, Tomomi Tani
    Developmental Neurobiology 71 7 634 - 649 2011年07月 [査読有り][通常論文]
     
    Intracellular transport of neurotrophin receptors together with neurotrophins is one of the key events of neurotrophin signaling for the growth and the survival of neurons. However, the involvement of neurotrophin signaling in the regulation of intracellular transport of neurotrophin receptors has been remained unclear. We visualized the behavior of TrkA, a receptor of nerve growth factor (NGF), by labeling with GFP in PC12 cells. We found remarkable changes of the behavior of TrkA-GFP upon the application of NGF. Before the application, only ~37% of the fluorescent dots of TrkA showed translocations along neurites of PC12 cells. After the application, number of the dots showing the directional movement increased to ~65%. The averaged velocities of the directional movement of TrkA-GFP dots became higher after the application of NGF. We tested the idea whether NGF binding accelerated the translocations of TrkA by simultaneously observing TrkA-GFP and fluorescently labeled NGF, Cy3.5-NGF. The velocity of TrkA-GFP dots associated with Cy3.5-NGF was remarkably higher than that of TrkA-GFP dots without Cy3.5-NGF. On the basis of these observations, we hypothesize that there is a signaling mechanism within a single vesicle that facilitates the intracellular transport of each vesicle containing the activated TrkA. © 2011 Wiley Periodicals, Inc.
  • Saito, K., Arai, Y., Zhang, J., Kobayashi, K., Tani, T., Nagai, T.
    Cell Structure and Function 36 2 237 - 246 2011年 [査読有り][通常論文]
     
    Laser-scanning confocal microscopy has been employed for exploring structures at subcellular, cellular and tissue level in three dimensions. To acquire the confocal image, a coherent light source, such as laser, is generally required in conventional single-point scanning microscopy. The illuminating beam must be focused onto a small spot with diffraction-limited size, and this determines the spatial resolution of the microscopy system. In contrast, multipoint scanning confocal microscopy using a Nipkow disk enables the use of an incoherent light source. We previously demonstrated successful application of a 100 W mercury arc lamp as a light source for the Yokogawa confocal scanner unit in which a microlens array was coupled with a Nipkow disk to focus the collimated incident light onto a pinhole (Saito et al., Cell Struct. Funct., 33: 133-141, 2008). However, transmission efficiency of incident light through the pinhole array was low because off-axis light, the major component of the incident light, was blocked by the non-aperture area of the disk. To improve transmission efficiency, we propose an optical system in which off-axis light is able to be transmitted through pinholes surrounding the pinhole located on the optical axis of the collimator lens. This optical system facilitates the use of not only the on-axis but also the off-axis light such that the available incident light is considerably improved. As a result, we apply the proposed system to high-speed confocal and multicolor imaging both with a satisfactory signal-to-noise ratio.
  • Yamada, Y., Michikawa, T., Hashimoto, M., Horikawa, K., Nagai, T., Miyawaki, A., Häusser, M., Mikoshiba, K.
    Frontiers in Cellular Neuroscience 5 SEPTEMBER 18  2011年 [査読有り][通常論文]
     
    Genetically encoded Ca2+ indicators (GECIs) are promising tools for cell type-specific and chronic recording of neuronal activity. In the mammalian central nervous system, however, GECIs have been tested almost exclusively in cortical and hippocampal pyramidal cells, and the usefulness of recently developed GECIs has not been systematically examined in other cell types. Here we expressed the latest series of GECIs, yellow cameleon (YC) 2.60, YC3.60, YC-Nano 15, and GCaMP3, in mouse cortical pyramidal cells as well as cerebellar Purkinje cells using in utero injection of recombinant adenoviral vectors. We characterized the performance of the GECIs by simultaneous two-photon imaging and whole-cell patch-clamp recording in acute brain slices at 33 +/- 2 degrees C. The fluorescent responses of GECIs to action potentials (APs) evoked by somatic current injection or to synaptic stimulation were examined using rapid dendritic imaging. In cortical pyramidal cells, YC2.60 showed the largest responses to single APs, but its decay kinetics were slower than YC3.60 and GCaMP3, while GCaMP3 showed the largest responses to 20APs evokedat 20 Hz. In cerebellar Purkinje cells, only YC2.60 and YC-Nano 15 could reliably report single complex spikes (CSs), and neither showed signal saturation over the entire stimulus range tested (1-10CSs at 10Hz). The expression and response of YC2.60 in Purkinje cells remained detectable and comparable for at least over 100 days. These results provide use ful information for selecting an optimal GECI depending on the experimental requirements: in cortical pyramidal cells, YC2.60 is suitable for detecting sparse firing of APs, whereas GCaMP3 is suitable for detecting burst firing of APs; in cerebellar Purkinje cells, YC2.60 as well as YC-Nano 15 is suitable for detecting CSs.
  • Yang, L., Matsuda, T., Raviraj, V., Ching, Y.W., Braet, F., Nagai, T., Soon, L.L.
    Journal of Microscopy 242 3 250 - 261 2011年 [査読有り][通常論文]
     
    Cybr/Reduced On-random Motile (ROM) is a scaffold protein, containing a postsynaptic density protein-95/discs-large/ZO-1 (PDZ) domain, a LEU region and a PDZ domain binding region at the C-terminus. In the immune system, Cybr/ROM was found to localize in vesicles and at the plasma membrane, through interactions with cytohesin-1. In this investigation, we reported Cybr/ROM as occurring in vesicles, the cytoplasm and at membrane ruffles of H1299 lung cancer cells. Its localization at the ruffles was dependent on intact actin structures as indicated by latrunculin A treatment, which abrogated ruffle formation and staining of Cybr/ROM at the cells' periphery. Transfection of truncation mutants consisting of either the PDZ or LEU domain showed that the LEU domain of ROM was localized to membrane ruffles, vesicles and the cytoplasm, whereas, the PDZ domain localized to the membrane ruffles and cytoplasm only. There was therefore, domain/molecular segregation of Cybr/ROM in different cellular compartments. Cybr/ROM was subcloned into a plasmid carrying the photoactivation-mediated resonance energy transfer (Phamret) protein. The photoconversion experiments demonstrated the diffusion of ROM from the cytoplasm to the membrane ruffling sites and conversely from membrane ruffles to the cytoplasm. Large variances in the transport velocity of Cybr/ROM in the cytoplasm suggested that its movements were facilitated by other mechanisms in addition to diffusion.
  • Takemoto, K., Matsuda, T., McDougall, M., Klaubert, D.H., Hasegawa, A., Los, G.V., Wood, K.V., Miyawaki, A., Nagai, T.
    ACS Chemical Biology 6 5 401 - 406 2011年 [査読有り][通常論文]
     
    Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen (O-1(2)) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of O-1(2) than fluorescein and about 5-fold efficiency in CALI of beta-galactosidase by using an eosin labeled anti-beta-galactosidase antibody compared With the fluorescein labeled one To covalently label target protein with eosin, we synthesize a membrane permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-gamma and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells.
  • Kazuki Horikawa, Yoshiyuki Yamada, Tomoki Matsuda, Kentarou Kobayashi, Mitsuhiro Hashimoto, Toru Matsu-ura, Atsushi Miyawaki, Takayuki Michikawa, Katsuhiko Mikoshiba, Takeharu Nagai
    Nature Methods 7 9 729 - 32 2010年09月 [査読有り][通常論文]
     
    We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60. Their high Ca(2+) affinities (K(d) = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca(2+) transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.
  • Henry Lütcke, Masanori Murayama, Thomas Hahn, David J. Margolis, Simone Astori, Stephan Meyer Zum Alten Borgloh, Werner Göbel, Ying Yang, Wannan Tang, Sebastian Kügler, Rolf Sprengel, Takeharu Nagai, Atsushi Miyawaki, Matthew E. Larkum, Fritjof Helmchen, Mazahir T. Hasan
    Frontiers in Neural Circuits 4 APR 2010年04月29日 [査読有り][通常論文]
     
    Fluorescent calcium (Ca2+) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiberoptic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 - in combination with various optical techniques - thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations. © 2010 Lütcke, Murayama, Hahn, Margolis, Astori, zum Alten Borgloh, Göbel, Yang, Tang, Kügler, Sprengel, Nagai, Miyawaki, Larkum, Helmchen and Hasan.
  • Jin Hee Hong, Cheol Hong Min, Byeongha Jeong, Tomoyoshi Kojiya, Eri Morioka, Takeharu Nagai, Masayuki Ikeda, Kyoung J. Lee
    PLoS ONE 5 3 2010年03月10日 [査読有り][通常論文]
     
    Background: Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca 2+ spikes" (i.e., [Ca2+]c transients having a bandwidth of 10-100 seconds) in SCN neurons, but it is unclear if these SCN Ca2+ spikes are related to the slow circadian rhythms. Methodology/Principal Findings: We addressed this issue based on a Ca 2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca2+ spike was barely observed (< 3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleonexpressing SCN cultures, however, the number of cells exhibiting Ca2+ spikes was increased to 13-14%. Conclusions/Significance: Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+] c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca 2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca 2+ spikes in the function of SCN. © 2010 Hong et al.
  • Ippei Kotera, Takuya Iwasaki, Hiromi Imamura, Hiroyuki Noji, Takeharu Nagai
    ACS Chemical Biology 5 2 215 - 222 2010年02月19日 [査読有り][通常論文]
     
    Fluorescent protein (FP)-based Förster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of Aequorea FPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range. © 2010 American Chemical Society.
  • Saito, K., Hatsugai, N., Horikawa, K., Kobayashi, K., Matsu-Ura, T., Mikoshiba, K., Nagai, T.
    PLoS ONE 5 4 2010年 [査読有り][通常論文]
     
    Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. We have applied this strategy to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. With this BRAC, we succeeded visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca2+-independent signal drifts due to change in cell shape, focus shift, etc. Taking advantage of the bioluminescence imaging property that does not require external excitation light, BRAC might become a powerful tool applicable in conjunction with so-called optogenetic technology by which we can control cellular and protein function by light illumination..
  • Hiromi Imamura, Kim P. Huynh Nhat, Hiroko Togawa, Kenta Saito, Ryota Iino, Yasuyuki Kato-Yamada, Takeharu Nagai, Hiroyuki Noji
    Proceedings of the National Academy of Sciences of the United States of America 106 37 15651 - 15656 2009年09月15日 [査読無し][通常論文]
  • Megumi Iwano, Tetsuyuki Entani, Hiroshi Shiba, Mituru Kakita, Takeharu Nagai, Hideaki Mizuno, Atsushi Miyawaki, Tsubasa Shoji, Kenichi Kubo, Akira Isogai, Seiji Takayama
    Plant Physiology 150 3 1322 - 1334 2009年07月 [査読有り][通常論文]
     
    Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca2+ concentra-tion ([Ca2+]) gradient and regular oscillations of the cytosolic [Ca2+] ([Ca2+]cyt) in the tip region. Whether this [Ca2+] gradient and/ or [Ca2+]cyt oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca2+]cyt dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca2+]cyt gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca2+]cyt, however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca2+]cyt in the tip region is essential for pollen tube growth, whereas a regular [Ca2+] oscillation is not. Next, we monitored [Ca2+] dynamics in the endoplasmic reticulum ([Ca2+]ER) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca2+compared with yellow cameleon 3.60. The [Ca2+]ER in pollen tubes grown under the semi-in vivo condition was between 100 and 500 mM. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca2+-ATPases, inhibited growth and decreased the [Ca2+]ER. Our observations suggest that the ER serves as one of the Ca2+ stores in the pollen tube and cyclopiazonic acid-sensitive Ca2+ -ATPases in the ER are required for pollen tube growth. © 2009 American Society of Plant Biologists.
  • Wataru Tomosugi, Tomoki Matsuda, Tomomi Tani, Tomomi Nemoto, Ippei Kotera, Kenta Saito, Kazuki Horikawa, Takeharu Nagai
    Nature Methods 6 5 351 - 3 2009年05月 [査読有り][通常論文]
     
    We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca(2+) concentration and caspase-3 activation in the same apoptotic cells.
  • Hiroyuki Ichijo, Takeharu Nagai, Michito Hamada, Makoto Kobayashi, Satoru Takahashi
    Neuroscience Research 65 Suppl. 1 S45 - S45 2009年 [査読無し][通常論文]
  • Yamamot, T., Akira, K., Saito, K., Nagai, T.
    Journal of Nanoscience and Nanotechnology 9 1 670 - 672 2009年 [査読有り][通常論文]
     
    The two-photon excitation action cross-sections of four kinds of co-oligomers of thiophene and pyridine (e.g., Py-(Th)(n)-Py; Py = 2-pyridyl, Th = thiophene-2,5-diyl; n = 3, 4) and a copolymer constituted of alternating pyridazine and 4,4'-dihexyl- 2,2'-bithiophene units have been measured by using a reported value ((sigma(2PE) at 780 nm = 10 GM) of green fluorescent protein as the reference. The co-oligomers give sigma(2PE) values of 14-26 GM and the copolymer shows sigma(2PE) of 23 GM.
  • Ippei Kotera, Takeharu Nagai
    Journal of Biotechnology 137 1-4 1 - 7 2008年10月10日 [査読有り][通常論文]
     
    Type IIS restriction enzymes have been successfully used as "universal" restriction enzymes in DNA manipulations. We took a step further to develop a rapid technique for recombining DNA fragments, fully automatic single-tube recombination (FASTR), which enables multiple-fragment DNA recombination in a single step. Crude PCR products are directly mixed with both type IIS restriction endonuclease and DNA ligase to initiate a spontaneous and one-way recombination reaction. Highly efficient DNA recombination can be achieved by an inhibition of DNA polymerase with aphidicolin and a selective digestion of template DNAs by DpnI, a restriction enzyme to digest hemi-methylated DNA in the reaction solution thereby the entire procedure takes less than 15 min. Owing to its simplicity, efficiency and rapidity, one-step FASTR can be applied to a wide range of DNA manipulations including those involving high-throughput applications where significant reduction in time and cost is expected. © 2008 Elsevier B.V. All rights reserved.
  • Takehiko Sunabori, Akinori Tokunaga, Takeharu Nagai, Kazunobu Sawamoto, Masaru Okabe, Atsushi Miyawaki, Yumi Matsuzaki, Takaki Miyata, Hideyuki Okano
    Journal of Cell Science 121 8 1204 - 1212 2008年04月15日 [査読有り][通常論文]
     
    During brain development, neural progenitor cells extend across the thickening brain wall and undergo mitosis. To understand how these two completely different cellular events are coordinated, we focused on the transcription pattern of the nestin gene (Nes), which encodes an intermediate filament protein strongly expressed in neural progenitor cells. To visualize nestin expression in vivo, we generated transgenic mice that expressed a destabilized fluorescent protein under the control of Nes second intronic enhancer (E/nestin:dVenus). During the neurogenic stage, when the brain wall thickens markedly, we found that nestin was regulated in a cell-cycle-dependent manner. Time-lapse imaging showed that nestin gene expression was upregulated during G1-S phase, when the neural progenitor cells elongate their fibers. However, nestin expression dramatically declined in G2-M phase, when progenitor cells round up to undergo mitosis. The cell-cycle-dependent phosphorylation of an upstream regulator class III POU transcription factor (Pou3f2 or Brn2) reduced its binding activity to the nestin core enhancer element and was therefore responsible for the decreased Nes transcription in G2-M phase. Collectively, these findings demonstrate precisely orchestrated gene regulation that correlates with the 3D morphological changes in neural progenitor cells in vivo.
  • Saito Kenta, Kobayashi Kentaro, Tani Tomomi, Nagai Takeharu
    Cell Structure and Function 33 1 133 - 141 Japan Society for Cell Biology 2008年 [査読有り][通常論文]
     
    Multi-point scanning confocal microscopy using a Nipkow disk enables the acquisition of fluorescent images with high spatial and temporal resolutions. Like other single-point scanning confocal systems that use Galvano meter mirrors, a commercially available Nipkow spinning disk confocal unit, Yokogawa CSU10, requires lasers as the excitation light source. The choice of fluorescent dyes is strongly restricted, however, because only a limited number of laser lines can be introduced into a single confocal system. To overcome this problem, we developed an illumination system in which light from a mercury arc lamp is scrambled to make homogeneous light by passing it through a multi-mode optical fiber. This illumination system provides incoherent light with continuous wavelengths, enabling the observation of a wide range of fluorophores. Using this optical system, we demonstrate both the high-speed imaging (up to 100 Hz) of intracellular Ca2+ propagation, and the multi-color imaging of Ca2+ and PKC-γ dynamics in living cells.
  • Matsuda, T., Miyawaki, A., Nagai, T.
    Nature Methods 5 4 339 - 345 2008年 [査読有り][通常論文]
     
    All biological reactions depend on the diffusion and re-localization of biomolecules. Our understanding of biological processes requires accurate measurement of biomolecule mobility in living cells. Currently, approaches for investigating the mobility of biomolecules are generally restricted to measuring either fast or slow diffusion kinetics. We describe the development and application of a photoconvertible fluorescent protein, Phamret, that can be highlighted by UV light stimulation inducing a change in fluorescence emission from cyan fluorescent protein (CFP) to photoactivated GFP (PA-GFP). Phamret can be monitored by single excitation-dual emission mode for visualization of molecular dynamics for a broad range of kinetics. We also devised a microscopy-based method to measure the diffusion coefficient from the fluorescence decay after photostimulation of Phamret, enabling analysis of diffusion kinetics ranging from less than 0.1 mu m(2)/s up to similar to 100 mu m(2)/s, and found significant changes in free protein movement during cell-cycle progression.
  • Takemoto, K, Kuranaga, E, Tonoki, A, Nagai, T, Miyawaki, A, Miura, M
    Proceedings of the National Academy of Sciences of the United States of America 104 33 13367 - 13372 2007年 [査読有り][通常論文]
  • An-A Kazuno, Kae Munakata, Takeharu Nagai, Satoshi Shimozono, Masashi Tanaka, Makoto Yoneda, Nobumasa Kato, Atsushi Miyawaki, Tadafumi Kato
    PLoS Genetics 2 8 1167 - 1177 2006年 [査読有り][通常論文]
     
    Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics. © 2006 Kazuno et al.
  • Kohyama, J., Tokunaga, A., Fujita, Y., Miyoshi, H., Nagai, T., Miyawaki, A., Nakao, K., Matsuzaki, Y., Okano, H.
    Developmental Biology 286 1 311 - 325 2005年 [査読有り][通常論文]
     
    Notch signaling plays various key roles in cell fate determination during CNS development in a context-dependent fashion. However, its precise physiological role and the localization of its target cells remain unclear. To address this issue, we developed a new reporter system for assessing the RBP-J-mediated activation of Notch signaling target genes in living cells and tissues using a fluorescent protein Venus. Our reporter system revealed that Notch signaling is selectively activated in neurosphere-initiating multipotent neural stem cells in vitro and in radial glia in the embryonic forebrain in vivo. Furthermore, the activation of Notch signaling occurs during gliogenesis and is required in the early stage of astroglial development. Consistent with these findings, the persistent activation of Notch signaling inhibits the differentiation of GFAP-positive astrocytes. Thus, the development of our RBP-J-dependent live reporter system, which is activated upon Notch activation, together with a stage-dependent gain-of-function analysis allowed us to gain further insight into the complexity of Notch signaling in mammalian CNS development. (c) 2005 Elsevier Inc. All rights reserved.
  • Takao, K., Okamoto, K.-I., Nakagawa, T., Neve, R.L., Nagai, T., Miyawaki, A., Hashikawa, T., Kobayashi, S., Hayashi, Y.
    Journal of Neuroscience 25 12 3107 - 3112 2005年 [査読有り][通常論文]
     
    Ca2+/calmodulin-dependent protein kinase II ( CaMKII) is highly enriched in excitatory synapses in the CNS and critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, because of a lack of specific methods. We tried to address this by optically detecting the conformational change in CaMKII during activation using fluorescence resonance energy transfer (FRET). The engineered FRET probe Camui alpha detects calmodulin binding and autophosphorylation at threonine 286 that renders the enzyme constitutively active. In combination with two-photon microscopy, we demonstrate that Camui alpha can be used to observe temporal and spatial regulation of CaMKII activity in living neurons.
  • Lin, X., Várnai, P., Csordás, G., Balla, A., Nagai, T., Miyawaki, A., Balla, T., Hajnóczky, G.
    Journal of Biological Chemistry 280 13 12820 - 12832 2005年 [査読有り][通常論文]
     
    Cytosolic Ca2+ ([Ca2+](c)) signals triggered by many agonists are established through the inositol 1,4,5-trisphosphate (IP3) messenger pathway. This pathway is believed to use Ca2+-dependent local interactions among IP3 receptors (IP3R) and other Ca2+ channels leading to coordinated Ca2+ release from the endoplasmic reticulum throughout the cell and coupling Ca2+ entry and mitochondrial Ca2+ uptake to Ca2+ release. To evaluate the role of IP3 in the local control mechanisms that support the propagation of [Ca2+](c) waves, storeoperated Ca2+ entry, and mitochondrial Ca2+ uptake, we used two IP3-binding proteins (IP3BP): 1) the PH domain of the phospholipase C-like protein, p130 (p130PH); and 2) the ligand-binding domain of the human type-IIP3R (IP3R224-605). As expected, p130PH-GFP and GFP-IP3R224-605 behave as effective mobile cytosolic IP3 buffers. In COS-7 cells, the expression of IP3BPs had no effect on store-operated Ca2+ entry. However, the IP3-linked [Ca2+] c signal appeared as a regenerative wave and IP3BPs slowed down the wave propagation. Most importantly, IP3BPs largely inhibited the mitochondrial [Ca2+] signal and decreased the relationship between the [Ca2+](c) and mitochondrial [Ca2+] signals, indicating disconnection of the mitochondria from the [Ca2+](c) signal. These data suggest that IP3 elevations are important to regulate the local interactions among IP3Rs during propagation of [Ca2+](c) waves and that the IP3-dependent synchronization of Ca2+ release events is crucial for the coupling between Ca2+ release and mitochondrial Ca2+ uptake.
  • Ikeda, M., Ikeda-Sagara, M., Okada, T., Clement, P., Urade, Y., Nagai, T., Sugiyama, T., Yoshioka, T., Honda, K., Inoué, S.
    Neuroscience 130 4 1029 - 1040 2005年 [査読有り][通常論文]
     
