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Papers - Hotta, Kohji

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CIPRO 2.5: Ciona intestinalis Protein Database, a unique integrated repository of large-scale omics data, bioinformatic analyses, and curated annotation, with user rating and reviewing functionality

Endo, Toshinori; Ueno, Keisuke; Yonezawa, Kouki; Mineta, Katsuhiko; Hotta, Kohji; Satou, Yutaka; Yamada, Lixy; Ogasawara, Michio; Takahashi, Hiroki.; Nakajima, Ayako; Nakachi, Mia; Nomura, Mamoru; Yaguchi, Junko; Sasakura , Yasunori; Yamasaki, Chisato; Sera, Miho; Yoshizawa, Akiyasu; Imanishi, Tadashi; Taniguchi, Hisaaki; Inaba, Kazuo

Nucleic Acid Research 39 ( 1 ) 807-814 2011.01

Research paper (scientific journal), Joint Work, Accepted

Newly developed Mg ²⁺-selective fluorescent probe enables visualization of Mg ²⁺ dynamics in mitochondria

Shindo Yutaka, Fujii Tomohiko, Komatsu Hirokazu, Citterio Daniel, Hotta Kohji, Suzuki Koji, Oka Kotaro

PLoS One 6 ( 8 ) 2011

Mg ²⁺ plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg ²⁺ regulation and the Mg ²⁺ concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg ²⁺ in mitochondria in intact cells. Here, we have developed a novel Mg ²⁺-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg ²⁺ concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg ²⁺, KMG-104, enabled us to compare the dynamics of Mg ²⁺ in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg ²⁺ mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg ²⁺ concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg ²⁺ and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP ⁺). A gradual decrease in the Mg ²⁺ concentration in mitochondria was observed in response to MPP ⁺ in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg ²⁺ dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)). © 2011 Shindo et al.

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Peptidomic analysis of the central nervous system of the protochordate, Ciona intestinalis: homologs and prototypes of vertebrate peptides and novel peptides

Tsuyoshi Kawada, Michio Ogasawara, Toshio Sekiguchi, Masato Aoyama, Kohji Hotta, Kotaro Oka, Honoo Satake

Endocrinology Epub ( ahead ) of print 2011

Research paper (scientific journal), Joint Work, Accepted

Newly developed Mg2+-selective fluorescent probe enables visualization of Mg2+ dynamics in mitochondria.

Shindo Yutaka, Fujii Tomohiko, Komatsu Hirokazu, Citterio Daniel, Hotta Kohji, Suzuki Koji, Oka Kotaro

PLoS One 6 ( 8 ) 2011

Mg(2+) plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg(2+) regulation and the Mg(2+) concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg(2+) in mitochondria in intact cells. Here, we have developed a novel Mg(2+)-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg(2+) concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg(2+), KMG-104, enabled us to compare the dynamics of Mg(2+) in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg(2+) mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg(2+) concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg(2+) and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP(+)). A gradual decrease in the Mg(2+) concentration in mitochondria was observed in response to MPP(+) in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg(2+) dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).

Chromatophore activity during natural pattern expression by the squid Sepioteuthis lessoniana

Suzuki Mamiko, Kimura Tetsuya, Ogawa Hiroto, Hotta Kohji, Oka Kotaro

PLoS One 6 ( 4 ) 2011

Squid can rapidly change the chromatic patterns on their body. The patterns are created by the expansion and retraction of chromatophores. The chromatophore consists of a central pigment-containing cell surrounded by radial muscles that are controlled by motor neurons located in the central nervous system (CNS). In this study we used semi-intact squid (Sepioteuthis lessoniana) displaying centrally controlled natural patterns to analyze spatial and temporal activities of chromatophores located on the dorsal mantle skin. We found that chromatophores oscillated with miniature expansions/retractions at various frequencies, even when the chromatic patterns appear macroscopically stable. The frequencies of this miniature oscillation differed between "feature" and "background" areas of chromatic patterns. Higher frequencies occurred in feature areas, whereas lower frequencies were detected in background areas. We also observed synchronization of the oscillation during chromatic pattern expression. The expansion size of chromatophores oscillating at high frequency correlated with the number of synchronized chromatophores but not the oscillation frequency. Miniature oscillations were not observed in denervated chromatophores. These results suggest that miniature oscillations of chromatophores are driven by motor neuronal activities in the CNS and that frequency and synchrony of this oscillation determine the chromatic pattern and the expansion size, respectively. © 2011 Suzuki et al.

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ACBD: Database for Ascidian Chemical Genomics.

Hira, Yuichiro, Terai, Jun, Imoto, Masaya, Tashiro, Etsu, and Hotta, Kohji.