    CNS activity is generally coupled to the vigilance state, being primarily active during wakefulness and primarily inactive during deep sleep. During periods of high neuronal activity, a significant volume of oxygen is used to maintain neuronal membrane potentials, which subsequently produces cytotoxic reactive oxygen species (ROS). Glutathione, a major endogenous antioxidant, is an important factor protecting against ROS-mediated neuronal degeneration. Glutathione has also been proposed to be a sleep-promoting substance, yet the relationship between sleep and cerebral oxidation remains unclear. Here we report that i.c.v. infusion of the organic peroxide t-butyl-hydroperoxide at a concentration below that triggering neurodegeneration (0.1 mumol/ 100 mul/10 h) promotes sleep in rats. Also, microinjection (2 nmol, 2 mul) or microdialysis (100 muM, 20 min) of t-butyl-hydroperoxide into the preoptic/anterior hypothalamus (POAH) induces the release of the sleep-inducing neuromodulators, nitric oxide and adenosine, without causing neurodegeneration. Nitric oxide and adenosine release was inhibited by co-dialysis of the N-methyl-D-aspartate receptor antagonist, d(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 mM), suggesting that glutamate-induced neuronal excitation mediates the peroxide-induced release of nitric oxide and adenosine. Indeed, Ca2+ release from mitochondria and delayed-onset Ca2+ influx via N-methyl-D-aspartate receptors was visualized during peroxide exposure using Ca2+ indicator proteins (YC-2.1 and mitochondrial-targeted Pericam) expressed in organotypic cultures of the POAH. In the in vitro models, t-butyl-hydroperoxide (50 muM) causes dendritic swelling followed by the intracellular Ca2+ mobilization, and D-AP5 (100 muM) or glutathione (500 muM) inhibited t-butyl-hydroperoxide-induced intracellular Ca2+ mobilization and protected POAH neurons from oxidative stress. These data suggest that low-level subcortical oxidation under the control of an antioxidant system may trigger sleep via the Ca2+-dependent release of sleep-inducing neuromodulators in the POAH, and thus we propose that a moderate increase of ROS during wakefulness in the neuronal circuits regulating sleep may be an initial trigger in sleep induction. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Nagai, T, Yamada, S, Tominaga, T, Ichikawa, M, Miyawaki, A
    Proceedings of the National Academy of Sciences of the United States of America 101 29 10554 - 10559 2004年 [査読有り][通常論文]
  • Mazahir T. Hasan, Rainer W. Friedrich, Thomas Euler, Matthew E. Larkum, Günter Giese, Matthias Both, Jens Duebel, Jack Waters, Hermann Bujard, Oliver Griesbeck, Roger Y. Tsien, Takeharu Nagai, Atsushi Miyawaki, Winfried Denk
    PLoS Biology 2 6 2004年 [査読有り][通常論文]
     
    Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.
  • Okamoto, K.-I., Nagai, T., Miyawaki, A., Hayashi, Y.
    Nature Neuroscience 7 10 1104 - 1112 2004年 [査読有り][通常論文]
     
    The synapse is a highly organized cellular specialization whose structure and composition are reorganized, both positively and negatively, depending on the strength of input signals. The mechanisms orchestrating these changes are not well understood. A plausible locus for the reorganization of synapse components and structure is actin, because it serves as both cytoskeleton and scaffold for synapses and exists in a dynamic equilibrium between F-actin and G-actin that is modulated bidirectionally by cellular signaling. Using a new FRET-based imaging technique to monitor F-actin/G-actin equilibrium, we show here that tetanic stimulation causes a rapid, persistent shift of actin equilibrium toward F-actin in the dendritic spines of rat hippocampal neurons. This enlarges the spines and increases postsynaptic binding capacity. In contrast, prolonged low-frequency stimulation shifts the equilibrium toward G-actin, resulting in a loss of postsynaptic actin and of structure. This bidirectional regulation of actin is actively involved in protein assembly and disassembly and provides a substrate for bidirectional synaptic plasticity.
  • Iwano, M., Shiba, H., Miwa, T., Che, F.-S., Takayama, S., Nagai, T., Miyawaki, A., Isogai, A.
    Plant Physiology 136 3 3562 - 3571 2004年 [査読有り][通常論文]
     
    Ca2+ dynamics in the growing pollen tube have been well documented in vitro using germination assays and Ca2+ imaging techniques. However, very few in vivo studies of Ca2+ in the pollen grain and papilla cell during pollination have been performed. We expressed yellow cameleon, a Ca2+ indicator based on green fluorescent protein, in the pollen grains and papilla cells of Arabidopsis (Arabidopsis thaliana) and monitored Ca2+ dynamics during pollination. In the pollen grain, [Ca2+](cyt) increased at the potential germination site soon after hydration and remained augmented until germination. As in previous in vitro germination studies, [Ca2+](cyt) oscillations were observed in the tip region of the growing pollen tube, but the oscillation frequency was faster and [Ca2+](cyt) was higher than had been observed in vitro. In the pollinated papilla cell, remarkable increases in [Ca2+](cyt) occurred three times in succession, just under the site of pollen-grain attachment. [Ca2+](cyt) increased first soon after pollen hydration, with a second increase occurring after pollen protrusion. The third and most remarkable [Ca2+](cyt) increase took place when the pollen tube penetrated into the papilla cell wall.
  • Nagai, T., Miyawaki, A.
    Biochemical and Biophysical Research Communications 319 1 72 - 77 2004年 [査読有り][通常論文]
     
    SCAT3 is a fluorescence resonance energy transfer (FRET)-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3-sensitive linker, and an enhanced yellow fluorescent protein with efficient maturation property (Venus). Despite its considerable promise, however, greater responsivity of fluorescence to the proteolysis has been desired for better understanding of spatio-temporal pattern of the activation of caspase-3 during apoptosis. In the present study, the length of linker regions of SCAT3 has been thoroughly optimized by use of a PCR technique. The bacterial colonies expressing the constructs were screened for high FRET efficiency using our home-made fluorescence image analyzer. The FRET signal of an improved SCAT3 changed by about tenfold during apoptotic events in mammalian cells, enabling visualization of caspase-3 activation with better spatial resolution than before. This new high-throughput method will be applicable to development and improvement of FRET-based indicators for proteolysis. (C) 2004 Elsevier Inc. All rights reserved.
  • Karasawa, S., Araki, T., Nagai, T., Mizuno, H., Miyawaki, A.
    Biochemical Journal 381 1 307 - 312 2004年 [査読有り][通常論文]
     
    GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) technology has facilitated the exploration of the spatio-temporal patterns of cellular signalling. While most studies have used cyan- and yellow-emitting FPs (fluorescent proteins) as FRET donors and acceptors respectively, this pair of proteins suffers from problems of pH-sensitivity and bleeding between channels. In the present paper, we demonstrate the use of an alternative additional donor/acceptor pair. We have cloned two genes encoding FPs from stony corals. We isolated a cyan-emitting FP from Acropara sp., whose tentacles exhibit cyan coloration. Similar to GFP from Renilla reniformis, the cyan FP forms a tight dimeric complex. We also discovered an orange-emitting FP from Fungia concinna. As the orange FP exists in a complex oligomeric structure, we converted this protein into a monomeric form through the introduction of three amino acid substitutions, recently reported to be effective for converting DsRed into a monomer (Clontech). We used the cyan FP and monomeric orange FP as a donor/acceptor pair to monitor the activity of caspase 3 during apoptosis. Due to the close spectral overlap of the donor emission and acceptor absorption (a large Forster distance), substantial pH-resistance of the donor fluorescence quantum yield and the acceptor absorbance, as well as good separation of the donor and acceptor signals, the new pair can be used for more effective quantitative FRET imaging.
  • Hasan, M.T., Friedrich, R.W., Euler, T., Larkum, M.E., Giese, G., Both, M., Duebel, J., Waters, J., Bujard, H., Griesbeck, O., Tsien, R.Y., Nagai, T., Miyawaki, A., Denk, W.
    PLoS Biology 2 6 763 - 775 2004年 [査読有り][通常論文]
     
    Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.
  • Hino, J., Nishimatsu, S.-I., Nagai, T., Matsuo, H., Kangawa, K., Nohno, T.
    Developmental Biology 260 1 138 - 157 2003年 [査読有り][通常論文]
     
    Bone morphogenetic proteins (BMPs) and their antagonists are involved in the axial patterning of vertebrate embryos. We report that both BMP-3b and BMP-3 dorsalize Xenopus embryos, but act as dissimilar antagonists within the BMP family. BMP-3b injected into Xenopus embryos triggered secondary head formation in an autonomous manner, whereas BMP-3 induced aberrant tail formation. At the molecular level, BMP-3b antagonized nodal-like proteins and ventralizing BMPs, whereas BMP-3 antagonized only the latter. These differences are due to divergence of their pro-domains. Less BMP-3b than BMP-3 precursor is proteolytically processed in embryos. BMP-3b protein associated with a monomeric form of Xnrl, a nodal-like protein, whereas BMP-3 did not. These molecular features are consistent with their expression profiles during Xenopus development. XBMP-3b is expressed in the prechordal plate; while xBMP-3 is expressed in the notochord. Using antisense morpholino oligonucleotides, we found that the depletion of both xBMP-3b and cerberus, a head inducer, caused headless Xenopus embryos, whereas the depletion of both xBMP-3 and cerberus affected the size of the somite. These results revealed that xBMP-3b and cerberus are essential for head formation regulated by the Spemann organizer, and that xBMP-3b and perhaps xBMP-3 are involved in the axial patterning of Xenopus embryos. (C) 2003 Elsevier Science (USA). All rights reserved.
  • Takemoto, K., Nagai, T., Miyawaki, A., Miura, M.
    Journal of Cell Biology 160 2 235 - 243 2003年 [査読有り][通常論文]
     
    Indicator molecules for caspase-3 activation have been reported that use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (the donor) and enhanced yellow fluorescent protein (EYFP; the acceptor). Because EYFP is highly sensitive to proton (H+) and chloride ion (Cl-) levels, which can change during apoptosis, this indicator's ability to trace the precise dynamics of caspase activation is limited, especially in vivo. Here, we generated an H+- and Cl--insensitive indicator for caspase activation, SCAT, in which EYFP was replaced with Venus, and monitored the spatio-temporal activation of caspases in living cells. Caspase-3 activation was initiated first in the cytosol and then in the nucleus, and rapidly reached maximum activation in 10 min or less. Furthermore, the nuclear activation of caspase-3 preceded the nuclear apoptotic morphological changes. In contrast, the completion of caspase-9 activation took much longer and its activation was attenuated in the nucleus. However, the time between the initiation of caspase-9 activation and the morphological changes was quite similar to that seen for caspase-3, indicating the activation of both caspases occurred essentially simultaneously during the initiation of apoptosis.
  • Rekas, A., Alattia, J.-R., Nagai, T., Miyawaki, A., Ikura, M.
    Journal of Biological Chemistry 277 52 50573 - 50578 2003年 [査読有り][通常論文]
     
    Yellow emission variants of green fluorescent protein (GFP) have been found useful in a variety of applications in biological systems due to their red-shifted emission spectrum and sensitivity to environmental parameters, such as pH and ionic strength. However, slow maturation properties and new requirements for more intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). In this paper, we present the crystal structure of Venus at 2.2 Angstrom resolution, which enabled us to correlate its novel features with these mutation points. The rearrangement of several side chains near the chromophore, initiated by the F46L mutation, was found to improve maturation at 37degreesC by removing steric and energetic constraints, which may hinder folding of the polypeptide chain, and by accelerating the oxidation of the Calpha-Cbeta bond of Tyr(66) during chromophore formation. M153T, V163A, and S175G were also found to improve the rate of maturation by creating regions of greater flexibility. F64L induced large conformational changes in the molecule, leading to the removal of halide sensitivity by preventing ion access to the binding site.
  • Tadafumi Kato, Mizuho Ishiwata, Takeharu Nagai
    Life Sciences 71 5 581 - 590 2002年06月21日 [査読有り][通常論文]
     
    Human lymphoblastoid cell line (LCL) transformed by Epstein-Barr Virus (EBV) is a unique cellular model for the study of human diseases. Although pathophysiological significance of mitochondrial calcium regulation is drawing attention, it is not known whether or not mitochondria in LCLs play a role in intracellular calcium signaling. In this study, role of mitochondria of the lymphoblastoid cell line in calcium signaling was examined. Intra-mitochondrial calcium concentration ([Ca2+]m) was successfully measured using dihydro-Rhod-2, revealed by the decrease of fluorescence after application of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and intracellular localization patterns imaged by florescent microscope. Platelet activating factor (PAF) concentration-dependently increased cytosolic calcium concentration ([Ca2+]i), while no increase of [Ca2+]m was observed. In contrast, 10 μM thapsigargin increased [Ca2+]i as well as [Ca2+]m. LCLs may be used for the study of possible pathophysiological role of mitochondrial calcium regulation in human diseases. © 2002 Elsevier Science Inc. All rights reserved.
  • Takeharu Nagai, Keiji Ibata, Eun Sun Park, Mie Kubota, Katsuhiko Mikoshiba, Atsushi Miyawaki
    Nature Biotechnology 20 1 87 - 90 2002年01月 [査読有り][通常論文]
  • Fujii, H., Nagai, T., Shirasawa, H., Doi, J.-Y., Yasui, K., Nishimatsu, S.-I., Takeda, H., Sakai, M.
    Developmental Biology 252 1 15 - 30 2002年 [査読有り][通常論文]
     
    Two distinct types of axis lacking embryos resulted from partial deletion of the vegetal part of early one-cell-stage embryos. When the deleted volume was 20-40% (relative surface area), the embryos underwent ventral-type gastrulation and formed ventral mesodermal tissues. When the deleted volume was more than 60%, the embryo did not gastrulate nor make mesodermal structures (M. Sakai, 1996, Development 122, 2207-2214). We have designated these two types of embryos as "gastrulating nonaxial embryos (GNEs)" and "permanent blastula-type embryos (PBEs)," respectively. Using these embryos as recipients, a series of Einsteck transplantation experiments were carried out to investigate mechanisms controlling anteroposterior patterning during early Xenopus development. GNEs receiving dorsal marginal zone (DMZ) transplants (GNE/DMZs) elongated and formed posteriorized phenotypes, which had muscle cells, melanocytes, and tail fins. In contrast, PBE/DMZs did not elongate but formed cement glands and brain-like structures showing strong anteriorization. Simultaneous transplantation of the cells from various regions of normal embryos with the DMZ into PBEs revealed that the entire vegetal half of normal embryos, except for the DMZ, showed posteriorizing activity. These results strongly suggest that anteroposterior patterning in Xenopus is not achieved solely by the dorsal marginal zone (the Spemann organizer), but instead by a synergistic mechanism of the dorsalizing domain (DMZ) and the posteriorizing domain (the entire vegetal half except for the DMZ). (C) 2002 Elsevier Science (USA).
  • Shimozono, S., Fukano, T., Nagai, T., Kirino, Y., Mizuno, H., Miyawaki, A.
    Science's STKE : signal transduction knowledge environment 2002 125 2002年 [査読有り][通常論文]
  • Valérie Robert, Pamela Gurlini, Valeria Tosello, Takeharu Nagai, Atsushi Miyawaki, Fabio Di Lisa, Tullio Pozzan
    EMBO Journal 20 17 4998 - 5007 2001年09月03日 [査読有り][通常論文]
     
    The Ca2+-sensitive photoprotein aequorin and the new green fluorescent protein-based fluorescent Ca2+ indicators 'ratiometric-pericam' were selectively expressed in the mitochondria, cytosol and/or nucleus of spontaneously beating ventricular myocytes from neonatal rats. This combined strategy reveals that mitochondrial [Ca2+] oscillates rapidly and in synchrony with cytosolic and nuclear [Ca2+]. The Ca2+ oscillations were reduced in frequency and/or amplitude by verapamil and carbachol and were enhanced by isoproterenol and elevation of extracellular [Ca2+]. An increased frequency and/or amplitude of cytosolic Ca2+ spikes was rapidly mirrored by similar changes in mitochondrial Ca2+ spikes and more slowly by elevations of the interspike Ca2+ levels. The present data unequivocally demonstrate that in cardiac cells mitochondrial [Ca2+] oscillates synchronously with cytosolic [Ca2+] and that mitochondrial Ca2+ handling rapidly adapts to inotropic or chronotropic inputs.
  • Naoki Mochizuki, Shigeko Yamashita, Kazuo Kurokawa, Yusuke Ohba, Takeharu Nagai, Atsushi Miyawaki, Michiyuki Matsuda
    Nature 411 6841 1065 - 1068 2001年06月 [査読有り][通常論文]
  • Tetsuya Kitaguchi, Katsunori Nakata, Takeharu Nagai, Jun Aruga, Katsuhiko Mikoshiba
    Development Genes and Evolution 211 6 309 - 314 2001年06月01日 [査読有り][通常論文]
  • T. Nagai, A. Sawano, Eun Sun Park, A. Miyawaki
    Proceedings of the National Academy of Sciences of the United States of America 98 6 3197 - 3202 2001年03月13日 [査読無し][通常論文]
  • Takeharu Nagai, Jun Aruga, Osamu Minowa, Takashi Sugimoto, Yoshiki Ohno, Tetsuo Noda, Katsuhiko Mikoshiba
    Proceedings of the National Academy of Sciences of the United States of America 97 4 1618 - 1623 2000年02月15日 [査読有り][通常論文]
  • T. Kitaguchi, T. Nagai, K. Nakata, J. Aruga, K. Mikoshiba
    Development 127 22 4787 - 4795 2000年 [査読有り][通常論文]
     
    Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin β or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.
  • Katsunori Nakata, Takeharu Nagai, Jun Aruga, Katsuhiko Mikoshiba
    Mechanisms of Development 75 1-2 43 - 51 1998年07月 [査読有り][通常論文]
     
    We characterized two new members of the Zic family, Xenopus Zic1 and Zic2. They are very similar to mouse Zic1 and Zic2 in the protein coding region including the zinc finger domain. In early gastrula, Zic1 expression was restricted to the prospective neural plate region whereas Zic2 was expressed widely in the ectoderm. We observed enhanced neural and neural crest-derived tissue formation in the Zic1 or Zic2 overexpressed embryos and neural and neural crest marker induction in the Zic1 or Zic2 overexpressed animal cap explants. Our findings suggest that Zic1 and Zic2 have essentially the same properties as Zic3 and that the Xenopus Zic family may act cooperatively in the initial phase of neural and neural crest development.
  • Jun Aruga, Takeharu Nagai, Katsunori Nakata, Tetsuya Kitaguchi, Kiyomi Mizugishi, Katsuhiko Mikoshiba
    Neuroscience Research 31 S42  Elsevier {BV} 1998年01月 [査読有り][通常論文]
  • Jun Aruga, Osamu Minowa, Hiroyuki Yaginuma, Junko Kuno, Takeharu Nagai, Tetsuo Noda, Katsuhiko Mikoshiba
    Journal of Neuroscience 18 1 284 - 293 1998年 [査読有り][通常論文]
     
    Zic genes encode zinc finger proteins, the expression of which is highly restricted to cerebellar granule cells and their precursors. These genes are homologs of the Drosophila pair-rule gene odd-paired. To clarify the role of the Zic1 gene, we have generate mice deficient in Zic1. Homozygous mice showed remarkable ataxia during postnatal development. Nearly all of the mice died within 1 month. Their cerebella were hypoplastic and missing a lobule in the anterior lobe. A bromodeoxyuridine labeling study indicated a reduction both in the proliferating cell fraction in the external germinal layer (EGL), from 14 d postcoitum, and in forward movement of the EGL. These findings suggest that Zic1 may determine the cerebellar folial pattern principally via regulation of cell proliferation in the EGL.
  • Katsunori Nakata, Takeharu Nagai, Jun Aruga, Katsuhiko Mikoshiba
    Proceedings of the National Academy of Sciences of the United States of America 94 22 11980 - 11985 1997年10月28日 [査読有り][通常論文]
  • Takeharu Nagai, Jun Aruga, Shinji Takada, Thomas Günther, Ralf Spörle, Klaus Schughart, Katsuhiko Mikoshiba
    Developmental Biology 182 2 299 - 313 1997年02月 [査読有り][通常論文]
  • Jun Aruga, Osamu Minowa, Junko Kuno, Hiroyuki Yaginuma, Takeharu Nagai, Tetsuo Noda, Katsuhiko Mikoshiba
    Neuroscience Research 28 S118  Elsevier {BV} 1997年01月 [査読有り][通常論文]
  • Jun Aruga, Takeharu Nagai, Tsutomu Tokuyama, Yoshihide Hayashizaki, Yasushi Okazaki, Verne M. Chapman, Katsuhiko Mikoshiba
    Journal of Biological Chemistry 271 2 1043 - 1047 1996年01月12日 [査読有り][通常論文]
     
    The mouse Zic gene, which encodes a zinc finger protein, is expressed in the developing or matured central nervous system in a highly restricted manner. We identified two novel Zic-related genes (Zic2, Zic3) through genomic and cDNA cloning. Both genes are highly similar to Zic(1), especially in their zinc finger motif. A comparison of genomic organization among the three Zic genes showed that they share common exon-intron boundaries and belong to the same gene family. Ziel, Zic2, and Zic3 were determined to mouse chromosome 9, 14, and X using an interspecific backcross panel. Northern blotting and ribonuclease protection showed that Zic2 and Zic3 are expressed in a restricted manner in the cerebellum at the adult stage. However, the temporal profile of the mRNA expression in the developing cerebella differ in the three Zic genes. Furthermore, we found that the Drosophila pair-rule gene, odd-paired is highly homologous to the Zic gene family. The similarity was not only the zinc finger motif, but also the exonintron boundary was the same as those of mouse Zic gene family. These findings suggest that the Zic gene family and Drosophila odd-paired are derived from a common ancestral gene.
  • Sadao Aoki, Ikuo Yoneda, Takeharu Nagai, Naoto Ueno, Kazuo Murakami
    Japanese Journal of Applied Physics 33 4 L556 - L558 1994年 [査読有り][通常論文]
     
    Nondestructive high-resolution imaging of frog (Xenopus laevis) embryos has been developed by X-ray microtomography. Shadow-projection X-ray microtomography with a brilliant fine focus laboratory X-ray source could image fine structures of Xenopus embryos which were embedded in paraffin wax. The imaging system enabled us to not only distinguish endoderm from ectoderm at the gastrula stage, but also to obtain a cross-section view of the tail bud embryo showing muscle, notochord and neural tube without staining. Furthermore, the distribution of myosin was also imaged in combination with whole-mount immunohistochemistry. © 1994 Japanese Journal of Applied Physics. All rights reserved.
  • A. Suzuki, T. Nagai, S. I. Nishimatsu, H. Sugino, Y. Eto, H. Shibai, K. Murakami, N. Ueno
    Biochemical Journal 298 2 275 - 280 1994年 [査読有り][通常論文]
     
    Activin exhibits a potent mesoderm inducing activity towards the ectodermal tissue (animal cap) of Xenopus laevis blastulae. Thus in order to investigate the role of activin in morphogenesis of early Xenopus embryos, activation of genes for activin βA and βB was examined by the reverse transcription polymerase chain reaction. In vivo, activin βB mRNA appears to be present in embryonic stage 1 whereas βA mRNA is undetectable prior to gastrulation. βB and βA mRNAs were noted to accumulate after stages 9 and 15 respectively. Activin gene expression in Xenopus animal caps was examined after treatment with various concentrations of activin A. Under these treatment conditions, both activin βA and βB mRNAs accumulated in a dose-dependent fashion after 24 h. The same effect was noted for treatment with similar concentrations of activin B. Accumulation of mRNAs was inhibited by the addition of cycloheximide to the culture medium, consistent with the proposition that activin gene expression requires certain protein factors. In total, therefore, these data suggest that an autoinduction mechanism is involved in the regulation of activin mRNA levels in normal Xenopus embryos and that this mechanism may play a pivotal role during early embryonic development.
  • S. Nishimatsu, M. Iwao, T. Nagai, S. Oda, A. Suzuki, M. Asashima, K. Murakami, N. Ueno
    FEBS Letters 312 2-3 169 - 173 1992年11月09日 [査読有り][通常論文]
     
    The function of a carboxyl-terminal truncated version of the Xenopus activin receptor, encoded by a previously isolated gene XSTK2, was investigated in early embryos. The transcript corresponding to the truncated receptor gene was detected throughout embryonic development although the temporal expression pattern was different from that of an intact receptor. Injection of X5TR2 mRNA into early embryos resulted in the formation of a duplicated body axis. Mesoderm induction as evaluated by the activation of the α-actin gene in presumptive ectoderm (animal cap) treated with exogenous activin was significantly enhanced by the injection of XSTK2 mRNA. These results suggest that the truncated receptor is capable of transmitting the activin signal to the same extent as the native receptor. © 1992.

書籍

講演・口頭発表等

  • 第6回医薬品毒性機序研究会 2023年12月 つくばカピオ 一般社団法人日本毒性学会 医薬品毒性機序研究部会
  • 永井 健治
    UVSOR シンポジウム 2023 2023年12月 岡崎コンファレンスセンター 自然科学研究機構分子科学研究所、UVSOR 利用者懇談会
  • Takeharu Nagai
    5+2レクチャーシリーズ 2023年11月 National Yang Ming Chiao Tung University National Yang Ming Chiao Tung University
  • Takeharu Nagai
    5+2レクチャーシリーズ 2023年11月 公開講演，セミナー，チュートリアル，講習，講義等 National Yang Ming Chiao Tung University National Yang Ming Chiao Tung University
  • Multicolor autoluminescent proteins for long term visualization of various biological events  [招待講演]
    Takeharu Nagai
    OptoRevolution: Exploring the Frontiers of Physiology with Light 2023年11月 Sanya, China 
    https://www.syiab.org.cn/#/active?id=5
  • Nagai T
    Advances in Optical Interrogation of Living Cells and Organisms: Focus on the Brain 2023年10月 Suzhou, China Cold Spring Harbor Asia
  • 永井 健治, 出村 拓, 寺川 輝彦
    会いに行ける科学者フェス 2023年10月 秋葉原UDX 日本科学振興協会
  • 永井 健治
    シンギュラリティ生物学×放射線医科学研究会 2023年10月 広島ガーデンパレス・鶴亀
  • 永井 健治
    第82回日本癌学会学術総会 2023年09月 パシフィコ横浜
  • Takeharu Nagai
    The 20th International Microscopy Congress (IMC20) 2023年09月 BEXCO, Busan, Korea Korean Society of Microscopy (KSM) , International Federation of Societies for Microscopy (IFSM)
     