Nature Precedings - ( - ) - 2010.12

Research paper (scientific journal), Joint Work

Glutamate-induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons

Shindo Yutaka, Fujimoto Ai, Hotta Kohji, Suzuki Koji, Oka Kotaro

Journal of Neuroscience Research 88 ( 14 ) 3125 - 3132 2010.11

ISSN 0360-4012

Excess administration of glutamate is known to induce Ca²⁺ overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg²⁺ in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg²⁺ in the excitotoxicity, we simultaneously measured intracellular Ca²⁺ and Mg²⁺, using fluorescent dyes, Fura red, a fluorescent Ca²⁺ probe, and KMG-104, a highly selective fluorescent Mg²⁺ probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg²⁺ concentration ([Mg²⁺]_i). Extracellular Mg²⁺ was not required for this glutamate-induced increase in [Mg²⁺]_i, and no increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) or [Mg²⁺]_i was observed in neurons in nominally Ca²⁺-free medium. Application of 5 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca²⁺]_i and [Mg²⁺]_i. Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg²⁺]_i but did induce an additive increase in [Ca²⁺]_i. Moreover, the glutamate-induced increase in [Mg²⁺]_i was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg²⁺ efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, prevented this [Mg²⁺]_i increase. These results indicate that glutamate-induced increases in [Mg²⁺]_i result from the Mg²⁺ release from mitochondria and that Ca²⁺ accumulation in the mitochondria is required for this Mg²⁺ release. © 2010 Wiley-Liss, Inc.

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Glutamate-Induced Calcium Increase Mediates Magnesium Release from Mitochondria in Rat Hippocampal Neurons

Yutaka Shindo, Ai Fujimoto, Kohji Hotta, Koji Suzuki and Kotaro Oka

Journal of Neuroscience Research. 88 ( 14 ) 3125-32 2010.11

Research paper (scientific journal), Joint Work, Accepted

The ANISEED database

Tassy Olivier, Dauga Delphine, Daian Fabrice, Sobral Daniel, Robin François, Khoueiry Pierre, Salgado David, Fox Vanessa, Caillol Danièle, Schiappa Renaud, Laporte Baptiste, Rios Anne, Luxardi Guillaume, Kusakabe Takehiro, Joly Jean Stéphane, Darras Sébastien, Christiaen Lionel, Contensin Magali, Auger Hélène, Lamy Clément, Hudson Clare, Rothbächer Ute, Gilchrist Michael J., Makabe Kazuhiro W., Hotta Kohji, Fujiwara Shigeki, Satoh Nori, Satou Yutaka, Lemaire Patrick

Genome Research 20 ( 10 ) 1459 - 1468 2010.10

ISSN 1088-9051

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions. © 2010 by Cold Spring Harbor Laboratory Press.

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The ANISEED database: digital representation, formalisation and elucidation of a chordate developmental program

O. Tassy, D. Dauga, F. Daian, D. Sobral, F. Robin, P. Khoueiry, D. Salgado, V. Fox, D. Caillo, M. Contensin, Renaud Schiappa, Anne Rios, Guillaume Luxardi, M. Gilchrist, K. Makabe, K. Hotta, Fujiwara, T. Kusakabe, N. Satoh, Y. Satou and P. Lemaire

Genome Research. 20 ( 10 ) 1459-68 2010.10

Research paper (scientific journal), Single Work, Accepted

Network structure of projections extending from peripheral neurons in tunic of ascidian larva

Hiroshi Q.Terakubo, Yoko Nakajima, Yasunori Sasakura, Takeo Horie, Alu Konno, Hiroki Takahashi, Kazuo Inaba, Kohji Hotta and Kotaro Oka

Developmental Dynamics. 239 ( 8 ) 2278-87 2010.08

Research paper (scientific journal), Single Work, Accepted

Network structure of projections extending from peripheral neurons in the tunic of ascidian larva

Terakubo Hiroshi Q., Nakajima Yoko, Sasakura Yasunori, Horie Takeo, Konno Alu, Takahashi Hiroki, Inaba Kazuo, Hotta Kohji, Oka Kotaro

Developmental Dynamics 239 ( 8 ) 2278 - 2287 2010.08

ISSN 1058-8388

In ascidian Ciona intestinalis, a subset of trunk epidermal neurons were shown to possess external network of neural projections. To characterize a more complete network in naturally hatched (chorionated) larvae, we visualized the structure with a confocal laser scanning microscope. High resolution images revealed the huge network consisting of several subnetworks in whole-larval tunic. We named this network the ASNET (ascidian dendritic network in tunic). The ASNET was dynamically generated and collapsed during larval stages. Interestingly, one of the subnetworks found around apical trunk epidermal neurons was bilaterally asymmetric. In caudal epidermal neurons, transmission electron microscopy revealed that 9+2 axonemes were accompanied by a vesicle-containing mass in the ASNET arbor, but the distal end of the arbor contained only the vesicle-containing fibrous mass and no 9+2 axonemes. The characteristics of the ASNET suggest that it forms a unique outer body network in the ascidian larval tunic. © 2010 Wiley-Liss, Inc.