    As chairperson.（セッションオーガナイザー）
  • 永井健治
    第1回 研究者をつなぐ研究フォーラム ～ライフサイエンスの交差点～, オンライン 2023年08月
  • 永井健治
    上原国際シンポジウム2023, ハイアットリージェンシー東京＆オンライン 2023年06月
  • 永井健治
    第18回 大阪大学・ニコンイメージングセンター シリーズセミナー, 大阪大学・ニコンイメージングセンター&オンライン 2023年04月
  • 発光植物ビジネス  [招待講演]
    永井健治
    ⼤阪ベンチャー研究会 第203回研究会・第18回総会, ⽇刊⼯業新聞社 ⼤阪⽀社&オンライン 2023年04月
  • Luminescent protein technologies  [招待講演]
    永井健治
    滋賀県立彦根東高等学校 サイエンス国際フォーラム 2023年03月
  • 永井健治
    アライアンス G3 分科会,北海道大学,札幌
  • 永井 健治
    第15回 先端シーズフォーラム, 梅田スカイビル&オンライン 2023年02月
  • 永井 健治
    The 26 th SANKEN international symposium, Osaka univeristy SANKEN
  • 永井 健治
    理研シンポジウム：第 10 回「光量子工学研究」, 理化学研究所 和光事業所 鈴木梅太郎記念ホール＆オンライン 2022年12月
  • 永井 健治
    けいはんなサイエンスカフェ, Online 2022年12月
  • New trends in life science opened up by means of trans-scale-scope-Science of Outliers  [招待講演]
    Nagai T
    Seminar, Tampere University, Finland, 2022年12月
  • 永井 健治
    第 41 回 日本マグネシウム学会学術集会, Online 2022年12月
  • 永井 健治
    JMAC 第 152 回定例会, Online 2022年11月
  • Nagai T
    ICRP2022 Sessions (19th International Conference on Retinal Proteins), Royton Sapporo 2022年11月
  • Singularity created by bioluminescent protein technologies  [招待講演]
    Nagai T
    Lecture on IVC2022, Online, 2022年07月
  • Singularity created by fluorescent protein technologies  [招待講演]
    Nagai T
    Lecture on IVC2022, Online 2022年07月
  • 発光するタンパク質の応用研究  [招待講演]
    永井 健治
    科学工学技術委員会, Online 2022年07月
  • 永井 健治
    神戸大学次世代光散乱イメージング科学研究センターキックオフシンポジウム, 神⼾⼤学百年記念館(神⼤会館)六甲ホール 2022年06月
  • 永井 健治
    第 78 回日本顕微鏡学会学術講演会 シンポジウム, ビッグパレットふくしま 2022年05月
  • Nagai T
    Focus on microscopy 2022, online 2022年04月
  • 永井 健治
    第127回日本解剖学会総会・全国学術集会, Online 2022年03月
  • 永井 健治
    第21回日本再生医療学会総会, On Demand 2022年03月
  • 永井 健治
    第95回日本薬理学会年会, 福岡国際会議場 2022年03月
  • Nagai T
    Nano Thailand 2021, Online 2021年12月 その他
  • 永井 健治
    第59回日本生物物理学会年会バイオフィジックスセミナー 2021年11月
  • 永井 健治
    未来医学研究会マンスリーセミナーvol.20, Online 2021年10月
  • 永井 健治
    ARO協議会第8回学術集会, Online 2021年09月
  • 永井 健治
    第3回細胞農業会議, Online (日本細胞農業協会主催 2021年08月
  • 「蛍光タンパク質を改変して機能指示薬を作成する方法」  [招待講演]
    永井 健治
    第46回組織細胞化学講習会, Web配信 (日本組織細胞化学会主催) 2021年08月
  • 永井 健治
    第60回日本生体医工学会大会, Online (日本生体医工学会) 2021年06月
  • Nagai T
    8th Japan-China Symposium on Nanomedicine 2021, Online 2021年06月 その他
  • 「競争的研究資金を得るための申請書作成 "虎の巻"」  [招待講演]
    永井 健治
    R03科研費セミナー 科研費獲得実践講習会, Online (大阪産業技術研究所) 2021年06月
  • Nagai T
    Osaka University Anniversary Lecture Series 7 Advanced Biotechnology and Technology, Online 2021年06月 その他
  • Nagai T
    The 11th BRI International Symposium, From single cell to systems neuroscience, Online 2021年02月 その他
  • Nagai T
    A3 Foresight & 5 Star Alliance Joint Workshop on Organic/Inorganic Hybrid Nano Materials and Bio Imaging, Online (Host: A3 Foresight & 5 Star Alliance） 2020年12月 その他
  • 永井 健治
    高分子・ハイブリッド材料研究センター 2020PHyMシンポジウム, 東北大学南総合研究棟 2020年10月
  • 永井 健治
    日本学術会議公開シンポジウム 「次世代統合バイオイメージングと数理の協働の展望」, オンライン（主催：日本学術会議） 2020年10月
  • 永井 健治
    第93回日本生化学会大会 シンポジウム「生物発光イメージングの最前線」, オンライン（主催：日本生化学会） 2020年09月
  • 「光るたんぱく質が拓く未来の社会」  [招待講演]
    永井 健治
    先端科学研修特別講義, 滋賀県立彦根東高等学校 2020年09月
  • 「トランススケールスコープが拓くシンギュラリティ生物学」  [招待講演]
    永井 健治
    鳥取大学特別講義Ⅵ, オンライン（主催：鳥取大学医学部） 2020年08月
  • How to come up with ideas on breakthrough research  [招待講演]
    Nagai T
    ForTi_World Class Scientists 2020, Online (Host: ForMIND Institute) 2020年07月 その他
  • 永井 健治
    日本顕微鏡学会第76回学術講演会, 紙上開催（大阪国際交流センター） 2020年05月
  • 永井 健治
    北海道大学 公共政策大学院 第6回文理融合セミナー,北海道大学 人文・社会科学総合教育研究棟 2020年02月
  • 永井 健治
    近畿化学協会機能性色素部会第１００回記念例会 , 大阪科学技術センター 2020年01月
  • Earth-shattering Application of Super-duper Bioluminescent Proteins for Sustainable Development Presenter  [招待講演]
    Nagai T
    Public Lecture, SITH Guest-Professor Talk, SITH - ITB Venue, Institut Teknologi Bandung 2019年12月
  • 「Trans-scale imaging toward singularity biology」  [招待講演]
    永井 健治
    17th IPR Retrea;大阪大学銀杏会館 2019年11月
  • Nagai T
    ICSB2019 Workshop (The 20th International Conference on Systems Biology), Okinawa Institute of Science and Technology Graduate University 2019年10月
  • 「発光タンパク質が拓く未来社会」  [招待講演]
    永井 健治
    大阪大学名誉教授会;リーガロイヤルホテル 2019年10月
  • 永井 健治
    理化学研究所;合同シンポジウム;イメージングから理論;東広島キャンパス;学士会館;レセプションホール 2019年10月
  • 永井 健治
    第60回日本組織細胞化学会総会・学術集会, 神戸商工会議所 2019年09月
  • 永井 健治
    第321回千里ライフサイエンスフォーラム, 千里ライフサイエンスセンター 2019年09月
  • Nagai T
    AIBBC Conference;Africa International Biotechnology;Biomedical Conference;PrideInn Paradise;Beach Resor;Mombasa 2019年08月
  • Nagai T
    AIBBC Workshops (4th Africa International Biotechnology and Biomedical Conference), Institute of Primate Research, Nairobi 2019年08月
  • 永井 健治
    第44回組織細胞化学講習会 2019年08月
  • Development of fluorescent/ bioluminescent probes toward singularity biology  [招待講演]
    Nagai T
    TOPICAL PROBLEMS OF BIOPHOTONICS, "Konstantin Korotkov" boat, Russia 2019年07月
  • 永井 健治
    2019年度第12回高校生事業 ライフサイエンスセミナー 2019年07月
  • 永井 健治
    関西経済連合会評議員会, リーガロイヤルホテルNCB 2019年06月
  • 「マルチモーダルトランススケールイメージングの展望」  [招待講演]
    永井 健治
    光ネットワークシステム技術第171委員会第67回研究会, 主婦会館プラザエフ 2019年05月
  • Singularity biology  [招待講演]
    Nagai T
    OIST Mini-Symposium The;h International Membrane Research Forum 2019年03月
  • 永井 健治
    第38回マグネシウム学会, 京都大学楽友会館 2018年12月
  • 「新学術領域「シンギュラリティ生物学」」  [招待講演]
    永井 健治
    生命機能;物質;デバイスシステムG;分科会;東京工業大学鈴掛キャンパス 2018年11月
  • 「シンギュラリティ生物学」  [招待講演]
    永井 健治
    生理学研究所研究会, 生理学研究所 2018年09月
  • 永井 健治
    第３回JKiCイメージングセミナー, 慶応義塾大学 2018年08月
  • 永井 健治
    第20回日本光生物協会年会, 京都大学 2018年08月
  • 「蛍光・化学発光ライブイメージングの現状と展望」  [招待講演]
    永井 健治
    組織細胞化学講習会, 奈良県立医科大学 2018年08月
  • Nagai T
    The 41st Annual Meeting of the Japan Neurosience Society, Kobe International Exhibition Hall 2018年07月
  • Nagai T
    World Congress of Basic and Clinical Pharmacology, Kyoto International Conference Center 2018年07月
  • Bioluminescent Probes for Vivid Visualization of Biological Phenomena  [招待講演]
    Nagai T
    Symposium Next Generation Technologies for Neuroscience, Brown University 2018年06月
  • 永井 健治
    第74回 知の拠点セミナー, 京都大学東京オフィス 2018年05月
  • 永井 健治
    日本化学会 第98春季年会, 日本大学理工学部船橋キャンパス 2018年03月
  • 「光る生きものの研究 -研究って面白い！-」  [招待講演]
    永井 健治
    特別講義, 奈良県立青翔子高等学校 2018年03月
  • Nagai T
    Bioengineering Seminar Series, Georgia Institute of Technology 2018年02月
  • Nagai T
    SPIE Photonic West BiOS 2018 plenary session, San Francisco 2018年01月
  • Nagai T
    Nanoscope technologies, Dallas 2018年01月
  • 永井 健治
    第10回つくばがん研究会, 筑波大学 2018年01月
  • 永井 健治
    IT連携フォーラムOACIS 第33回シンポジウム「ICT産学連携フェア2017」, 大阪大学 2017年12月
  • 永井 健治
    2017年度生命科学系学会合同年次大会, 神戸ポートアイランド 2017年12月
  • Nagai T
    From bioimaging to glowing plants, 29th Annual Meeting of Thai Society for Biotechnology and International Conference, Swissotel Le Concorde, Bangkok, Thailand 2017年11月
  • Bioluminescent probes for multi-purpose use in wide range of bioimaging  [招待講演]
    Nagai T
    8th Asia and Oceania Conference on Photobiology, Imperial Palace Seoul Hotel, Korea 2017年11月
  • 「外れ値から見出す新しい生命現象」  [招待講演]
    永井 健治
    研究会「理論と実験」2017, 広島大学 2017年10月
  • 永井 健治
    第55回日本生物物理学会年会, 熊本大学 2017年09月
  • 2017年応用物理学会秋季学術講演会;福岡国際会議場 2017年09月
  • 永井 健治
    2017年光化学討論会・市民公開講座「バイオイメージング最前線講演会」, 東北大学青葉山キャンパス 2017年09月
  • 「高光度化学発光タンパク質が可能にする次世代バイオイメージング」  [招待講演]
    永井 健治
    第36回分子病理学研究会「フェニックスシンポジウム in 宮崎」, フェニックス・シーガイア・リゾート「コテージヒムカ」 2017年07月
  • Nagai T
    IBCS seminar, RILD Auditorium, University of Exeter (Exeter, Devon UK) 2017年07月
  • 「高光度発タンパク質を利用したバイオイメージング」  [招待講演]
    永井 健治
    北海道大学医学研究院
  • 「高光度発光タンパク質が拓く未来社会」  [招待講演]
    永井 健治
    特別講義, 和歌山県立医科大学医学部 2017年06月
  • Nagai T
    the Biophysical Society Thematic Meeting Single-Cell Biophysics: Measurement, Modulation, and Modeling, National Taiwan University, Taiwan 2017年06月
  • Various Application of Super-duper Chemiluminescent Proteins:From Bioimaging to Glowing Plants  [招待講演]
    Nagai T
    Special Lecture, National Chiao Tung University, Taiwan 2017年06月
  • 「蛍光・化学発光タンパク質の開発と社会実装への展開」  [招待講演]
    永井 健治
    第 59 回公開講演会（繊維技術）, 大阪産業創造館 2017年05月
  • Nagai T
    Joint Symposium on Bioimaging between Singapore and Bioimaging Society of Japan, National University of Singapore, Singapore 2017年05月
  • Nagai T
    (Plenary Presentation) OPTICS & PHOTONICS International Congress 2017, Pacifico Yokohama 2017年04月
  • Super-Easy Superresolution Imaging by Spontaneously Photoswitchable Fluorescent Protein  [招待講演]
    Nagai T
    Palais de Congress, Bordeaux, France 2017年04月
  • Nagai T
    Janelia Research Campus/HHMI, Ashburn, Virginia, USA 2017年04月
  • 永井 健治
    薬学会第137年会, 仙台国際センター 2017年03月
  • 永井 健治
    第112回分子科学フォーラム, 岡崎コンファレンスセンター 2017年03月
  • 永井 健治
    第8回OCARINA国際シンポジウム, 大阪市立大学 2017年03月
  • 永井 健治
    総合生命科学部シンポジウム 2017年03月
  • 「高光度発光タンパク質の開発と社会実装への展開」  [招待講演]
    永井 健治
    多元技術融合光プロセス研究会, AIST, 東京 2017年03月
  • 「蛍光・化学発光タンパク質プローブの開発と生命科学研究への応用」  [招待講演]
    永井 健治
    特別講演会,広島大学大学院理学研究科 2017年02月
  • Nagai T
    SPIE BIOS, San Fransisco 2017年01月
  • 永井 健治
    先端医学トピックス, 神戸大学院 医学研究科 2016年12月
  • 永井 健治
    第142回微小光学研究会, 大阪大学吹田キャンパス 2016年12月
  • 永井 健治
    第39回日本分子生物学会年会,パシフィコ横浜 2016年12月
  • 永井 健治
    機能物性セミナー, 東京大学物性研究所, 千葉県柏市 2016年11月
  • Nagai T
    Janelia Fluorescent Proteins and Biological Sensors V Conference, Virginia, USA 2016年11月
  • Nagai T
    World Life Science Conference, National Convention Center, Beijing, China 2016年11月
  • Nagai T
    Beijing University, China, 2016年11月
  • Revolutionary bioimaging with bright biolulminescent proteins  [招待講演]
    Nagai T
    Emory University, Atlanta 2016年09月
  • 「発光性タンパク質を利用した産学民供創によるオープンイノベーションへの取り組み」  [招待講演]
    永井 健治
    岡山県総合研究センター研修講座(生物), 岡山県総合教育センター 2016年07月
  • 永井 健治
    光産業技術マンスリーセミナー ,東京都文京区 光産業技術振興協会 2016年07月
  • Nagai T
    Gordon Research Conference(GRC) Single Molecule Approaches to Biology, Hong Kong 2016年07月
  • Nagai T
    FASEB Calcium and Cell Function, Lisbon Marriott Hotel, Lisbon, Portugal 2016年06月
  • Bioluminescent probes capable of video rate functional imaging at various spatial level ranging from single cell to whole body  [招待講演]
    Nagai T
    International Symposium on Chemiluminescence and Bioluminesence, Tukuba, Japan 2016年05月
  • 「バイオイメージング-蛍光vs化学発光」  [招待講演]
    永井 健治
    第五回バイオイメージング研究会, 東京大学大学院薬学系研究科 5Wセミナー室 2016年05月
  • 永井 健治
    講義, 岡山大学医歯薬総合研究科 2016年05月
  • Genetically Encoded Bioluminescent Probes for Multi-purpose Use in Wide Range of Bioimaging  [招待講演]
    Nagai T
    The Fourth Japan-China Symposium on Nanomedicine, Kitakyushu International Conference Center, Kitakyushu, Japan 2016年05月
  • 永井 健治
    第７８回（平成27年度第4回）産研テクノサロン 『未来を拓くサイエンス －産研教授が語る研究の夢－ 』, 富国生命ビル まちラボ,大阪市 2016年02月
  • Super-duper luminescent proteins applicable to wide range of research  [招待講演]
    Nagai T
    2016 IMCE International Symposium, ITO-IMCE, ITO Campus, Kyushu University, Fukuoka, Japan 2016年01月
  • Super-duper chemilumiescent proteins  [招待講演]
    Nagai T
    University of Bordeaux- Osaka University, Osaka City University, Mini-Symposium,on Synthetic, ISIR, Osaka University, Ibaraki, Osaka, Japan 2016年01月
  • Prospect of minority biology  [招待講演]
    Nagai T
    Pacifichem2015, Hawaii 2015年12月
  • Genetically-encoded luminescent indicator applicable in millisecond voltage phenomena  [招待講演]
    Nagai T
    Pacifichem2015, Hawaii 2015年12月
  • Bioimaging with bright luminescent proteins: Comparing pros and cons of fluorescent and luminescence  [招待講演]
    Nagai T
    Pacifichem2015,Hawaii 2015年12月
  • 永井 健治
    第49回光学五学会, 大阪市立大学文化交流センター・ホール 2015年12月
  • 永井 健治
    BMB2015, 神戸ポートアイランド 2015年12月
  • Genetically-Ecoded Tools to Optically Control and Image Ca2+ Dynamics  [招待講演]
    Nagai T
    The 2nd East-Asia Microscopy Conference, The Himeji Chamber of Commerce and Industry 2015年11月
  • 永井 健治
    北海道大学ニコンイメージングセンター学術講演会, 北海道大学電子科学研究所 2015年11月
  • Revolutionary bioimaging with super-duper luminescent proteins  [招待講演]
    Nagai T
    Physical Biology Lecture at SINAP, Chinese Academy of Sciences, Shanghai, P. R. China 2015年11月
  • 永井 健治
    第8回関西発仕事コンソーシアム;やってみなはれ勉強会;富国生命ビル;まちラボ;大阪市 2015年10月
  • 「高光度発光タンパク質によるバイオイメージングと多用途利用」  [招待講演]
    永井 健治
    岡山県医用工学研究会, 岡山大学鹿田キャンパス 2015年10月
  • Genetically-encoded tools to optically control and image neuronal activity  [招待講演]
    Nagai T
    4th International Frontiers in Neurophotonics Symposium, the Musée de la civilization, Québec city, Canada 2015年10月
  • 永井 健治
    第24回 日本バイオイメージング学会 学術集会公開講座「私たちのくらしとバイオイメージング～見えるからわかるバイオの世界～」 , 東京理科大学 葛飾キャンパス 2015年09月
  • Nagai T
    PRESTO-Harvard Joint Symposium, Harvard University 2015年09月
  • 永井 健治
    第53回日本生物物理学会年会シンポジウム, 金沢大学角間キャンパス自然科学本館 2015年09月
  • 永井 健治
    第53回日本生物物理学会年会・浜松ホトニクス株式会社ランチョンセミナー, 金沢大学角間キャンパス自然科学本館 2015年09月
  • 永井 健治
    第53回日本生物物理学会年会・市民講演会, 石川県教育会館 ホール 2015年09月
  • 「細胞の構造・動態・機能を可視化するための遺伝子ツール」  [招待講演]
    永井 健治
    第３１回分析電子顕微鏡討論会 , 幕張メッセ国際会議場 2015年09月
  • 永井 健治
    市民参加シンポジウム 未来を拓く植物バイオのチカラ, 大阪富国生命ビル 2015年08月
  • Single Molecule/Nanoparticle Spectroscopy and Imaging  [招待講演]
    Nagai T
    The 4th Hsinchu Summer Workshop ,National Chiao Tung University, Taiwan 2015年07月
  • Single Molecule/Nanoparticle Spectroscopy and Imaging "Fluorescent bio-probes"  [招待講演]
    Nagai T
    The 4th Hsinchu Summer Course, National Chiao Tung University, Taiwan 2015年07月
  • 「蛍光から発光へ：バイオイメージングの新潮」  [招待講演]
    永井 健治
    BioMecForum 第 77 回研究会, 大阪大学 2015年07月
  • Revolutionary bioimaging with bright chemiluminescent proteins  [招待講演]
    Nagai T
    Biophysical Society Thematic Meeting: New Biological Frontiers Illuminated by Molecular Sensors and Actuators ,National Taiwan University, Taipei, Taiwan 2015年06月
  • Revolutionary bioimaging with bright luminescent proteins  [招待講演]
    Nagai T
    The 3rd China-Japan Nanomedicine Symposium, Institute of Basic Medical Sciences Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China 2015年06月
  • 永井 健治
    2015年 先端光化学若手研究会, 早稲田大学 2015年06月
  • 永井 健治
    第10回日本ケミカルバイオロジー学会, 東北大学 2015年06月
  • Revolutionary bioimaging with bright luminescent proteins -Comparing pros and cons of fluorescence and luminescence  [招待講演]
    Nagai T
    The 1st Infectious Disease Imaging Symposium -Basics and Applications-, Nagasaki University, Japan 2015年06月
  • Nagai T
    19th International Symposium on Calcium Binding Proteins and Calcium Function in Health and Disease, Vanderbilt University,USA 2015年05月
  • 永井 健治
    日本顕微鏡学会第71回学術講演会, 国立京都国際会館 2015年05月
  • A fast- and positively photoswitchable fluorescent protein for ultralos-laser power RESOLFT nanoscopy  [招待講演]
    Nagai T
    Hangzhor, China 2015年05月
  • 「街路樹を照明灯に！遺伝子組み換え植物の可能性」  [招待講演]
    永井 健治
    市民参加シンポジウム「未来を拓く植物バイオのチカラ」, 大阪富国生命ビル４F（大阪市） 2015年03月
  • 永井 健治
    第120回日本解剖学会・第92回日本生理学会合同大会, 神戸国際会議場 2015年03月
  • Genetically-encoded tools to optically control and image Ca2+ and Mg2+ dynamics  [招待講演]
    Nagai T
    The 2nd International Symposium on Plant Environmental Sensing, AIST Tokyo Waterfront, Tokyo, Japan 2015年03月
  • Toward long term single molecule imaging in live cells with luminescent probes  [招待講演]
    Nagai T
    The 15th International Membrane Research Forum、iCeMS, Kyoto University 2015年03月
  • Genetically-encoded tools to optically control and image Ca2+ dynamics  [招待講演]
    Nagai T
    International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama, the 50th Anniversary Hall, Okayama University (Okayama City) 2015年02月
  • 「Ca2+動態を光で操作し可視化するための遺伝子ツール」  [招待講演]
    永井 健治
    新学術領域 柔らかな分子系 第8回ワークショップ、岡山いこいの村(岡山県瀬戸内市) 2015年01月
  • 「遺伝子にコードされた分子スパイによる最先端バイオイメージング －蛍光vs化学発光ー」  [招待講演]
    永井 健治
    講演会、筑波大学 生物・農林学系F棟506（つくば市） 2015年01月
  • 「少数性生物学概説および少数性分子の可視化・操作技術の開発」  [招待講演]
    永井 健治
    第14回名古屋大学・遺伝子実験施設公開セミナー、名古屋大学 坂田・平田ホール、名古屋市 2014年12月
  • Genetically-encoded chemiluminescent sensor for membrane voltage to monitor neuronal activity  [招待講演]
    Nagai T
    2nd Conference of SANKEN Core to Core, 3rd imec Handai International Symposium, Knowledge Capital Congrès Convention Center, Osaka, Japan 2014年12月
  • Basics of genetically-encoded fluorescent/chemiluminescentprobes  [招待講演]
    Nagai T
    2nd AIST International Imaging Workshop, DAILAB, Biomedical Research Institute, AIST, Tsukuba, Japan 2014年12月
  • Bioimaging by means of engineered fluorescent/chemiluminescent proteins  [招待講演]
    Nagai T
    Special Lecture, Indian Institute of Technology Madras, Chennai, India 2014年12月
  • 「今にのめり込む～超サイエンス術～」  [招待講演]
    永井 健治
    第３回TRCイメージングシリーズセミナー、愛媛大学医学部、愛媛県東温市 2014年10月
  • 「生命現象を光で視て弄る技術の開発」  [招待講演]
    永井 健治
    大学院講義 基礎研究方法論、大学院医学系研究科、愛媛県東温市 2014年10月
  • Genetically-encoded tools to optically control and image Ca2+ dynamics  [招待講演]
    Nagai T
    the 16th International Conference on Retinal Proteins (ICRP2014), Nagahama, Japan, Nagahama Royal Hotel 2014年10月
  • Expanded palette of bright luminescent proteins for real-time multi-color luminescence imaging  [招待講演]
    Nagai T
    Janelia Conference: Fluorescent Proteins and Biological Sensors IV, HHMI's Janelia Research Campus, Ashburn, USA 2014年09月
  • 「生物発光がもたらす未来のほっこり生活」  [招待講演]
    永井 健治
    第52回日本生物物理学会年会 第一回総会ワークショップ「生物物理が拓く未来社会」、札幌コンベンションセンター、札幌市 2014年09月
  • 「Genetically-encoded tools to optically control and image cellular events」  [招待講演]
    永井 健治
    京都大学生命動態システム科学推進拠点：発生・細胞生物学・システム生物学コース、医学部記念講堂、京都市 2014年06月
  • Genetically-encoded photosensitizer for light-dependent perturbation of biological function  [招待講演]
    Nagai T
    iCeMS International Symposium: "Light Control in Cell Biology", Kyoto University iCeMS, Kyoto, Japan 2014年06月
  • Genetically-encoded tools to optically control and image calcium dynamics  [招待講演]
    Nagai T
    the 2014 FASEB SRC on Calcium and Cell Function, Meliã Nassau Beach, Nassau, Bahamas 2014年06月
  • 「in vivo ライブイメージングによる高次生命現象の可視化と応用」  [招待講演]
    永井 健治
    日本実験動物科学技術さっぽろ2014、札幌コンベンションセンター、札幌市 2014年05月
  • 「少数性生物学って何？」  [招待講演]
    永井 健治
    静岡県立大学 市民勉強会「環境・生命・宇宙 －わたしたちの星、地球、月そして太陽ー」 、静岡県立大学、静岡市 2014年03月
  • 「少数性生物学概説および少数生体分子の可視化・操作技術の開発」  [招待講演]
    永井 健治
    生体システム専攻バイオサイエンスシンポジウム、東京工業大学すずかけ台キャンパス、横浜市 2014年02月
  • 「特別講演会」  [招待講演]
    永井 健治
    Meet the Mentors 2.0、岡山大学大学院歯学部棟4 階 第3 講義室、岡山市 2014年02月
  • 「蛍光から化学発光へ―バイオイメージングの新潮流―」  [招待講演]
    永井 健治
    平成25年度第2回次世代バイオナノ研究会「マイクロ・ナノ空間の生体計測・イメージング」、東京ビッグサイト会議棟6階606会議室、東京都 2014年01月
  • 「発光性タンパク質を利用した様々な技術開発」  [招待講演]
    永井 健治
    特許庁 平成25年度技術研修、独立行政法人工業所有権情報・研修館、東京都 2014年01月
  • 「蛍光・化学発光タンパク質エンジニアリングによる生理機能の操作と可視化」  [招待講演]
    永井 健治
    応用物理学会・量子エレクトロニクス研究会、上智大学軽井沢セミナーハウス、北佐久郡軽井沢町 2013年12月
  • Manipulation and visualization of biological function with genetically encoded molecular spies  [招待講演]
    Nagai T
    2013 ASCB Annual Meeting, Morial Convention Center, New Orleans, USA 2013年12月
  • 「生命現象を弄って視る分子スパイの開発」  [招待講演]
    永井 健治
    第26回産研技術室報告会、大阪大学産業科学研究所、茨木市 2013年12月
  • 「少数性生物学〜タンパク質分子に個性はあるのか」  [招待講演]
    永井 健治
    日本分子生物学会 公開プレゼンテーション「生命世界を問う」、神戸国際会議場 ポートピアホール、神戸市 2013年12月
  • 「SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation」  [招待講演]
    永井 健治
    第36回日本分子生物学会年会シンポジウム、神戸国際会議場、神戸市 2013年12月
  • Luminescent protein able to high speed imaging at single cell and whole body level  [招待講演]
    Nagai T
    First Osaka University-EPFL International Symposium, Icho-Kaikan, Osaka University, Suita, Osaka, Japan 2013年12月
  • 「細胞機能操って観る技術の開発」  [招待講演]
    永井 健治
    シンポジウム「細胞を創る操る」、奈良先端科学技術大学院大学 ミレニアムホール、生駒市 2013年11月
  • 「生物発光を利用した未来産業」  [招待講演]
    永井 健治
    大阪大学産業科学研究所第69回学術講演会シンポジウム「産業科学の未来戦略」、大阪大学産業科学研究所、茨木市 2013年11月
  • 「ぱっぱらぱーになって、科学革命を起こしませんか？」  [招待講演]
    永井 健治
    遠賀中間歯科医師会学術講演会、中間市ハーモニーホール、福岡県中間市 2013年11月
  • Genetically-encoded functional probes applicable in conjunction with photo-manipulation technologies  [招待講演]
    Nagai T
    7th International Symposium on Nanomedicine (ISNM2013), Nakamura Centenary Hall of Kyushu Institute of Technology, Kitakyushu, Japan 2013年11月
  • 「ハイパワーLED光源の特徴を活かした、生命科学研究へのアプローチ例とその可能性」  [招待講演]
    永井 健治
    日本生物物理学会第51回年会 オプトラインランチョンセミナー、国立京都国際会館、京都市 2013年10月
  • Genetically-encoded functional probes applicable in conjunction with photo-manipulation technologies  [招待講演]
    Nagai T
    Optogenetics2013, Keio University, Mita Campus, Tokyo, Japan 2013年09月
  • 「蛍光から化学発光へ －バイオイメージングの新潮流－」  [招待講演]
    永井 健治
    第55回歯科基礎医学会学術大会・総会、岡山コンベンションセンター、岡山市 2013年09月
  • 「高輝度化学発光タンパク質によるリアルタイムバイオイメージング」  [招待講演]
    永井 健治
    第65回日本生物工学会大会、広島国際会議場、広島市 2013年09月
  • Superstrong luminescent protein for high speed imaging at single cell and whole body level  [招待講演]
    Nagai T
    JSAP-OSA Joint Symposia 2013, the 74th JSAP Autumn Meeting 2013, Kyotanabe Campus, Doshisha University, Kyotanabe, Japan 2013年09月
  • 「細胞・個体イメージング用光学プローブの開発」  [招待講演]
    永井 健治
    日本学術会議公開シンポジウム 「医学・生命科学の革新的発展に資する統合バイオイメージングの展望」、日本学術会議講堂、東京都 2013年09月
  • 「高輝度発光タンパク質の医学応用」  [招待講演]
    永井 健治
    化学工学会 第45回秋季大会、岡山大学 津島(東)キャンパス、岡山市 2013年09月
  • Revolutionary bioimaging with super-duper luminescent proteins  [招待講演]
    Nagai T
    Special Lecture, Department of Pharmacology, University of Oxford, Oxford, United Kingdom 2013年08月
  • 「光で拓く生命科学」  [招待講演]
    永井 健治
    上宮高等学校講演会、上宮高等学校、大阪市 2013年07月
  • 「蛍光から化学発光へ：バイオイメージングの新潮流」  [招待講演]
    永井 健治
    CRSET・さきがけ光科学技術合同シンポジウム、東京大学 弥生講堂・一条ホール、東京都 2013年06月
  • 「超高輝度発光タンパク質による革命的バイオイメージング」  [招待講演]
    永井 健治
    日本顕微鏡学会第69回学術講演会、ホテル阪急エキスポパーク、吹田市 2013年05月
  • 「蛍光から化学発光へ~バイオイメージングの新潮流～」  [招待講演]
    永井 健治
    木原記念財団学術賞応用科学賞記念講演会、横浜市立大学木原生物学研究所３階ホール、横浜市 2013年05月
  • 「超高輝度発光タンパク質による革命的バイオイメージング」  [招待講演]
    永井 健治
    第90回日本生理学会年会、タワーホール船堀、東京都 2013年03月
  • 「蛍光から化学発光へ：バイオイメージングの新潮流」  [招待講演]
    永井 健治
    先端的光イメージング拠点形成プロジェクト・成果報告シンポジウム、北海道大学、札幌市 2013年03月
  • Revolutionary bioimaging with super-duper luminescent proteins  [招待講演]
    Nagai T
    The Fourteenth International Membrane Research Forum, iCeMS Main Building, Kyoto University, Kyoto, Japan 2013年03月
  • 「蛍光から化学発光へ：バイオイメージングの新潮流」  [招待講演]
    永井 健治
    神戸バイオメディカル学術交流会、理研CDB、神戸市 2013年02月
  • 「バイオイメージング光源の最新技術」  [招待講演]
    永井 健治
    平成24年度第4回テクノサロン 「バイオサイエンスの応用展開」、大阪大学産業科学研究所、茨木市 2013年02月
  • 「LED を利用したバイオサイエンス技術」  [招待講演]
    永井 健治
    レーザー学会第33回年次大会、姫路商工会議所、姫路市 2013年01月
  • Revolusionary bioimaging with super-duper luminescent proteins  [招待講演]
    Nagai T
    Special Work Shop for Oxford University, ISIR, Osaka University, Ibaraki, Osaka, Japan 2012年12月
  • Genetically-encodable functional indicator and manipulator for deciphering biological events  [招待講演]
    Nagai T
    The 25th Annual and International Meeting of the Japanese Association for Animal Cell Technology (JAACT2012), the Nagoya Congress Center, Nagoya, Japan 2012年11月
  • Revolutionary Bioimaging with Super-Duper Luminescent Proteins  [招待講演]
    Nagai T
    International Joint Symposium on Single-Cell Analysis (The 6th International Workshop on Approaches to Single-Cell Analysis & The 8th International Forum on Post-Genome Technologies), Kyoto Research Park, Kyoto, Japan 2012年11月
  • 「オワンクラゲの研究から」  [招待講演]
    永井 健治
    講演会、北三瓶小学校、中学校、大田市三瓶町 2012年11月
  • 「遺伝子にコードされた分子スパイによる生命現象の解明に向けて」  [招待講演]
    永井 健治
    33回日本レーザー医学会総会、大阪大学吹田キャンパス、茨木市 2012年11月
  • Revolutionary bioimaging with super-duper luminescent proteins  [招待講演]
    Nagai T
    Fluorescent Proteins and Biological Sensors III, Janelia Farm, Ashburn, USA 2012年11月
  • Genetically-encoded functional probes applicable in conjunction with photo-manipulation technologies”  [招待講演]
    Nagai T
    Pradigm Innovation in Biology-Novel Strategy and Thinking-Academia Sinica, Taipei, Taiwan 2012年10月
  • 「生体組織内の任意の細胞で機能イメージングを可能にする光活性化型Ca2+センサーの開発」  [招待講演]
    永井健治, 松田知己, 堀川一樹
    生理研研究会「超階層シグナル伝達研究の新展開」,岡崎コンファレンスセンター、岡崎 2012年10月
  • 「生体組織内の任意の細胞での機能イメージングを可能にする光活性化型 Ca2+センサーの開発」  [招待講演]
    永井 健治
    生理学研究所研究会「超階層シグナル伝達研究の新展開」、自然科学研究機構・生理学研究所、岡崎市 2012年10月
  • 「遺伝子にコードされた発光プローブ」  [招待講演]
    永井 健治
    第50回日本生物物理学会年会シンポジウム「1分子生物学と生化学の狭間に潜むナノシステム動作力学の理解を目指して」,名古屋大学、名古屋 2012年09月
  • 「遺伝子にコードされた発光型プローブ」  [招待講演]
    永井 健治
    第50回日本生物物理学会年会、名古屋大学 東山キャンパス、名古屋市 2012年09月
  • Revolutionary bioimaging with super-duper luminescent proteins  [招待講演]
    Nagai T
    4th International Symposium on Photonic Bioimaging 2012, Hokkaido University, Sapporo, Japan 2012年09月
  • Genetically-encoded functional probes applicable in conjunction with photo-manipulation technologies  [招待講演]
    Nagai T
    The 14th International Congress of Histochemistry and Cytochemistry (ICHC 2012), Kyoto International Conference Center, Kyoto, Japan 2012年08月
  • Genetically-encoded tools for chromophore-assisted light inactivation of biomolecule functions  [招待講演]
    Nagai T
    The 14th International Congress of Histochemistry and Cytochemistry (ICHC 2012), Kyoto International Conference Center, Kyoto, Japan 2012年08月
  • 「最先端バイオイメージング技術で何を解き明かすのか？」  [招待講演]
    永井 健治
    第52回生命科学夏の学校、西浦温泉ホテルたつき、蒲郡市 2012年08月
  • 「遺伝子にコードされた分子スパイによる生命現象の解明に向けて」  [招待講演]
    永井 健治
    第24回高遠シンポジウム 生命の制御系の進化を探る 、高遠さくらホテル、伊那市 2012年08月
  • 「FRETの基礎」  [招待講演]
    永井 健治
    第１９回細胞生物学ワークショップ、神戸 2012年08月
  • 「蛍光タンパク質の基礎」  [招待講演]
    永井 健治
    第１９回細胞生物学ワークショップ、神戸 2012年08月
  • 「システム機械設計」  [招待講演]
    永井 健治
    大阪府立大学集中講義、堺
  • Revolutionary bioimaging with super-duper lluminescent proteins.  [招待講演]
    Nagai T
    Special Lecture, Department of Chemistry, Alberta University, Alberta Canada 2012年07月
  • 「遺伝子にコードされた分子スパイによる生命現象の解明に向けて」  [招待講演]
    永井 健治
    超高速バイオアセンブラ第1回若手研究者シンポジウム、京都大学iPS細胞研究所(CiRA)、京都市 2012年07月
  • 「光で拓くナノバイオテクノロジー」  [招待講演]
    永井 健治
    札幌 2012年06月
  • 「少数性生物学って何？」  [招待講演]
    永井 健治
    新学術領域「秩序形成ロジック」2012年度班会議、ルスツ・リゾート、北海道虻田郡 2012年06月
  • 「LEDを利用したバイオサイエンス技術」  [招待講演]
    永井 健治
    第１回「レーザーバイオ医療」技術専門委員会、北海道大学 大学院地球環境科学研究院、札幌市 2012年06月
  • 「バイオイメージングで何を観るのか？」  [招待講演]
    永井 健治
    第64回細胞生物学会、神戸 2012年05月
  • 「What do we see with bioimaging?」  [招待講演]
    永井 健治
    第４５回日本発生生物学会大会・第64回日本細胞生物学会大会 合同大会、Kobe International Conference Center、神戸市 2012年05月
  • 「生理機能の光操作と可視化技術」  [招待講演]
    永井 健治
    九州大学先導物質科学研究所セミナー、九州大学先導物質化学研究所、春日市 2012年05月
  • 「可視化と光操作で読み解く少数性生物学」  [招待講演]
    永井 健治
    北大・生化学特別講義Ｉ 2012年05月
  • 「改変蛍光タンパク質を利用した生理機能の可視化」  [招待講演]
    永井 健治
    学術振興会第167委員会、台場 2012年04月
  • 「改変蛍光タンパク質を利用した生理機能の可視化」  [招待講演]
    永井 健治
    ナノプローブテクノロジー第１６７委員会第６６回研究会テーマ「バイオイメージング・計測技術の最先端」、学振１６７委員会第66回研究会「バイオイメージング・計測技術の最先端」、東京都 2012年04月
  • Genetically-encoded technologies to quantitatively visualize and control bio-function in living cells  [招待講演]
    Nagai T
    International Symposium on Molecular Imaging and Systems Biology (Tokyo University, Tokyo, Japan) 2012年01月
  • 「光遺伝学ツールを含む光操作技術と組み見合わせて利用可能な自動発光型機能プローブ」  [招待講演]
    永井 健治
    第4回未来創薬・医療イノベーションセミナー、札幌 2012年01月
  • 「光技術が拓く生命科学の新潮流」  [招待講演]
    永井 健治
    東京大学医学研究科大学院講義、東京 2012年01月
  • 「個と多数の狭間が織りなす生命現象の解明を目指して」  [招待講演]
    永井 健治
    数理連携１０の根本問題の発掘（理化学研究所、和光） 2011年12月
  • 「Auto-luminescent functional probes applicable in conjunction with photo-manipulation technology including optogenetic tools」  [招待講演]
    永井 健治
    Promega Dynamic Connection（パシフィコ横浜、横浜） 2011年12月
  • 「Genetically-encoded technologies to quantitatively visualize and manipulate biomolecule in living cells」  [招待講演]
    永井 健治
    第34回日本分子生物学会（パシフィコ横浜、横浜） 2011年12月
  • Auto-luminescent genetically encoded indicators for real time in vivo imaging  [招待講演]
    Nagai T
    SPIE 2011 Smart Nano+Micro material and Devices(Swainburne Univ. of Technology, Melbourne, Australia) 2011年12月
  • Spying biological events in living cells by genetically-encoded functional indicators  [招待講演]
    Nagai T
    The third RIES-CIS international symposium (NCTU, Sinchu, Taiwan) 2011年10月
  • Expanding the repertory of genetically-encoded Ca2+ indicators  [招待講演]
    Nagai T
    3rd International Symposium on Photonic Bioimaging(Keio Plaza Hotel, Sapporo, Japan) 2011年10月
  • 「Imaging tools applicable in conjunction with optogenetic technology」  [招待講演]
    永井 健治
    生理研研究所研究会「超階層シグナル伝達研究の新展開」（岡崎コンファレンスセンター、岡崎） 2011年09月
  • 「蛍光・化学発光タンパク質を用いたバイオイメージング技術の現状と展望」  [招待講演]
    永井 健治
    第20回日本バイオイメージング学会学術集会（千歳科学技術大学、千歳） 2011年09月
  • 「１分子生物学と生化学の狭間に潜むナノシステム動作力学の理解を目指して」  [招待講演]
    永井 健治
    第84回日本生化学大会（国立京都国際会館，京都） 2011年09月
  • 「Halo Tag テクノロジーによる分子機能の時空間的光不活性化と超高輝度化学発光タンパク質によるリアルタイムイメージング」  [招待講演]
    永井 健治
    2011 Promega New Technology Seminar（キャンパスプラザ京都、京都） 2011年09月
  • What should we learn from light emitting organisms? Efficient energy transfer and its application for bioimaging  [招待講演]
    Nagai T
    The 49th Annual Meeting of the Biophysical Society of Japan(University of Hyogo, Hyogo, Japan) 2011年09月
  • 「What should we learn from light emitting organisms? Efficient energy transfer and its application for bioimaging」  [招待講演]
    永井健治
    第49回日本製物物理学会年会（兵庫県立大学姫路書写キャンパス，姫路） 2011年09月
  • 「蛍光タンパク質を使用した分子動態計測と化学発光タンパク質による次世代バイオイメージング」  [招待講演]
    永井 健治
    第49回日本生物物理学会年会（兵庫県立大学姫路書写キャンパス，姫路） 2011年09月
  • 「蛍光・化学発光タンパク質を用いたバイオイメージング技術の現状と展望」  [招待講演]
    第 20 回日本バイオイメ ージング学会学術集会 （千歳科学技術大学、千歳） 2011年09月
  • 「大自然からの素晴らしい贈り物－緑色蛍光タンパク質とイクオリン－」  [招待講演]
    第20回日本バイオイメージング学会学術集会（千歳市民文化センター、千歳） 2011年08月
  • Engineering green fluorescent proteins to visualize and manipulate biological functions  [招待講演]
    Nagai T
    5th COE symposium, 14th MPC(Hamamatsu University School of Medicine, Shizuoka, Japan) 2011年08月
  • 「１分子生物学と生化学の狭間に潜むナノシステム動作力学の理解を目指して」  [招待講演]
    第 84 回日本生化学大会 （国立京都国際会館，京都） 2011年08月
  • Engineering green fluorescent proteins to visualize andmanipulate biological functions  [招待講演]
    Nagai T
    5th COE symposium, 14th MPC (Hamamatsu University School of Medicine, Shizuoka, Japan) 2011年08月
  • Imaging Probes for Neuronal Cell Biology  [招待講演]
    Nagai T
    Developmental Neurobiology Course(OIST, Okinawa, Japan) 2011年07月
  • Toward invention of high performance genetically-encoded luminescent indicators for functional imaging in living organisms  [招待講演]
    Nagai T
    Symposium to commemorate 150 years of German-Japan friendship(Mirai CAN Hall, Tokyo, Japan) 2011年07月
  • Invention of high performance bright luminescent proteins used as a nanolight source  [招待講演]
    Nagai T
    AS-JST joint workshop on innovative use of light and nano/bio materials (Academia Sinica, Taipei, Taiwan) 2011年05月
  • Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level  [招待講演]
    Nagai T
    IUPAC international congress on analytical science 2011(Kyoto international conference center, Kyoto, Japan) 2011年05月
  • Auto-luminescent imaging tools for combination use with optogenetic technology  [招待講演]
    Nagai T
    44th Annual meeting of the Japanese Society of Developmental Biology(Okinawa Convention Center, Okinawa, Japan) 2011年05月
  • Towards Understanding Biological Phenomena by Genetically-Encoded Molecular Spies  [招待講演]
    Nagai T
    NCCR CHEMICAL BIOLOGY MINI-SYMPOSIUM (SV1717a, École polytechnique fédérale de Lausanne) 2011年04月
  • Toward invention of high performance genetically-encoded luminescent probes for functional imaging in living organisms  [招待講演]
    Nagai T
    Photonic Bioimaging(Hilton Niseko Village, Hokkaido, Japan)
  • How to engineer fluorescent proteins to construct high performance FRET-based indicators for biological functions  [招待講演]
    Nagai T
    Chemistry Seminar (Chemistry Gazebo, Department of Chemistry, Stanford University) 2011年01月
  • Development of ultra-sensitie Ca2+ indicators  [招待講演]
    Nagai T
    yellow cameleon-nano” Japan-Taiwan joint workshop on Bioelectronics (National Cheng Kung University, Tainan, Taiwan) 2011年01月
  • Imaging and manipulation of biomoloecules using engineered fluorescent and chemiluminescent proteins  [招待講演]
    永井 健治
    BMB2010（神戸国際会議場，神戸） 2010年12月
  • 蛍光・化学発光タンパク質のエンジニアリングによるバイオイメージング用ツール開発の現状と展望  [招待講演]
    永井 健治
    第36回日本生体エネルギー研究会（大阪大学銀杏会館，大阪） 2010年11月
  • How to engineer fluorescent proteins to construct high performance FRET-based indicators for biological functions  [招待講演]
    Nagai T
    3rd Advanced bioimaging workshop-tracking molecules with light (Sydney, Australia) 2010年11月
  • Wonderful gifts from light-emitting jelly fish-Green Fluorescent Protein and Aequorin  [招待講演]
    Nagai T
    3rd Advanced bioimaging workshop- tracking molecules with light (Sydney, Australia) 2010年11月
  • New generation of genetically-encoded Ca2+ indicators for real-time imaging at single cell level  [招待講演]
    Nagai T
    Invited seminar in Institute of Biotechnology, UNAM(Cuernavaca, Mexico) 2010年11月
  • Deciphering enigma of biological function by a genetically-encoded molecular spy  [招待講演]
    Nagai T
    UNAM(Cuernavaca, Mexico) 2010年11月
  • Toward invention of high performance genetically-encoded luminescent probes for functional imaging in living organisms  [招待講演]
    Nagai T
    Annual meeting of Mexican Biochemistry Society(Chiapas, Mexico) 2010年11月
  • 何が活き活きした科学研究にとって大切か？－私の人生秘話に学ぶ－  [招待講演]
    永井 健治
    総合研究大学院大学・生命科学合同セミナー（ヤマハリゾートつま恋，静岡） 2010年11月
  • Auto-luminescent Genetically-Encoded Ratiometric Indicator for Real-time Ca2+ Imaging at the Single Cell Level  [招待講演]
    Nagai T
    KSBMB2010 (Seoul, Korea) 2010年10月
  • Auto-luminescent Genetically-Encoded Ratiometric Indicator for Real-time Ca2+ Imaging at the Single Cell Level  [招待講演]
    Nagai T
    Invited seminar in KAIST (Daegon, Korea) 2010年10月
  • 蛍光・化学発光タンパク質によるバイオイメージング技術の動向と展望  [招待講演]
    永井 健治
    第2回Bio Opto Japan カンファレンス（パシフィコ横浜，横浜） 2010年09月
  • 光遺伝学的手法との併用を可能にする持続的自励発光型Ca2+指示薬  [招待講演]
    永井 健治
    シグナル伝達の動的理解を目指す新戦略（岡崎カンファレンスセンター，岡崎） 2010年09月
  • A Novel Way to Expand the Dynamic Range of Genetically-encoded FRET-based Indicators  [招待講演]
    Nagai T
    Bio Korea2010(Seoul, Korea)
  • A Novel Way to Expand the Dynamic Range of Genetically-encoded FRET-based Indicators  [招待講演]
    Nagai T
    KIST-Tohoku Joint Symposium on Nanobiomedical Engineering (Seoul, Korea) 2010年08月
  • Toward Elucidation of Biological Enigma by Genetically-encoded Molecular Spies  [招待講演]
    永井 健治
    新学術領域「システム分子行動学，イメージングワークショッププログラム（九州大学箱崎キャンパス・福岡） 2010年08月
  • 蛍光・化学発光タンパク質の基礎と応用  [招待講演]
    永井 健治
    浜松医科大学第19回メディカル・ホトニクスコース（浜松医科大学・浜松） 2010年08月
  • Crusing inside cells by genetically-encoded fluorescent sensor  [招待講演]
    Nagai T
    RIES-CIS Joint Symposium (Sapporo, Japan) 2010年07月
  • 光による分子機能コントロール  [招待講演]
    永井 健治
    平成22年度レーザー顕微鏡研究会（独立行政法人理化学研究所・和光）
  • 結晶構造情報に基づく蛍光タンパク質の改変とその生物学的応用  [招待講演]
    永井 健治
    第10回日本蛋白質科学会年会（札幌コンベンションセンター・札幌） 2010年06月
  • 先端的バイオセンシング技術-蛍光タンパク質間FRET法の新たな展開  [招待講演]
    永井 健治
    10th Symposium of Gene and Drug Delivery（北海道大学・札幌）
  • A Novel Way to Expand the Dynamic Range of Genetically-encoded FRET-based Indicators  [招待講演]
    Nagai T
    International Symposium for the Bio Imaging and Cell Dynamics (Gwangiu, Korea) 2010年05月
  • 神経細胞の機能イメージング  [招待講演]
    永井 健治
    何をどこまで可視化できるようになっているのか，研究会（自然科学研究機構 基礎生物学研究所・岡崎）
  • 改変蛍光タンパク質による神経機能の可視化と操作  [招待講演]
    永井 健治
    重点共同利用研究研究会－ニューロンのネットワーク構築における細胞極性の生物学的意義（基礎生物学研究所・岡崎） 2010年04月
  • 蛍光タンパク質技術が拓いたもの  [招待講演]
    永井 健治
    第115回日本解剖学会学術集会（岩手県民会館・盛岡） 2010年03月
  • 蛍光・発光物質が切り開く未来の生物学  [招待講演]
    永井 健治
    目で見る生命科学の面白さ～ノーベル賞受賞者 下村脩博士と一緒に実験をしませんか～（岩手医科大学・盛岡） 2010年03月
  • Approaches to in vivo functional imaging by engineering bacterial luminescent protein  [招待講演]
    永井 健治
    第83回日本細菌学会（パシフィコ横浜・横浜） 2010年03月
  • 結晶構造情報に基づく蛍光タンパク質の改変とその生物学的応用  [招待講演]
    永井 健治
    ソフトナノ材料研究ステーションシンポジウム（東北大・仙台） 2010年03月
  • Toward elucidation of biological enigma by genetically-encoded molecular spies  [招待講演]
    Nagai T
    International Symposium on Waching Biomolecules in Action(Senri Life Science Center, Osaka, Japan) 2009年12月
  • 蛍光タンパク質テクノロジーの展望 －観て触って探る生命の不思議－  [招待講演]
    永井 健治
    平成21年度生理学研究所シナプス研究会「シナプス機能と病態」（生理学研究所，岡崎） 2009年12月
  • 蛍光タンパク質性機能プローブの精密設計とバイオイメージングへの応用  [招待講演]
    永井 健治
    第7回脳科学研究教育センターシンポジウム（北海道大学，札幌） 2009年12月
  • Toward understanding biological phenomena by genetically-encoded molecular spies  [招待講演]
    Nagai T
    Molecular Imaging for Syetems Biology (Okazaki Conference Center, Okazaki, Japan) 2009年12月
  • Toward understanding biological phenomena by genetically-encoded molecular spies  [招待講演]
    Nagai T
    BIT’s 3rd Annual Eorld Congress of Gene-2009(Foshan Sansui Golden Sun Hotel, China) 2009年12月
  • FRET指示薬  [招待講演]
    永井 健治
    第14回細胞生物学ワークショップ（北海道大学，札幌） 2009年11月
  • 蛍光・化学発光ハイブリッドが拓く次世代バイオイメージング  [招待講演]
    永井 健治
    第16回細胞生物学ワークショップ（北海道大学，札幌市） 2009年11月
  • 蛍光タンパク質テクノロジー  [招待講演]
    永井 健治
    第14回細胞生物学ワークショップ（北海道大学，札幌） 2009年11月
  • Toward understanding biological phenomena by genetically-encoded molecular spies  [招待講演]
    永井 健治
    2009年生物物理・生化学・分子生物学会北海道支部会合同年会（北海道大学，札幌） 2009年11月
  • 蛍光タンパク質を巧妙に用いた生理機能動態の可視化  [招待講演]
    永井 健治