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Blue fluorescent cGMP sensor for multiparameter fluorescence imaging

Niino Yusuke, Hotta Kohji, Oka Kotaro

PLoS One 5 ( 2 ) 2010.02

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca ²⁺. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca²⁺ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca ²⁺, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks. © 2010 Niino et al.

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Regulation of notochord-specific expression of Ci-Bra downstream genes in Ciona intestinalis embryos

Takahashi Hiroki, Hotta Kohji, Takagi Chiyo, Ueno Naoto, Satoh Nori, Shoguchi Eiichi

Zoological Science 27 ( 2 ) 110 - 118 2010.02

ISSN 0289-0003

Brachyury, a T-box transcription factor, is expressed in ascidian embryos exclusively in primordial notochord cells and plays a pivotal role in differentiation of notochord cells. Previously, we identified ∼450 genes downstream of Ciona intestinalis Brachyury (Ci-Bra), and characterized the expression profiles of 45 of these in differentiating notochord cells. In this study, we looked for cisregulatory sequences in minimal enhancers of 20 Ci-Bra downstream genes by electroporating region within ∼3 kb upstream of each gene fused with lacZ. Eight of the 20 reporters were expressed in notochord cells. The minimal enchancer for each of these eight genes was narrowed to a region ∼0.51.0-kb long. We also explored the genome-wide and coordinate regulation of 43 Ci-Bra-downstream genes. When we determined their chromosomal localization, it became evident that they are not clustered in a given region of the genome, but rather distributed evenly over 13 of the 14 pairs of chromosomes, suggesting that gene clustering does not contribute to coordinate control of the Ci-Bra downstream gene expression. Our results might provide Insights Into the molecular mechanisms underlying notochord formation in chordates. © 2010 Zoological Society of Japan.

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Regulation of notochord-specific expression of Ci-Bra downstream genes in Ciona intestinalis embryos

Hiroki Takahashi1, Kohji Hotta, Chiyo Takagi, Naoto Ueno, Nori Satoh, Eiichi Shoguchi

Zoological Sci., 27 ( 2 ) 110-118 2010.02

Research paper (scientific journal), Joint Work, Accepted

Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

Yusuke Niino, Kohji Hotta and Kotaro Oka

PLoS One. 5 ( 2 ) e9164 2010.02

Research paper (scientific journal), Single Work, Accepted

Distribution and structural diversity of cilia in tadpole larvae of the ascidian Ciona intestinalis

Konno Alu, Kaizu Maiko, Hotta Kohji, Horie Takeo, Sasakura Yasunori, Ikeo Kazuho, Inaba Kazuo

Developmental Biology 337 ( 1 ) 42 - 62 2010.01

ISSN 0012-1606

Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9 + 0 structure similar to vertebrate primary cilia, a 9 + 0 structure with electron-dense materials in the center, a 9 + 2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan. © 2009 Elsevier Inc. All rights reserved.

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Distribution and structural diversity of cilia in the tadpole larvae of the ascidian Ciona intestinalis

Alu Konno, Maiko Kaizu, Kohji Hotta, Takeo Horie, Yasunori Sasakura, Kazuho Ikeo and Kazuo Inaba

Developmental Biology 1 ( 337 ) 42-62 2010.01

Research paper (scientific journal), Joint Work, Accepted

FMRFamide elicits chromatophore expansion and retraction depending on its type and development in the squid, Sepioteuthis lessoniana

Suzuki Mamiko, Kimura Tetsuya, Ogawa Hiroto, Hotta Kohji, Oka Kotaro

Invertebrate Neuroscience 9 ( 3-4 ) 185 - 193 2009.12

ISSN 1354-2516

To examine chromatophore control by FMRFamide-related peptide (FaRP), we investigated the pharmacological effect of FMRFamide on the chromatophores and the FMRFamide-immunoreactivity of nerves surrounding the muscles in the coastal squid, Sepioteuthis lessoniana. Applications of FMRFamide elicited expansion of black chromatophores and retraction of yellow chromatophores in the adult squid. FMRFamide-immunoreactive terminals were distributed along black chromatophore muscles but were not observed around the yellow ones. This means that FMRFamide functions differently for each of the two types of chromatophores in the adult squid. Moreover, the pharmacological effect of FMRFamide on the black chromatophores differed between adults and hatchlings; application of FMRFamide retracted black chromatophores in hatchlings but not in adults. These results indicate that certain squid species have an FaRP system for controlling the chromatophores in their skin and that the system changes during development. © 2010 Springer-Verlag.

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Simultaneous live cell imaging using dual FRET sensors with a single excitation light

Niino Yusuke, Hotta Kohji, Oka Kotaro

PLoS One 4 ( 6 ) 2009.06

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca²⁺ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility. © 2009 Niino et al.

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