    第47回日本生物物理学会年会（アスティ徳島，徳島） 2009年10月
  • Toward elucidation of dynamic living system by hierarchical molecular imaging  [招待講演]
    Nagai T
    82th Annual meeting of JBS(Kobe Convention Center, Kobe) 2009年10月
  • Vivid visualization of biological functions using fluorescent protein-based molecular spies  [招待講演]
    Nagai T
    91th G-COE seminar (Kyusyu University, Fukuoka) 2009年10月
  • Toward understanding biological enigma by genetically-encoded molecular spies  [招待講演]
    Nagai T
    Hokkaido University-Academia Sinica Joint Symposium(Hokkaido University, Sapporo, Japan) 2009年10月
  • GFP は何故光るか？-機能指示薬作成法と生理機能の可視化/フェルスター共鳴エネルギー移動(FRET)を利用したバイオセンサー作成法  [招待講演]
    永井 健治
    第5回ライブセルイメージング講習会(産業技術総合研究所つくばセンター，つくば) 2009年10月
  • Visualization of Cellular Functions and Dynamics by the Smart Use of Fluorescent Protein  [招待講演]
    永井 健治
    30th iCeM Seminer(京都大学吉田キャンパス，京都) 2009年09月
  • 新規蛍光タンパク質の開発とバイオイメージングへの応用  [招待講演]
    永井 健治
    BioOpto Japan 2009 (パシフィコ横浜，横浜) 2009年09月
  • Toward elcidation of biological enigma by genetically-encoded molecular spies and snipers  [招待講演]
    Nagai T
    2nd Swiss-Japan Chemical Biology Symposium(Tokyo, Japan) 2009年09月
  • Perspective of bioimaging and biomanipulation techniques  [招待講演]
    Nagai T
    International Symposium of post-silicon materials and devices research alliance projecInternational Symposium "Innovative Nanoscience of Supermolecular Motor Proteins" (Kyoto, Japan) 2009年09月
  • Molecular Nano-Mechanics & Bio-Mechanics Research Group(G3)  [招待講演]
    Nagai T
    International Symposium of post-silicon materials and devices research alliance project (Osaka, Japan) 2009年09月
  • Toward understanding biological phenomena by genetically-encoded molecular spies  [招待講演]
    Nagai T
    Invited seminar in NIH ( Bethesda, USA) 2009年08月
  • Rational design of photoswitchable fluorescent probes and its use for bioimaging  [招待講演]
    238th American Chemical Society Meeting (Washington Convention Center, Washington DC, USA) 2009年08月
  • Deciphering Enigma of Biological Function by Genetically-encoded Molecular Spies  [招待講演]
    Nagai T
    International Symposium on Photonic Bioimaging(Sappro, Japan) 2009年08月
  • 蛍光タンパク質テクノロジーの最前線  [招待講演]
    永井 健治
    第34回組織細胞化学会講習会(徳島大学長井記念ホール，徳島) 2009年07月
  • Tune-up of FRET-based functional indicators for high sensitive bioimaging  [招待講演]
    Nagai T
    Application Trens of Live Cell Imaging(Ewha Woman University, Soul, Korea) 2009年07月
  • Deciphering enigma of biological function by a genetically-encoded fluorescent indicator  [招待講演]
    Nagai T
    Gordon Research Conferences-Fertilization & Activation of Development (the Holderness School in Plymouth, NH,USA) 2009年07月
  • タンパク質でできた分子スパイによる細胞内非平衡現象の可視化  [招待講演]
    永井健治
    ソフトマター物理第;回領域研究会(北海道大学，札幌) 2009年07月
  • 生理機能・動態を計測するための蛍光タンパク質技術  [招待講演]
    永井 健治
    第9回日本蛋白質科学会年会 (熊本全日空ホテルニュースカイ，熊本) 2009年05月
  • Measurement of;diffusion coefficient of biomolecules by;fluorescence decay after photostimulation of;photoswichable fluorescen;proteins in living cells  [招待講演]
    Nagai T
    9th NIBB-EMBLSymposium(Okazaki, Japan) 2009年04月
  • Multifunctional imaging by genetically-encoded homo-FRET-based indicator  [招待講演]
    Focus On Microscopy 2009（Jagiellonian University Auditorium Maximum Krakow, Poland） 2009年04月
  • 光活性化型バイオセンサータンパク質の開発  [招待講演]
    永井 健治
    特定領域研究マルチスケール操作によるシステム細胞工学（バイオ操作）第7回公開シンポジウム(東京エレクトロンホール宮城，宮城)
  • Deciphering enigma of biological function by genetically-encoded molecular spies  [招待講演]
    Nagai T
    (ENS Joliot Curie Lab，Lyon, France)
  • Deciphering enigma of biological function by genetically-encoded molecular spies  [招待講演]
    Nagai T
    Current Advances in Live Cell Imaging Workshop(Seoul National University Hospital，Seoul, Korea)
  • 改変蛍光タンパク質を利用した生理機能の可視化  [招待講演]
    未踏・ナノテクノロジー第151委員会 第86回(物質材料研究機構，つくば)
  • GFPを巧妙に用いた分子機能・動態の可視化  [招待講演]
    永井 健治
    第8回学術講演会(京都府立医科大学 研究開発センター，京都)
  • Deciphering enigma of biological function by a genetically-encoded molecular spy  [招待講演]
    Nagai T
    The 4th LSW symposium (Hokkaido University, Sapporo) 2009年01月
  • バイオテクノロジー／生命システムの階層間をまたぐイメージング技術  [招待講演]
    永井 健治
    第31回日本分子生物学会年会(ポートアイランド，神戸)
  • Engineering fluorescent proteins to visualize and manipulate biological functions  [招待講演]
    Nagai T
    11th JAFoS Symposium(Irvine, USA)
  • Engineering fluorescent proteins to visualize and manipulate biological functions  [招待講演]
    Nagai T
    The 11th Japan and America Frontier of Science(Beckman center, Irvine, USA) 2008年12月
  • Engineering fluorescent proteins to visualize biological functions  [招待講演]
    Nagai T
    8th JAFoE Symposium(Kobe, Japan)
  • 蛍光タンパク質を巧妙に用いた分子機能・動態の可視化  [招待講演]
    永井 健治
    薬学会関東支部会 (長井記念館，東京) 2008年11月
  • GFP技術とFRET  [招待講演]
    永井 健治
    第12回細胞生物学ワークショップ(北海道大学，札幌) 2008年11月
  • Engineering fluorescent proteins to visualize biological functions  [招待講演]
    Nagai T
    The 8th Japan and America Frontier of Engineering(Kobe convention center, Kobe) 2008年11月
  • GFPは何故光る？  [招待講演]
    永井 健治
    第４回ライブセルイメージング講習会(産総研，筑波) 2008年10月
  • Imagaing biological functions by using FRET-based sensor proteins  [招待講演]
    ICCB2008(COEX, Seoul, Korea) 2008年10月
  • Engineering fluorescent and bioluminescent proteins to visualize biological functions  [招待講演]
    Nagai T
    IBRO (I Neurolatam)(Buzios, Brazil) 2008年09月
  • Engineering fluorescent and bioluminescent proteins to visualize biological functions  [招待講演]
    Nagai T
    Catolica de Chile(Catolica de Chile university, Chile) 2008年09月
  • Engineering fluorescent and bioluminescent proteins to visualize biological functions  [招待講演]
    Nagai T
    NEWroscience2008(de Sao Paulo university, Brazil) 2008年09月
  • 蛍光プロープの利用  [招待講演]
    永井 健治
    FRETの基礎，第11回細胞生物学ワークショップ(情通研未来ICT研究センター，神戸) 2008年08月
  • 改変蛍光タンパク質によるタンパク質機能・動態のリアルタイム可視化  [招待講演]
    永井 健治
    岩手医科大学先端医療研究センター公開シンポジウム「バイオイメージングと分子生物学による脳・血管解明ー健康増進に向けた最先端研究(岩手県医師会館ホール，盛岡) 2008年07月
  • GFPは何故光る？機能指示薬作成法と生理機能の可視化  [招待講演]
    永井 健治
    第17回メディカル・ホトニクスコース(浜松医科大学，浜松) 2008年07月
  • バイオイメージングの効能  [招待講演]
    永井 健治
    京都大学薬学研究科集中講義・生命薬科学特論Ⅲ/創薬リード探索理論（京都大学，京都） 2008年06月
  • Direct measurement of protein dynamics in single living cells using a rationally designed photoconvertible fluorescent protein？  [招待講演]
    永井 健治
    第60回日本細胞生物学会大会 (パシフィコ横浜，横浜) 2008年06月
  • バイオイメージングの効能  [招待講演]
    永井健治
    大阪大学理学研究科集中講義・生物科学特別講義Ⅰ/生物科学特別講義E（大阪大学，豊中） 2008年06月
  • Engineering fluorescent and bioluminescent proteins for biological research  [招待講演]
    Nagai T
    Institute of Neuroscience(Chinese Academy of Sciences, Shanghai, China) 2008年05月
  • Development of a fluorescent protein with deep blue color  [招待講演]
    Nagai T
    FOM2008(Awaji Yume Butai, Osaka, Japan) 2008年04月
  • Direct measurement of protein dynamics in living cells using a rationally designed photoconvertible fluorescent protein  [招待講演]
    Nagai T
    NIPS-JST international workshop(Okazaki conference center, Okazaki) 2008年04月
  • バイオイメージングの難しさ  [招待講演]
    永井 健治
    癌研セミナー(癌研究所，東京) 2008年03月
  • 蛍光タンパク質のエンジニアリングによる生理機能指示薬作成とライブイメージング  [招待講演]
    永井 健治
    東北大学多元物質化学研究所特別講演会ー細胞機能を探るインテリジェントマテリアルの開拓と医療展開(東北大，仙台) 2008年02月
  • 私の研究観 －ビジュアルバイオロジーへの道－  [招待講演]
    永井 健治
    阪大グローバルCOE若手・学生研究交流会(関西セミナーハウス，京都) 2008年02月
  • 天然ナノ発光粒子GFPを用いた生理機能センサーの開発とバイオイメージング  [招待講演]
    永井 健治
    通研共同プロジェクト研究会「フォトニック結晶の光産業技術への展開(東北大，仙台) 2008年02月
  • 最新のバイオイメージング技術  [招待講演]
    永井 健治
    北海道バイオ産業クラスター・フォーラム，シーズン公開会 (ホテルモントレエーデルホフ，札幌) 2008年01月
  • GFP技術とFRET  [招待講演]
    永井 健治
    第10回細胞生物学ワークショップ(北海道大学，札幌) 2008年01月
  • Perspective of Bioimaging Technologies using Engineered Fluorescent Proteins  [招待講演]
    Nagai T
    Joint symposium of Seoul University and Hokkaido University(Seoul Univ, Korea) 2008年01月
  • Imaging Biological Events Using FRET-based Sensor Proteins  [招待講演]
    Nagai T
    International Symposium on Advance Techniques for Molecular Imaging (Taichu, Taiwan) 2007年12月
  • イメージングの現在と未来  [招待講演]
    永井 健治
    視る生物学２-イメージングの現在と未来- (奈良先端大学院大学，奈良) 2007年11月
  • 合理的に設計した光変換蛍光タンパク質による生きた細胞内のタンパク質  [招待講演]
    永井 健治
    OPJ2007(大阪大学，大阪) 2007年11月
  • Engneering fluorescent proteins to visualize biological functions  [招待講演]
    Nagai T
    Joint Symposium for the Promotion of Academic exchange on Nanotechnology and Nanobiology(Sapporo, Japan) 2007年11月
  • 蛍光プロープの利用  [招待講演]
    永井 健治
    FRETの基礎，第9回細胞生物学ワークショップ(情通研未来ICT研究センター，神戸) 2007年10月
  • GFPは何故光る？  [招待講演]
    永井 健治
    第3回ライブセルイメージング講習会(産総研，筑波) 2007年10月
  • Engineering fluorescent and bioluminescent proteins to visualize biological functions  [招待講演]
    Nagai T
    Annual Meeting of Korean Society for Molecular Biology and Biochemistry (Seoul, Korea) 2007年10月
  • Engineering fluorescent and bioluminescent jproteins to visualize biological functions  [招待講演]
    The 2nd International Workshop on Approaches to Single-Cell Analysis(Tokyo, Japan) 2007年09月
  • Engineering fluorescent and bioluminescent jproteins to visualize biological functions,  [招待講演]
    Nagai T
    The 5th International Forum on Post-Genome Technologies(Shujo, Chaina) 2007年09月
  • Engineering Fluorescence Proteins  [招待講演]
    Microscopia de Fluorescencia y Confocal Espectral(Mexico City, Mexico) 2007年09月
  • バイオイメージング技術に望むもの―「細胞は如何にして外界情報を知覚するか？の探求―  [招待講演]
    平成19年度科研費特定領域研究「細胞感覚，夏の班会議・若手の会（湘南国際村センター，葉山） 2007年08月
  • GFPは何故光る？‐機能指示薬作成法と生理機能の可視化‐  [招待講演]
    永井 健治
    第16回メディカル・ホトニクスコース (浜松医科大学，浜松) 2007年07月
  • Visualization of biological function by using FRET- and BRET-based indicator  [招待講演]
    Nagai T
    APBP2007 3rd Asian and Pacific Rim Symposium on Biophotonics & Biophotonics (Shangri-La Hotel, Cairns, Australia) 2007年07月
  • 蛍光・化学発光タンパク質を利用して何ができるか？  [招待講演]
    生物発光化学発光研究会第２５回学術講演会(北海道大学，札幌) 2007年06月
  • Various Applications of Fluorescent Proteins for Cell Biology  [招待講演]
    Nagai T
    The 16th Annual Meeting of the Korean Scociety for Smooth Muscle Research(Seoul, Korea) 2007年06月
  • バイオイメージング技術の展望  [招待講演]
    永井 健治
    第40回日本発生生物学会・第59回日本細胞生物学会合同大会(福岡国際会議場，福岡) 2007年05月
  • GFP and Quantum Dot  [招待講演]
    Nagai T
    Advanced Microscopy Course for Bio-Medical Research(Beijin, China) 2007年05月
  • 蛍光・発光タンパク質を利用した生理機能センサーの開発とライブイメージング  [招待講演]
    永井 健治
    第96回日本病理学会(大阪国際会議場，大阪) 2007年03月
  • 蛍光・化学発光タンパク質を利用した生細胞内のリアルタイム機能イメージング  [招待講演]
    永井 健治
    第127回日本薬理学会 (ヴォルフォートとやまホール，富山) 2007年03月
  • GFP技術とFRET  [招待講演]
    第8回細胞生物学ワークショップ(北海道大学，札幌) 2007年01月
  • 蛍光・発光タンパク質を利用した生理機能イメージング  [招待講演]
    永井 健治
    通研共同プロジェクト研究会，｢フォトニック結晶の光産業技術への展開(東北大学ナノスピン総合研究棟，仙台) 2006年12月
  • 蛍光・発光タンパク質を用いたバイオセンサーの開発と生理機能のライブイメージング  [招待講演]
    永井 健治
    第4回分野横断スクール「ナノバイオスクール，－生体細胞の形態・機能観察から動的診断，都市センターホテル(都市センターホテル，東京) 2006年12月
  • 近未来的バイオイメージング技術の展望  [招待講演]
    第15回バイオイメージング学術集会(岩手医科大学60周年記念館，盛岡) 2006年11月
  • 生きた細胞の中の生理反応をリアルタイムに可視化する技術の開発  [招待講演]
    永井 健治
    大阪大学蛋白質研究所セミナー：ケミカルバイオロジーの進展と生命科学研究の新たな展開，大阪大学蛋白質研究所(大阪大学，大阪) 2006年11月
  • GFPは何故光る？  [招待講演]
    永井 健治
    第2回ライブセルイメージング講習会(産総研，筑波) 2006年10月
  • Development of FRET- and BRET-based functional indicators with expanded dynamic range  [招待講演]
    Nagai T
    The 2nd Sapporo Conference (Hokkaido University, Sapporo, Japan) 2006年10月
  • 個体レベルでのニューロン・グリア機能解析に資するイメージング法の開発  [招待講演]
    永井 健治
    第18回生理研研究会Neuro-glio-vascular interaction におけるプリン作動性シグナリングの病態生理的機能（生理学研究所，岡崎） 2006年09月
  • 蛍光分子を利用して生体機能と情報をはかる  [招待講演]
    永井 健治
    生化学会近畿支部(大阪大学コンベンションセンター，大阪) 2006年09月
  • 蛍光・化学発光タンパク質を利用した生理機能イメージング  [招待講演]
    分子構造討論会２００６ (グランシップ，静岡) 2006年09月
  • Development of FRET- and BRET-based functional indicators with expanded dynamic range  [招待講演]
    16th International Microscopy Congress(Sapporo Convention Center, Sapporo, Japan) 2006年09月
  • 蛍光分子を利用して生体機能と情報をはかる  [招待講演]
    永井 健治
    生命をはかる研究会(日本コンベンションセンター，幕張) 2006年08月
  • 蛍光プロープの利用，FRETの基礎  [招待講演]
    永井 健治
    第7回細胞生物学ワークショップ(情通研未来ICT研究センター，神戸) 2006年08月
  • Vivid visualization of biological functions by using DNA-encodable indicators- Toward multifunctional imaging  [招待講演]
    7th Joint Meeting of The Histochemical Society & The Japan Society of Histochemistry and Cytochemistry (Hilton Waikoloa Village, Big Island of Hawaii, USA) 2006年08月
  • GFPは何故光る？‐機能指示薬作成法と生理機能の可視化  [招待講演]
    永井 健治
    第15回メディカル・ホトニクスコース(浜松医科大学，浜松) 2006年07月
  • Engineering green fluorescent proteins to visualize biological functions  [招待講演]
    Symposium on New Frontier in Live Cell Imaging(Yang Ming University, Taipei, Taiwan) 2006年07月
  • Engineering green fluorescent proteins to visualize and manipulate biological functions  [招待講演]
    Nagai T
    Microscopy workshop(National Cheng Kung University, Tainan) 2006年07月
  • Realtime imaging and manipulation of biological functions by fluorescent proteins  [招待講演]
    Nagai T
    20th IUBMB International Congress of Biochemistry and Molecular Biology(Kyoto, Japan) 2006年07月
  • Engineering fluorescent and bioluminescent jproteins to visualize biological functions  [招待講演]
    Nagai T
    The 2nd International Workshop on Approaches to Single-Cell Analysis(Tokyo, Japan) 2006年07月
  • バイオイメージング技術の現状と展望  [招待講演]
    永井 健治
    日本生体医工学会北海道支部第28回ME研究会(北海道大学，札幌) 2006年06月
  • 近未来的バイオイメージングの展望  [招待講演]
    永井 健治
    特定領域研究２領域合同公開シンポジウム，細胞内のダイナミックネットワーク：前進するオルガネラ研究，千里ライフサイエンスセンター(千里ライフサイエンスセンター，大阪) 2006年06月
  • 発光性タンパク質を利用した生理機能の可視化と操作～医療応用を目指して～  [招待講演]
    永井 健治
    臨床応用を目指した産学連携セミナー３分子細胞イメージングと疾患・創薬研究～ライブセルからin vivoへの展開 (コクヨホール，東京) 2006年05月
  • Molecular and cellular imaging of living organisms  [招待講演]
    Nagai T
    63rd KSBMB Annual Meeting, COEX(Seoul, Korea) 2006年05月
  • 個体レベルの機能イメージングを目指して  [招待講演]
    永井 健治
    第111回日本解剖学会シンポジウム(北里大学，相模原) 2006年03月
  • 個体レベルの機能イメージングを目指して  [招待講演]
    永井 健治
    第79回日本薬理学会シンポジウム(パシフィコ横浜，横浜) 2006年03月
  • 生理機能イメージング技術とその医学応用  [招待講演]
    永井 健治
    京都大学医学研究科集中講義・第130回細胞生物学セミナー（京都大学，京都） 2006年02月
  • 個体レベルの機能イメージングを目指して  [招待講演]
    永井 健治
    岡崎統合バイオサイエンスセンター創設５周年シンポジウム(岡崎カンファレンスセンター，岡崎) 2006年02月
  • GFP技術とFRET  [招待講演]
    永井 健治
    第6回細胞生物学ワークショップ (北海道大学，札幌) 2006年01月
  • 形態形成原理の解明を目指して－バイオイメージングによるアプローチー  [招待講演]
    永井 健治
    第28回日本分子生物学会バイオテクノロジーセミナー (JALリゾートシーホークホテル，福岡) 2005年12月
  • 生命の原理に迫るには？  [招待講演]
    永井 健治
    第５回先端研究交流セミナー (順天堂大学，東京) 2005年11月
  • GFPは何故光る？  [招待講演]
    永井 健治
    第1回ライブセルイメージング講習会 (産総研，筑波) 2005年10月
  • Application of GFP-based indicators to visualize biological functions  [招待講演]
    Japan-UK Symposium on Promotion of Regional Partnership on Nanotechnology Development (Hokkaido University, Sapporo, Japan) 2005年10月
  • Application of GFP-based indicators to visualize biological functions  [招待講演]
    Nagai T
    The TRF & Microscopy Workshop (Yang Ming University, Taipei, Taiwan) 2005年10月
  • Functional imaging of living cells by the GFP-based indicators  [招待講演]
    LSM CLUB 2005(Shanhai, China) 2005年09月
  • 細胞内の現象を見る・いじる  [招待講演]
    永井 健治
    第14回メディカル・ホトニクスコース(浜松医科大学，浜松) 2005年08月
  • Engineering green fluorescent proteins to visualize and manipulate biological functions  [招待講演]
    永井 健治
    浜松医科大学COE国際シンポジウム(浜松医科大学，浜松) 2005年08月
  • FRETの基礎  [招待講演]
    永井 健治
    第5回細胞生物学ワークショップ(情通研未来ICT研究センター，神戸) 2005年08月
  • Real-time detection of signal transduction pathway in living cells  [招待講演]
    Nagai T
    American Academy of Nanomedicine First Annual Meeting (Johns Hopkins University, Baltimore, USA) 2005年08月
  • ニポウ式共焦点によるFRETイメージング  [招待講演]
    永井 健治
    日本顕微鏡学会(エポカルつくば，つくば) 2005年06月
  • 蛍光タンパク質を利用した生体機能のライブイメージング  [招待講演]
    永井 健治
    日本顕微鏡学会(エポカルつくば，つくば) 2005年06月
  • 蛍光タンパク質が拓く光観察・操作技術  [招待講演]
    永井健治
    浜松医科大学セミナー(浜松) 2005年05月
  • 蛍光タンパク質が拓く光観察・操作技術  [招待講演]
    永井 健治
    慶応大学医学部セミナー (東京) 2005年05月
  • 蛍光タンパク質を用いた生体機能イメージングの現状と展望  [招待講演]
    永井 健治
    日本病理学会(パシフィコ横浜，横浜) 2005年04月
  • GFP技術とFRET  [招待講演]
    永井 健治
    第4回細胞生物学ワークショップ「大学院トレーニングコース：中級～上級，(北海道大学，札幌) 2005年01月
  • Fluorescence imaging technologies for visualizing biological functions  [招待講演]
    Nagai T
    The symposium on Imaging Technologies of Live Cells and Animals(Helsinki, Finland) 2004年11月
  • GFPを使ったバイオセンサーの作成法  [招待講演]
    永井 健治
    第29回組織細胞化学講習会（山梨大学，甲府） 2004年08月
  • FRETの基礎  [招待講演]
    永井 健治
    第3回細胞生物学ワークショップ「大学院トレーニングコース：基礎から中級（通信総合研究所，神戸） 2004年08月
  • GFPの基礎と種類  [招待講演]
    永井 健治
    第13回浜松医科大学メディカルホトニクスコース（浜松医科大学，浜松） 2004年07月
  • FRET観察の基礎および２光子顕微鏡による観察  [招待講演]
    永井 健治
    第１９回基礎生物学研究所バイオサイエンストレーニングコース（基礎生物学研究所，岡崎） 2004年06月
  • 蛍光タンパク質が拓くバイオイメージング技術  [招待講演]
    2004年度北海道大学大学院共通授業・生化学特別講義(北海道大学，札幌) 2004年05月
  • 可視化のためのReagentの開発  [招待講演]
    永井 健治
    基礎生物学研究所研究会－生体シグナルの可視化を目指して－（基礎生物学研究所，岡崎） 2004年03月
  • GFP技術とFRET  [招待講演]
    永井 健治
    第2回細胞生物学ワークショップ「大学院トレーニングコース：中級から上級 (北海道大学，札幌) 2003年11月
  • Development of FRET-based biosensors with expanded dynamic range  [招待講演]
    永井 健治, 宮脇敦史
    生理学研究所研究会「細胞内シグナル伝達機構の多角的・包括的理解(生理学研究所，岡崎) 2003年10月
  • GFPを使った方法の新たな発展  [招待講演]
    永井 健治
    第28回組織細胞化学講習会(愛知医科大学，名古屋) 2003年08月
  • GFPの基礎と種類  [招待講演]
    永井 健治
    第１２回浜松医科大学メディカルホトニクスコース（浜松医科大学，浜松） 2003年08月
  • FRETの基礎  [招待講演]
    永井 健治
    第1回細胞生物学ワークショップ「大学院トレーニングコース：初級から中級（通信総合研究所，神戸） 2003年08月
  • Opening new windows for future biology by using GFP technologies  [招待講演]
    Blue Seminar at EMBL(Heidelberg, Germany) 2003年08月
  • ミトコンドリアにおける遊離Ca2+動態  [招待講演]
    永井 健治, 宮脇敦史
    第26回日本神経科学学会シンポジウム（名古屋国際会議場，名古屋） 2003年07月
  • 顕微鏡技術によるFRET測定  [招待講演]
    永井 健治
    第32回千里ライフサイエンス技術講習会（千里ライフサイエンスセンター，吹田） 2003年06月
  • Dynamic and quantitative imaging of cell functions  [招待講演]
    Nagai T
    Seminars at the Hutchson/MRC research centre(Cambridge, UK) 2003年06月
  • Dynamic and quantitative imaging of cell functions EMBO practical course-MICROINJECTION AND PROBE VISUALISATION IN CELLS  [招待講演]
    Nagai T
    (Heidelberg, Germany) 2003年05月
  • 新技術融合が拓く Dynamical Biology  [招待講演]
    永井 健治
    産業技術総合研究所セミナー（産業技術研究所，つくば） 2003年03月
  • 新技術融合が拓く Dynamical Biology  [招待講演]
    永井 健治
    国立遺伝学研究所セミナー（国立遺伝学研究所，三島） 2003年01月
  • GFPを用いた細胞機能のライブイメージング  [招待講演]
    永井 健治
    第25回日本分子生物学会ワークショップ（パシフィコ横浜，横浜） 2002年12月
  • FRET microscopy  [招待講演]
    永井 健治
    EMBO practical course, Fluorescence Microscopy of Living Cells（通信総合研究所，神戸） 2002年11月
  • GFPの物理化学的性質とライブイメージング  [招待講演]
    永井 健治
    第12回日本サイトメトリー学会シンポジウム（愛知医科大学，名古屋） 2002年08月
  • バイオイメージングと計算機シミュレーションの相補性  [招待講演]
    永井 健治
    第35回日本発生生物学会ワークショップ（パシフィコ横浜，横浜） 2002年05月

その他活動・業績

特許

  • 特開2023-94290:超解像画像生成方法、超解像画像生成システム、蛍光プローブ、及び発現キット  2023年07月05日
    永井健治, 野間涼平, 原聡, 松田知己, 和沢鉄一, 鷲尾隆
  • 特開2023-43461:発光性タンパク質を用いた発光方法  2023年03月29日
    永井健治, ホサイン ナディム エムディ  大阪大学
  • 特許7241423:発光蛋白質，その基質，及びそれらの使用  
    永井健治, 岩野惠  大阪大学  
    PCT/JP2019/022198（出願日：2019年6月4日）
  • 特開2023-3158:蛍光イメージング装置  2023年01月11日
    永井健治, 市村垂生, 藤田克昌, 垣塚太志, 橋本均  大阪大学
  • 特許7148136:水の硬度の測定  
    永井健治, ホサイン ナディム エムディ, 石田竜一, 松田知己, 服部満  大阪大学
  • 特許7137842:デバイス、及びそれを用いた判定システム  
    永井健治, 新井由之, 岩野恵  国立大学法人大阪大学  
    11619628（登録日：2023年4月4日）米国 PCT/JP2018/002591（出願日：2018年1月26日） 優先権主張番号 特願2017-18773(P2017-18773)
  • 特願2022-128319:発光タンパク質  2022年08月10日
    永井健治, 田中奏希, 長部謙二, 圓谷徹之  国立大学法人大阪大学  
    出願国：ＷＩＰＯ 国際出願番号：PCT/JP2023/028990 国際出願日：2023年8月8日
  • 特許7014437:生体物質の検出方法、それに用いる化学発光指示薬  
    永井健治, 新井由之  国立大学法人大阪大学  
    PCT/JP2018/002587（出願日：2018年1月26日） 優先権主張番号 特願2017-13463(P2017-13463)
  • 商標登録 6342260:LEP  
  • 特許6764469:生体試料を生存状態で観察する顕微鏡および方法  
    永井健治, 新井由之, 鈴木和志  国立研究開発法人科学技術振興機構  
    PCT/JP2017/011837（出願日：2017年3月23日） 登録番号：3435135号（EPドイツ・登録日：令和2年9月23日）
  • 特許第6762069号:蛍光タンパク質  
    永井健治, 篠田肇, 松田知己, MA Yuanqing  国立大学法人大阪大学  
    PCT/JP2017/009759
  • 特願2019-177540:画像処理装置，ニューラルネットワークおよび画像処理方法  2019年09月27日
    鷲尾隆, 原聡, 和沢鉄一, 永井健治, 陳唯之  大阪大学
  • 特許6425483:細胞内の酸化還元状態をモニターするための蛍光タンパク質，ＤＮＡ，ベクター，形質転換体，及び方法  
    久堀 徹, 杉浦 一徳, 永井 健治  国立大学法人東京工業大学
  • 特許6115923:蛍光蛋白質  
    永井 健治, ティバリ ダーメンドラ クマール, 新井 由之  大阪大学  
    PCT/JP2014/074121 登録番号：9945784号（アメリカ・登録日：平成30年4月17日）
  • 特許6075963:蛍光観察方法及び蛍光観察装置  
    藤田 克昌, 永井 健治, 齊藤 健太, 山中 真仁, 瀧本 真一  オリンパス株式会社
  • 特許6044941:光学顕微鏡、および、光学顕微鏡のオートフォーカス装置  
    永井健治, 新井由之  国立大学法人大阪大学  
    PCT/JP2013/081259 登録番号：9632303（アメリカ・登録日：2017年4月25日）
  • 特許5943432:共焦点顕微鏡  
    永井 健治, 齊藤 健太, 新井 由之, 斉藤 浩二, 中村 洋介, 石井 淳一  国立大学法人大阪大学, 株式会社 オプトライン  
    登録番号：I534473号(台湾・平成28年5月21日)
  • 特開2015-186464:ｃＡＭＰ検出方法  2015年10月29日
    瓜生 幸嗣, 下野 健, 永井 健治  国立大学法人大阪大学, パナソニック株式会社
  • 特開2015-186463:ｃＡＭＰ検出方法  2015年10月29日
    瓜生 幸嗣, 下野 健, 永井 健治  国立大学法人大阪大学, パナソニック株式会社
  • 特開2015-139420:化学物質検出方法  2015年08月03日
    田中 真司, 村岡 仁, 下野 健, 永井 健治, 谷口 正輝, 川合 知二  国立大学法人大阪大学, パナソニック株式会社
  • 特開2015-94637:吸収顕微鏡  2015年05月18日
    永井 健治, 新井 由之, 宗形 豊, 寺尾 功生, 山田 洋平  国立大学法人大阪大学, 中央精機株式会社
  • 特願2017-517979:蛍光蛋白質  2015年05月12日
    永井健治, 高内大貴, 新井由之, 中野雅裕  国立大学法人大阪大学  
    PCT/JP2016/064132
  • 特開2013-78264:光増感性蛍光タンパク質  2013年05月02日
    永井 健治, 松田 知己, 高橋理佳, 竹本 研  国立大学法人北海道大学
  • 特開2012-177549:蛍光温度プローブおよびそれを用いた温度測定装置  2012年09月13日
    永井 健治, 小寺 一平, 岩崎 卓也  国立大学法人北海道大学
  • 特開2012-157306:光駆動カルシウムイオン制御タンパク質  2012年08月23日
    永井 健治, 松田 知己, 福田 憲隆  国立大学法人北海道大学
  • 特願2011-123041:共焦点顕微鏡  2011年06月01日
    永井 健治, 齊藤 健太  国立大学法人北海道大学  
    【国際出願番号】PCT/JP2012/002598 【国際出願日】平成24年4月13日(2012.4.13)
  • 特許4557685:蛍光タンパク質  
    永井 健治, 宮脇 敦史  独立行政法人理化学研究所  
    PCT/JP2005/020843
  • 特願2009-233826:光活性化生理機能センサータンパク質  2009年10月07日
    永井 健治, 松田 知己  国立大学法人北海道大学
  • 特許4288571:標的物質の生理的機能を解析する方法  
    永井 健治, 宮脇 敦史  独立行政法人理化学研究所, 独立行政法人科学技術振興機構  
    JCT/JP2004/007238
  • 特許4214206:ＦＲＥＴを利用した蛍光指示薬  
    宮脇 敦史, 永井 健治  独立行政法人理化学研究所, 独立行政法人科学技術振興機構  
    JCT/JP03/15790
  • 特許4014536:共焦点光スキャナ  
    御厨 健太, 田名網 健雄, 関 直樹, 永井 健治, 宮脇 敦史  横河電機株式会社, 独立行政法人 科学技術振興機構, 独立行政法人理化学研究所  
    外国出願番号：米国10/844421、ヨーロッパ04011431.6
  • 特願2009-529010:組換えＤＮＡの調製方法  2007年08月21日
    永井 健治, 小寺 一平  国立大学法人北海道大学  
    【国際出願番号】PCT/JP2008/064482 【国際出願日】平成20年8月12日
  • 特願2007-203300:群青色蛍光タンパク質  2007年08月03日
    永井 健治, 友杉 亘, 松田 知己  国立大学法人北海道大学  
    PCT/JP2008/064266
  • 特許3829252:蛍光蛋白質  
    宮脇 敦史, 永井 健治  独立行政法人理化学研究所  
    外国出願番号：米国10/162593、ヨーロッパ02012397.2
  • 特願2006-43211:ＤＮＡ塩基配列を決定する方法  2006年02月20日
    永井 健治, 谷 知己, 小寺 一平, 米田 悦啓  国立大学法人北海道大学  
    PCT/JP2007/053461
  • 特許3674744:神経発生誘導遺伝子  
    御子柴 克彦, 有賀 純, 永井 健治, 中田 勝紀  独立行政法人理化学研究所
  • 特願2006-549032:光増感物質で標識した蛍光蛋白質を利用した標的生理機能不活性化剤  2004年12月21日
    永井 健治, 宮脇 敦史  独立行政法人理化学研究所, 独立行政法人科学技術振興機構  
    PCT/JP2005/023508
  • 特願2003-355192:ＦＲＥＴを利用した蛍光指示薬  2003年10月15日
    宮脇 敦史, 永井 健治  独立行政法人理化学研究所, 独立行政法人科学技術振興機構  
    PCT/JP2004/015671
  • 特開2002-253261:蛍光タンパク質  2002年09月10日
    宮脇 敦史, 永井 健治  独立行政法人理化学研究所

受賞

  • 2023年06月 日本顕微鏡学会 第38回（2023年度）論文賞 応用研究（生物）部門
     "A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging". Microscopy, 70: 340–352, 2021 
    受賞者: 和沢鉄一;野間涼平;宇土周作;杉浦一徳;鷲尾隆;永井健治
  • 2023年02月 2022年度 中谷賞大賞
  • 2021年11月 第21回 山崎貞一賞
  • 2020年06月 第35回 日本顕微鏡学会論文賞 顕微鏡法基礎部門
     
    受賞者: 和沢鉄一, 新井由之, 河原吉伸, 高内大貴, 鷲尾隆, 永井健治
  • 2018年10月 第36回 大阪科学賞
  • 2018年 第3回 日本光生物学協会協会賞
  • 2015年 第4回 大阪大学総長顕彰賞
  • 2014年 第10回 日本学術振興会賞
  • 2014年 第3回 大阪大学総長顕彰賞
  • 2013年 第2回 大阪大学総長顕彰賞
  • 2012年 第21回 木原記念財団学術賞応用科学賞

共同研究・競争的資金等の研究課題

  • 蛍光/化学発光タンパク質による波長変換/自発光で誘起する光合成の理解と技術応用
    基盤研究(A)
    研究期間 : 2022年 -2025年
  • 超解像光照射分子不活性化法の開発
    挑戦的研究(萌芽)
    研究期間 : 2021年 -2023年
  • シンギュラリティ生物学
    新学術領域研究(研究領域提案型)
    研究期間 : 2018年 -2022年
  • シンギュラリティ細胞を探索・操作するための細胞機能3次元可視化・光操作技術の開発
    新学術領域研究(研究領域提案型)
    研究期間 : 2018年 -2022年
  • 電力非依存型発光生体分子の改変と樹木への実装
    NEDO マテリアル・バイオ革新技術先導研究プログラム
    研究期間 : 2022年
  • 細胞熱産生におけるジュール熱仮説の検証
    基盤研究(A)
    研究期間 : 2018年 -2020年
  • オールインワン化学発光顕微鏡システムの開発
    JST 先端計測分析技術・機器開発プログラム
    研究期間 : 2016年 -2020年
  • 超解像「生理機能」イメージング法の開発と細胞状態解析への応用
    JST 戦略的創造研究推進事業 CREST ナノテクノロジー・材料 新たな光機能や光物性の発現・利活用を基軸とする次世代フォトニクスの基盤技術
    研究期間 : 2015年 -2020年
  • 感染症を在宅で簡易診断する技術基盤の開発
    JST CREST 異分野融合による新型コロナウイルスをはじめとした感染症との共生に資する技術基盤の創生
    研究期間 : 2020年 -2023年
  • ケミルミノジェネティクスによる構成的エネルギー生合成系の創出
    挑戦的研究(萌芽)
    研究期間 : 2017年 -2019年
  • グローバル分子技術実装ネットワークの構築
    JSPS 人材育成事業・大学の教育研究機能の向上 人材育成事業 頭脳循環を加速する戦略的国際研究ネットワーク推進プログラム
    研究期間 : 2016年 -2018年
  • 脳内神経機能を自律的に非侵襲操作可能な細胞活動依存的化学発光遺伝学プローブの創成
    JSPS 学術国際交流事業 国際的な共同研究等の促進 二国間交流事業 共同研究・セミナー オープンパートナーシップ共同研究
    研究期間 : 2016年 -2017年
  • フレネル非干渉光相関ホログラムに基づく化学発光超解像高速3Dイメージング法の確立
    挑戦的萌芽研究
    研究期間 : 2015年 -2016年
  • 個体深部の生命機能を非侵襲的に操作可能なケミルミノジェネティクス技術の創成
    基盤研究(A)
    研究期間 : 2014年 -2016年
  • マルチモーダル発光イメージングシステムの開発
    JST 研究成果展開事業（企業化開発・ベンチャー支援・出資） 先端計測分析技術・機器開発プログラム 要素技術タイプ
    研究期間 : 2013年 -2016年
  • 少数性生物学ー個と多数の狭間が織りなす生命現象の探求ー
    JSPS 研究助成事業 KAKEN 新学術領域研究(研究領域提案型) 少数性生物学―個と多数の狭間が織りなす生命現象の探求―
    研究期間 : 2016年
  • 1粒子イメージングによる動態解析を基盤としたIgE非誘導性ワクチン創成への挑戦
    JSPS 研究助成事業 KAKEN 挑戦的萌芽研究
    研究期間 : 2014年 -2015年
  • 超解像機能イメージングを可能にする新規蛍光タンパク質プローブの開発
    JSPS 研究助成事業 KAKEN 特別研究員奨励費
    研究期間 : 2014年 -2015年
  • 新規インバースタイプのカルシウムプローブを用いた抑制性神経活動測定法の開発
    JSPS 研究助成事業 KAKEN 挑戦的萌芽研究
    研究期間 : 2014年 -2015年
  • 超高感度指示薬による細胞性粘菌発生過程の時空間カルシウムイオン観察
    JSPS 学術国際交流事業 国際的な共同研究等の促進 二国間交流事業 共同研究・セミナー 共同研究 アジア
    研究期間 : 2013年 -2015年
  • 少数性生物学ー個と多数の狭間が織りなす生命現象の探求ー
    JSPS 研究助成事業 KAKEN 新学術領域研究(研究領域提案型)
    研究期間 : 2011年 -2015年
  • 分子プローブと光摂動ツールの開発－少数生体分子の可視化・操作技術－
    JSPS 研究助成事業 KAKEN 新学術領域研究(研究領域提案型)
    研究期間 : 2011年 -2015年
  • 新しい原理に基づく吸収増幅顕微鏡の開発と生物研究応用
    NEDO 分野横断的公募事業 先導的産業技術創出事業(若手研究グラント) 4年型
    研究期間 : 2009年 -2012年
  • ナノサイズ高輝度バイオ光源の開発と生命機能計測への応用
    JST 戦略的創造研究推進事業 さきがけ ナノテクノロジー・材料 光の利用と物質材料・生命機能
    研究期間 : 2008年 -2012年
  • アブラナ科およびナス科植物の自家不和合性の分子機構解明
    JSPS 研究助成事業 KAKEN 基盤研究(A)
    研究期間 : 2009年 -2011年
  • 植物の病害抵抗性と細胞死機構の時空間的制御メカニズムの解明
    JSPS 研究助成事業 KAKEN 若手研究(A)
    研究期間 : 2009年 -2010年
  • 光スイッチングバイオセンサーの開発
    JSPS 研究助成事業 KAKEN 挑戦的萌芽研究
    研究期間 : 2008年 -2009年
  • 光操作によるマルチスケール機能イメージング法の開発
    JSPS 研究助成事業 KAKEN 特定領域研究
    研究期間 : 2008年 -2009年
  • 神経変性タンパク質の細胞局所場に於ける動態・フィブリル化のイメージングに基く効率的な医薬品評価系の開発
    MHLW 厚生労働省研究事業 厚生労働科学研究費補助金(厚生科研費)
    研究期間 : 2007年 -2009年
  • 細胞内現象をリアルタイムに可視化する技術と光で操作する技術の開発
    研究期間 : 2005年 -2009年
  • 構造変化感受性蛍光タンパク質を用いたTrk受容体リン酸化のライブイメージング
    JSPS 研究助成事業 KAKEN 特定領域研究
    研究期間 : 2007年 -2008年
  • ホモFRETによる複数機能の可視化技術開発
    JSPS 研究助成事業 KAKEN 特定領域研究
    研究期間 : 2007年 -2008年
  • 完全定量1分子共鳴エネルギー移動測定によるタンパク質構造動態解析法の確立
    JSPS 研究助成事業 KAKEN 特定領域研究
    研究期間 : 2007年 -2008年
  • 多機能ライブイメージング法の開発とその細胞生物学研究への応用
    JSPS 研究助成事業 KAKEN 特別研究員奨励費
    研究期間 : 2007年 -2008年
  • 少数分子による自己組織化過程の生体機能シグナル可視化による解析
    JSPS 研究助成事業 KAKEN 若手研究(A)
    研究期間 : 2006年 -2008年
  • 立体構造情報を利用した蛍光イメージング用バイオセンサーの設計法の開発
    JSPS 研究助成事業 KAKEN 若手研究(B)
    研究期間 : 2006年 -2007年
  • 植物の栄養感知に関わるシグナル伝達のリアルタイム可視化解析
    JSPS 研究助成事業 KAKEN 特定領域研究
    研究期間 : 2006年 -2007年
  • 吸収位相差顕微鏡の開発
    JSPS 研究助成事業 KAKEN 萌芽研究
    研究期間 : 2005年 -2006年
  • ナノレベルイメージングによる分子の機能および構造解析
    MHLW 厚生労働省研究事業 厚生労働科学研究費補助金(厚生科研費)
    研究期間 : 2002年 -2006年
  • 蛍光・化学発光タンパク質及びその変異体を用いた機能指示薬開発
    研究期間 : 2005年
  • 色素タンパク質による生体機能の時空間的な不活性化法の確立
    JST 戦略的創造研究推進事業 さきがけ ライフイノベーション 生体分子の形と機能
    研究期間 : 2001年 -2004年
  • cameleon1分子イメージングによる局所的カルシウム動態の解析
    JSPS 研究助成事業 KAKEN 特定領域研究
    研究期間 : 1999年 -2003年
  • 色素タンパク質を用いた生体機能の時空間的な破壊法の開発
  • 緑色蛍光タンパク質の改良およびタンパク質間相互作用指示薬の開発

大学運営

委員歴

  • 2023年10月 - 現在   JST創発 ”岡田パネル” アドバイザー   https://www.jst.go.jp/souhatsu/research2/panel_okada.html
  • 2021年 - 現在   International Biophysics Congress 2024 Kyoto 実行委員   https://www.c-linkage.co.jp/iupab2024-bsj-kyoto/board.html
  • 2020年04月 - 現在   JST さきがけ "多細胞システムにおける細胞間相互作用とそのダイナミクス" アドバイザー   https://www.jst.go.jp/kisoken/presto/research_area/ongoing/bunya2019-1.html
  • 2017年01月 - 現在   ACS Sensors誌 Editorial Advisory Board   https://pubs.acs.org/page/ascefj/editors.html
  • 2015年01月 - 現在   日本バイオイメージング学会評議員（2017年４月から理事）   https://j-bioimaging.org/officer/
  • 2014年10月 - 現在   日本学術会議連携会員   https://www.scj.go.jp/ja/scj/member/renkei.html#na
  • 2014年01月 - 現在   日本生物物理学会理事   https://www.biophys.jp/org/org03_01.html
  • 2013年05月 - 現在   Biophysics and Physicobiology誌 Associate Editor   https://www.biophys.jp/biophysics_and_physicobiology02.html
  • 2022年   第45回 日本分子生物学会年会 副大会長   https://www2.aeplan.co.jp/mbsj2022/general-info.html
  • 2022年   第31回 日本バイオイメージング学会学術集会 大会長   https://www.sanken.osaka-u.ac.jp/labs/bse/bioimaging/organization.html
  • 2016年06月 - 2021年   JST CREST "光の特性を活用した生命機能の時空間制御技術の開発と応用" 領域 領域アドバイザー   領域アドバイザー https://www.jst.go.jp/kisoken/crest/research_area/ongoing/bunyah28-1.html
  • 2012年11月 - 2020年03月   Microscopy誌 Editorial Board
  • 2016年 - 2020年   日本学術振興会160委員会委員   https://bsw3.naist.jp/jsps160c/meibo.html
  • 2019年09月   第57回生物物理学会 年会長   https://www2.aeplan.co.jp/bsj2019/kaisai.html
  • 2018年   First UK/JAPAN super-resolution bioimaging meeting, Oxford, Organizer
  • 2015年04月 - 2017年   日本顕微鏡学会代議員
  • 2011年08月 - 2016年03月   JST さきがけ "細胞機能の構成的な理解と制御" 研究領域アドバイザー   領域アドバイザー https://www.jst.go.jp/presto/synbio/
  • 2016年   新学術領域「少数性⽣物学」 PacifiChem 2015 Sumposium "Life at Small Copy Numbers", Organizer
  • 2012年11月   日米先端科学 ( JAFoS ) シンポジウム, 主査
  • 2012年   新学術領域「少数性⽣物学」第1回国際会議 2012 Paradigm Innovation in Biology: Novel Strategy and Thinking, Organizer
  • 2010年   International Symposium of Joint Research Network on Advanced Materials and Devices " 彫 " Organizing Chair   https://www.es.hokudai.ac.jp/symposium/2009/

社会貢献活動

  • 「光るタンパク質の研究と未来応用」
    期間 : 2023年09月27日
    役割 : 講師
    主催者・発行元 : 滋賀県立彦根東高等学校
  • Luminescent protein technologies
    期間 : 2023年03月22日
    役割 : 講師
    主催者・発行元 : 滋賀県立彦根東高等学校
    イベント・番組・新聞雑誌名 : サイエンス国際フォーラム
  • 「光るタンパク質の研究と未来応用」
    期間 : 2021年10月19日
    役割 : 講師
    イベント・番組・新聞雑誌名 : 先端科学研修, 滋賀県立彦根東高等学校
  • 「光るたんぱく質が拓く未来の社会」
    期間 : 2020年09月11日
    役割 : 講師
    主催者・発行元 : 先端科学研修特別講義, 滋賀県立彦根東高等学校
  • 「光るタンパク質の研究と未来応用」
    期間 : 2019年07月25日
    役割 : 講師
    主催者・発行元 : 2019年度第12回高校生事業 ライフサイエンスセミナー
  • 「光る生きものの研究 -研究って面白い！-」
    期間 : 2018年03月22日
    役割 : 講師
    主催者・発行元 : 特別講義, 奈良県立青翔子高等学校
  • 多彩なナノランタンで未来の世界を彩る」
    期間 : 2017年09月04日
    役割 : 講師
    主催者・発行元 : 2017年光化学討論会・市民公開講座「バイオイメージング最前線講演会」, 東北大学青葉山キャンパス
  • 「蛍光・化学発光タンパク質の開発と社会実装への展開」
    期間 : 2017年05月26日
    役割 : 講師
    主催者・発行元 : 第 59 回公開講演会（繊維技術）, 大阪産業創造館
  • 「生物発光が拓く未来の生活」
    期間 : 2015年09月26日
    役割 : 講師
    主催者・発行元 : 第 24 回 日本バイオイメージング学会 学術集会公開講座
  • 「生物発光が拓く未来社会」
    期間 : 2015年09月12日
    役割 : 講師
    主催者・発行元 : 第53回日本生物物理学会年会・市民講演会, 石川県教育会館 ホール
  • 「光る植物で明るい未来」
    期間 : 2015年08月29日
    役割 : 講師
    主催者・発行元 : 市民参加シンポジウム 未来を拓く植物バイオのチカラ, 大阪富国生命ビル
  • 「街路樹を照明灯に！遺伝子組み換え植物の可能性」
    期間 : 2015年03月28日
    役割 : 講師
    主催者・発行元 : 市民参加シンポジウム「未来を拓く植物バイオのチカラ」, 大阪富国生命ビル４F（大阪市）
  • 「少数性生物学概説および少数性分子の可視化・操作技術の開発」
    期間 : 2014年12月17日
    役割 : 講師
    主催者・発行元 : 第 14 回名古屋大学・遺伝子実験施 設公開セミナー、名古屋大学 坂田・平田ホール、名古屋市
  • 「少数性生物学って何？」
    期間 : 2014年03月15日
    役割 : 講師
    主催者・発行元 : 静岡県立大学 市民勉強会「環境・生命・宇宙 －わたしたちの星、地球、月そして太陽ー」 、静岡県立大学、静岡市
  • 「少数性生物学〜タンパク質分子に個性はあるのか」
    期間 : 2013年12月06日
    役割 : 講師
    主催者・発行元 : 日本分子生物学会 公開プレゼンテーション「生 命世界を問う」、神戸国際会議場 ポートピアホール、神戸市
  • 「細胞・個体イメージング用光学プローブの開発」
    期間 : 2013年09月17日
    役割 : 編集長
    主催者・発行元 : 日本学術会議公開シンポジウム 「医学・生命科学 の革新的発展に資する統合バイオイメージングの展望」、日本学術会議講堂、東京都
  • 「光で拓く生命科学」
    期間 : 2013年07月18日
    役割 : 講師
    主催者・発行元 : 上宮高等学校講演会、上宮高等学校、大阪市
  • 「オワンクラゲの研究から」
    期間 : 2012年11月16日
    役割 : 講師
    主催者・発行元 : 講演会、北三瓶小学校、中学校、大田市三瓶町
  • 「最先端バイオイメージング技術で何を解き明かすのか？」
    期間 : 2012年08月25日
    役割 : 講師
    主催者・発行元 : 第 52 回生命科学夏の学校、西浦温泉ホテ ルたつき、蒲郡市
  • 光活性化型バイオセンサータンパク質の開発，特定領域研究マルチスケール操作によるシステム細胞 工学（バイオ操作）
    期間 : 2009年03月05日 - 2009年03月07日
    役割 : 講師
    主催者・発行元 : 第 7 回公開シンポジウム，(東京エレクトロンホール宮城，宮城)
  • 改変蛍光タンパク質によるタンパク質機能・動態のリアルタイム可視化
    期間 : 2008年07月
    役割 : 講師
    主催者・発行元 : (岩手県医師会館ホール，盛岡)
    イベント・番組・新聞雑誌名 : 岩手医科大学先端医療研究 センター公開シンポジウム「バイオイメージングと分子生物学による脳・血管解明ー健康増進に向けた最先端研究
  • 最新のバイオイメージング技術
    期間 : 2008年01月
    役割 : 講師
    主催者・発行元 : (ホテルモントレエーデルホフ，札幌)
    イベント・番組・新聞雑誌名 : 北海道バイオ産業クラスター・フォーラム，シーズン公開会
  • 近未来的バイオイメージングの展望
    期間 : 2006年06月
    役割 : 講師
    主催者・発行元 : 特定領域研究２領域合同公開シンポジウム，細胞内のダイナミ ックネットワーク：前進するオルガネラ研究，千里ライフサイエンスセンター,大阪

メディア報道

  • 報道 : 2023年10月07日
    番組・新聞雑誌 : 毎日新聞
    読む写真　新聞・雑誌
  • 報道 : 2023年10月06日
    番組・新聞雑誌 : 中日新聞
    　新聞・雑誌
  • 報道 : 2023年10月03日
    発行元・放送局 : テレビ大阪
    番組・新聞雑誌 : やさしいニュース
    　テレビ・ラジオ番組
  • "自ら光る"樹木 ～いずれは電力を使わないイルミネーションに～
    報道 : 2023年09月26日
    発行元・放送局 : 関西テレビ
    番組・新聞雑誌 : newsランナー
    　テレビ・ラジオ番組
  • 電気を使わず明るく照らせ ～「光る樹木」実用化への挑戦～
    報道 : 2023年09月26日
    発行元・放送局 : 朝日放送
    番組・新聞雑誌 : newsおかえり
    　テレビ・ラジオ番組
  • 未来のタネ～光る植物で街を照らせ！～おはよう関西
    報道 : 2023年06月13日
    発行元・放送局 : NHK関西
    番組・新聞雑誌 : おはよう関西
    　テレビ・ラジオ番組
  • 発光技術が地球の未来照らす
    報道 : 2023年03月29日
    番組・新聞雑誌 : 産経新聞 2p
    　新聞・雑誌
  • 【びっくりサイエンス 】オワンクラゲから始まった生物発光技術の未来
    報道 : 2023年03月04日
    番組・新聞雑誌 : 産経新聞
    　インターネットメディア
  • 暗闇に浮かぶ光の花
    報道 : 2022年06月23日
    番組・新聞雑誌 : 読売新聞
    5p　新聞・雑誌
  • 光る植物、電源不要の照明にー遺伝子を改変、ポプラなどに応用へ
    報道 : 2022年02月04日
    番組・新聞雑誌 : 日経産業新聞
    　新聞・雑誌
  • 凝固能の評価はスマートフォンで簡単に!?
    報道 : 2021年12月06日
    発行元・放送局 : 日経メディカル
    　インターネットメディア
  • 阪大, スマホで血栓症酵素が測定できる指示薬開発
    報道 : 2021年12月03日
    発行元・放送局 : OPTRONICS
    　インターネットメディア
  • 血液凝固活性判定にスマホ利用 血栓予防に期待
    報道 : 2021年11月26日
    番組・新聞雑誌 : 科学新聞
    8p　新聞・雑誌
  • スマートフォンを利用した 血液凝固活性の判定法
    報道 : 2021年10月20日
    番組・新聞雑誌 : Research at Osaka University
    　新聞・雑誌
  • 令和3年度 第21回 山崎貞一賞 2分野4名の受賞者を決定
    報道 : 2021年09月28日
    番組・新聞雑誌 : 財形新聞
    　インターネットメディア
  • 阪大が光イメージング装置アマテラス開発
    報道 : 2021年09月03日
    番組・新聞雑誌 : 科学新聞
    4p　新聞・雑誌
  • 細胞の動態 同時観察
    報道 : 2021年08月24日
    番組・新聞雑誌 : 日刊工業新聞
    24p　新聞・雑誌
  • 黄疸の判定、スマホで撮るだけ生物発光を利用した指示薬を開発
    報道 : 2021年04月
    番組・新聞雑誌 : JST news
    　新聞・雑誌
  • 細胞光らせ生体観察
    報道 : 2021年03月03日
    番組・新聞雑誌 : 京都新聞(夕刊) その他全国の地方新聞に掲載
    2p　新聞・雑誌
  • 光る生体細胞 詳細に観察
    報道 : 2021年02月22日
    番組・新聞雑誌 : 信濃毎新聞
    7p　新聞・雑誌
  • 光で可視化 細胞を観察
    報道 : 2021年02月21日
    番組・新聞雑誌 : 中国新聞
    11p　新聞・雑誌
  • 細胞光らせ体内解析
    報道 : 2021年02月20日
    番組・新聞雑誌 : 河北新報
    14p　新聞・雑誌
  • 細胞光らせて生体観察
    報道 : 2021年02月20日
    番組・新聞雑誌 : 神戸新聞
    15p　新聞・雑誌
  • 細胞光らせ体内観察
    報道 : 2021年02月20日
    番組・新聞雑誌 : 北海道新聞(夕刊)
    2p　新聞・雑誌
  • 細胞光らせ生体 可視化
    報道 : 2021年02月19日
    番組・新聞雑誌 : 熊本新聞
    13p　新聞・雑誌
  • 細胞光らせ活動観察
    報道 : 2021年02月14日
    番組・新聞雑誌 : 高知新聞
    10p　新聞・雑誌
  • 「光るたんぱく質」研究で未来を照らす
    報道 : 2020年12月17日
    発行元・放送局 : テレビ東京
    番組・新聞雑誌 : 探究の階段
    　テレビ・ラジオ番組
  • 日本顕微鏡学会論文賞
    報道 : 2020年06月12日
    番組・新聞雑誌 : 科学新聞
    5p　新聞・雑誌
  • ノーベル賞研究引き継ぐ世界的権威
    報道 : 2020年03月11日
    発行元・放送局 : フジテレビ
    番組・新聞雑誌 : ホンマでっか！？TV
  • 傷ついたリソソームを修復する新たなメカニズムを発見
    報道 : 2020年
    番組・新聞雑誌 : Research at Osaka University
    　新聞・雑誌
  • 酸性下でも蛍光する緑色蛍光タンパク質「rsGamillus」が切り開く新領域とは?
    報道 : 2019年08月26日
    番組・新聞雑誌 : 財形新聞 他
    　インターネットメディア
  • 阪大, 酸性でも光刺激でon・offできる緑色蛍光タンパク質 rsGamillusを開発
    報道 : 2019年08月20日
    番組・新聞雑誌 : 日本経済新聞
    　インターネットメディア
  • 酸性でも光刺激でオン・オフできる緑色蛍光たんぱく質rsGamillus～生体内の酸性環境を高解像度で観察する新技術～
    報道 : 2019年08月20日
    番組・新聞雑誌 : BtoBプラットフォーム 業界チャネル
    　インターネットメディア
  • DNA最前線 大学や研究所を飛び出したDNA発見「肉味のトマトや輝く街路樹 スターの皮膚再生しバッグに」
    報道 : 2019年07月29日
    発行元・放送局 : 朝新聞出版
    番組・新聞雑誌 : AERA, No.35
    　新聞・雑誌
  • ステーキ味のトマト, 輝く街頭樹も…進化するDNA研究の最前線
    報道 : 2019年07月29日
    発行元・放送局 : NTTドコモ（docomo）他
    　インターネットメディア
  • 无线脑成像技术侵入性较小，在神经科学中具有广阔前景
    報道 : 2019年06月14日
    番組・新聞雑誌 : 中国光学期刊网
    　インターネットメディア
  • 憂楽帳：特異点生物学
    報道 : 2019年06月11日
    番組・新聞雑誌 : 毎日新聞
    　新聞・雑誌
  • Cable-Free Brain Imaging Technique Proves Less Invasive, Shows Promise In Neuroscience
    報道 : 2019年06月06日
    発行元・放送局 : Photonics.com
    　インターネットメディア
  • 大阪・関西万博は未来社会への実験場, 「万博の跡地を利用してオープンイノベーションを推進し，永続的なエコシステムを構築」
    報道 : 2019年06月
    発行元・放送局 : 伊藤忠商事株式会社
    番組・新聞雑誌 : 繊維日報, vol.710
  • New cable-free brain imaging method may take social neuroscience to the next level
    報道 : 2019年05月30日
    発行元・放送局 : Laboratory Equipment
    　インターネットメディア
  • New cable-free brain imaging method may take social neuroscience to the next level
    報道 : 2019年05月29日
    発行元・放送局 : ScienceDaily
    　インターネットメディア
  • Measuring brain activity associated with social behavior in mice
    報道 : 2019年05月28日
    発行元・放送局 : Medical Xpress
    　インターネットメディア
  • マウスの脳活動、複数匹を同時観察
    報道 : 2019年05月20日
    番組・新聞雑誌 : 日本経済新聞
    　新聞・雑誌
  • 生物発光で複数マウスの脳活動を同時にライブ観察～社会性行動をつかさどる脳機能や精神疾患研究分野での新たな展開に期待～
    報道 : 2019年05月17日
    発行元・放送局 : BtoBプラットフォーム 業界チャネル
    　インターネットメディア
  • 阪大など、複数の動物脳を無線計測 神経細胞の電気信号可視化
    報道 : 2019年05月17日
    番組・新聞雑誌 : 日刊工業新聞A
    　インターネットメディア
  • 阪大・東北大・理研・JST、「生物発光膜電位センサー」を用いた脳活動ライブ計測法を開発
    報道 : 2019年05月17日
    番組・新聞雑誌 : 日本経済新聞
    　インターネットメディア
  • 生物発光で複数マウスの脳活動を同時にライブ観察できるワイヤレス計測法開発－阪大ら
    報道 : 2019年05月15日
    番組・新聞雑誌 : 医療ニュース
    　インターネットメディア
  • 「光るタンパク質」で医療やエネルギー問題に貢献 未来社会を大きく変革する
    報道 : 2019年
    番組・新聞雑誌 : Osaka University brochure
    　新聞・雑誌
  • かがくアゴラ 細胞と個体 同時に詳細観察
    報道 : 2018年12月14日
    番組・新聞雑誌 : 日本経済新聞
    27p　新聞・雑誌
  • 新イメージング装置 阪大が開発へ 小動物全体の観察目指す
    報道 : 2018年11月01日
    番組・新聞雑誌 : 電子デバイス産業新聞
    　新聞・雑誌
  • 大阪科学賞に京大・白石教授ら
    報道 : 2018年09月21日
    番組・新聞雑誌 : 日経産業新聞
    　新聞・雑誌
  • 大阪科学賞に2教授
    報道 : 2018年09月13日
    番組・新聞雑誌 : 読売新聞
    35p　新聞・雑誌
  • 大阪科学賞に白石・永井両氏
    報道 : 2018年09月13日
    番組・新聞雑誌 : 朝日新聞
    29p　新聞・雑誌
  • 白石・永井両教授を選出 大阪科学賞
    報道 : 2018年09月13日
    番組・新聞雑誌 : 日本経済新聞
    　インターネットメディア
  • 京大の白石誠司教授と大阪大の永井健治教授に
    報道 : 2018年09月13日
    番組・新聞雑誌 : 毎日新聞
    26p　新聞・雑誌
  • 大阪科学賞に京大と阪大の教授
    報道 : 2018年09月12日
    番組・新聞雑誌 : NHK NEWS ニュースほっと関西
    　テレビ・ラジオ番組
  • 知られざるクラゲの世界 ～不老不死からノーベル賞まで～
    報道 : 2018年08月12日
    番組・新聞雑誌 : ガリレオX
    　テレビ・ラジオ番組
  • 輝く街路樹作り「生物発光が拓く生命科学と未来社会」
    報道 : 2018年06月10日
    番組・新聞雑誌 : 読売新聞
    　新聞・雑誌
  • 神経活動の抑制を抑える新規カルシウムセンサー開発
    報道 : 2018年05月18日
    番組・新聞雑誌 : 科学新聞
    2p　新聞・雑誌
  • 少数性生物学からアプローチする生命システム
    報道 : 2018年04月25日
    番組・新聞雑誌 : 情報誌NEXT45
    　新聞・雑誌
  • 耐酸性緑色蛍光タンパク質 ハナガサクラゲから開発
    報道 : 2018年02月02日
    番組・新聞雑誌 : 科学新聞
    6p
  • 大阪大学、JST、本産ハナガサクラゲより開発！耐酸性緑色蛍光タンパク質Gamillus
    報道 : 2018年01月04日
    発行元・放送局 : 日経バイオテクONLINE
    　インターネットメディア
  • 阪大など、ハナガサクラゲの触手から耐酸性で緑色の蛍光タンパク質Gamillusを開発
    報道 : 2018年01月04日
    番組・新聞雑誌 : 日本経済新聞 電子版
    　インターネットメディア
  • 酸性に強い緑色蛍光たんぱく質 阪大など開発 ハナガサクラゲから
    報道 : 2017年12月29日
    番組・新聞雑誌 : 朝日新聞
    　新聞・雑誌
  • 「IUPAB」日本開催決定 生理物理学のトップ研究者集結
    報道 : 2017年08月25日
    番組・新聞雑誌 : 科学新聞
    1p　新聞・雑誌
  • 御堂筋に輝く街路樹 発光たんぱく質で社会貢献を
    報道 : 2017年06月27日
    番組・新聞雑誌 : 毎日新聞
    　新聞・雑誌
  • 化学発光たんぱく質 改良 色彩5色で明るく
    報道 : 2016年12月20日
    番組・新聞雑誌 : 日刊工業新聞
    24p　新聞・雑誌
  • iPS心筋 エビの光で観察
    報道 : 2016年12月18日
    番組・新聞雑誌 : 読売新聞
    14p　新聞・雑誌
  • 細胞を10倍明るく光らせる
    報道 : 2016年12月15日
    番組・新聞雑誌 : NHK 関西 NEWS WEB
    　テレビ・ラジオ番組
  • 阪大の永井健治教授ら、明るい5色の発光蛋白質で細胞内の微細構造を同時計測 「阪大の専売特許」の1分子検出も、「木を見て森も見れるドローンの威力」
    報道 : 2016年12月15日
    発行元・放送局 : 日経バイオテクONLINE
    　インターネットメディア
  • 鮮やか５色発光タンパク質を開発 大阪大, ips研究にも
    報道 : 2016年12月14日
    番組・新聞雑誌 : 共同通信47NEWS
    　インターネットメディア
  • 発光たんぱく、５色で明るく＝多種類の観察可能に－阪大
    報道 : 2016年12月14日
    番組・新聞雑誌 : 時事ドットコム
    　インターネットメディア
  • 虹色に光るたんぱく質 阪大、街灯やがん研究に
    報道 : 2016年09月20日
    番組・新聞雑誌 : 日本経済新聞
    　インターネットメディア
  • 「未来の公園part3 華やかな公園の島」
    報道 : 2016年08月22日
    発行元・放送局 : テレビ東京
    番組・新聞雑誌 : 未来シティ研究所 #97
    　テレビ・ラジオ番組
  • 医療からエネルギー問題まで 幅広い分野に変革をもたらす「光るタンパク質」
    報道 : 2016年07月14日
    番組・新聞雑誌 : 蛍雪時代
    　新聞・雑誌
  • 「光るタンパク質」で医療やエネルギー問題に貢献 未来社会を大きく変革する
    報道 : 2016年07月14日
    番組・新聞雑誌 : 蛍雪時代
    　新聞・雑誌
  • 発光生物から、光る街路樹を作る
    報道 : 2016年03月
    番組・新聞雑誌 : at home 教授対談シリーズ
    　新聞・雑誌
  • 生体に優しい超解像イメージング
    報道 : 2016年02月01日
    番組・新聞雑誌 : 日経バイオテク
    　インターネットメディア
  • 本発の最新植物テクノロジー
    報道 : 2015年09月10日
    発行元・放送局 : 朝日放送
    番組・新聞雑誌 : ビーバップハイヒール
  • 10億分の1, ナノの世界を肉眼で 阪大、蛍光タンパク質の反応を高速化 超解像顕微鏡技術
    報道 : 2015年05月05日
    番組・新聞雑誌 : 産経WEST
    　インターネットメディア
  • 光スイッチング高速化 阪大蛍光たんぱく質開発
    報道 : 2015年04月21日
    番組・新聞雑誌 : 日刊工業新聞
    　新聞・雑誌
  • 光る植物 観賞用に
    報道 : 2015年04月20日
    番組・新聞雑誌 : 日経産業新聞
    11p　新聞・雑誌
  • 発光タンパク質に新色
    報道 : 2015年03月30日
    番組・新聞雑誌 : 産経新聞
    12p　新聞・雑誌
  • 肉眼でも観察可能 20倍光るタンパク質
    報道 : 2015年03月27日
    番組・新聞雑誌 : 科学新聞
    1p　新聞・雑誌
  • 光るたんぱく質開発
    報道 : 2015年03月24日
    番組・新聞雑誌 : 関西 NEWS WEB
    　テレビ・ラジオ番組
  • 阪大・理研 高光度発光たんぱく質 明るさ20倍に
    報道 : 2015年03月24日
    番組・新聞雑誌 : 日刊工業新聞
    21p　新聞・雑誌
  • 青・オレンジ発色たんぱく質開発
    報道 : 2015年03月24日
    番組・新聞雑誌 : 日本経済新聞
    16p　新聞・雑誌
  • 青緑・黄緑・オレンジに光るたんぱく質開発
    報道 : 2015年03月24日
    番組・新聞雑誌 : 毎日新聞
    31p
  • 光るたんぱく質 明るさ20倍
    報道 : 2015年03月24日
    番組・新聞雑誌 : 産経新聞
    30p　新聞・雑誌
  • 肉眼で見える「発光たんぱく質」＝再生医療研究への応用期待-阪大・理研
    報道 : 2015年03月24日
    番組・新聞雑誌 : 時事ドットコム
    　インターネットメディア
  • 究極のエコ 光る街路樹
    報道 : 2015年03月23日
    番組・新聞雑誌 : 読売新聞
    28p　新聞・雑誌
  • 明るく光るコケを開発
    報道 : 2014年12月02日
    番組・新聞雑誌 : 日経新聞
    16p　新聞・雑誌
  • 新しい化学発光タンパク質開発
    報道 : 2014年01月01日
    番組・新聞雑誌 : 科学新聞
    6p　新聞・雑誌
  • 光当て活性酸素 がん細胞を攻撃
    報道 : 2013年10月16日
    番組・新聞雑誌 : 日経産業新聞
    7p　新聞・雑誌
  • 少数分子の反応が生物を支配する？
    報道 : 2013年03月
    番組・新聞雑誌 : Nature ダイジェストVol. 10 No. 3
    　新聞・雑誌
  • 10倍で発光 タンパク質 阪大など開発 微小がん検出に道
    報道 : 2013年02月10日
    番組・新聞雑誌 : 日本経済新聞
    30p　新聞・雑誌
  • 体を傷つけずにがん観察 -光るたんぱく質組み合わせ-
    報道 : 2013年01月05日
    番組・新聞雑誌 : 毎日新聞
    6p　新聞・雑誌
  • Nano-lantern lights the way
    報道 : 2013年
    番組・新聞雑誌 : Nature Methods
    10, 104　新聞・雑誌
  • Brighter than ever
    報道 : 2013年
    番組・新聞雑誌 : Nature Photonics
    7, 166
  • 大阪大学、JST、動き回る小動物体内の組織や生理機能を高感度に検出可能な超高輝度発光タンパク質の開発に成功超早期癌の診断法確立に期待
    報道 : 2012年12月14日
    発行元・放送局 : 日経バイオテク
    　インターネットメディア
  • 阪大、輝度10倍の化学発光たんぱく質開発-励起光照射が不要
    報道 : 2012年12月14日
    番組・新聞雑誌 : 日刊工業新聞
    25p
  • がん細胞を肉眼で、光るたんぱく質開発-阪大
    報道 : 2012年12月14日
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    11p　新聞・雑誌
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    番組・新聞雑誌 : 西日本新聞
    31p　新聞・雑誌
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    報道 : 2010年08月10日
    番組・新聞雑誌 : 北海道新聞
    27p　新聞・雑誌
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    番組・新聞雑誌 : 化学工業日報
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    番組・新聞雑誌 : 読売新聞
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