-
[show abstract]
[hide abstract]
ABSTRACT: The application of a new rhodamine-based fluorescent probe, RH-NH(2) and an acyl transfer promoter, PBu(3), to Hela cells induced a time-dependent increase in fluorescence in the mitochondria, which was most likely due to acetylation of RH-NH(2) with activated acetyl-CoA by the artificial reaction promoter in living cells.
Chemical Communications 02/2013; · 6.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Although some interneurons in C. elegans have been shown to show unusual region-specific Ca2+ dynamics, the region-specific Ca2+ and membrane potential response properties of these neurons are largely unknown due to technical limitations. In this report, we focused on one of these neurons, AIY interneuron, where Ca2+ dynamics have been detected only in neurites, and not the soma, during odor and temperature stimulation to determine whether membrane potential and Ca2+ are region-specific dynamics and distinct from one another. To visualize voltage change both in the soma and neurites of AIY, we used voltage-sensitive fluorescent protein (VSFP) 2.42. First, we confirmed that the sensor protein worked correctly in C. elegans by depolarizing AIY interneuron with high concentrations of KCl. Next, we observed membrane potential depolarization during odor (isoamyl alcohol) stimulation in both neurites and the soma. Additionally, depolarization of membrane potential with direct application of high KCl induced a Ca2+ increase in the soma. From these results, we conclude that membrane potential behavior and Ca2+ dynamics in AIY differ in its subcellular regions and that VSFP2.42 can be a useful tool for studying information processing in single neurons.
Neuroscience Letters 02/2013; · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: During embryogenesis, chordates pass through a tailbud stage in which the larval tail is formed. Since acquisition of a tadpole-like tail during tailbud stage is one of the key events in the evolution of chordates, understanding the anatomy of the tailbud stage chordate embryo is of special interest. In this study, to understand comprehensively the anatomy of the tailbud embryo at single-cell-level, real microscopic image stacks of the tailbud embryo in Ciona intestinalis were reconstructed into a 3D computer model. This comprehensive 3D model of the ascidian tailbud embryo was based on real images of confocal laser scanning microscope (CLSM) and therefore, cell shape, location and cell arrangement reflect real geometries of the tailbud embryo. We found that the tailbud embryo consists of 1579 cells, including 836 epidermal cells, 228 cells in the central nervous system, 218 mesenchymal cells, four trunk ventral cells, two B/B(⁎)8.11 cells, 36 muscle cells, 40 notochord cells, four primordial germ cells, and 199 endodermal cells. Moreover, we identified for the first time two populations of previously undefined cells (a total of 12 cells) in Ciona: one located in the lateral trunk and the other located under the tail dorsal epidermis. This information provides a first step for understanding how the body plan of the chordate tailbud embryo formed and evolved.
Developmental Biology 09/2012; · 4.07 Impact Factor
-
Noriko Hiroi,
Michael Klann,
Keisuke Iba,
Pablo de Heras Ciechomski,
Shuji Yamashita,
Akito Tabira,
Takahiro Okuhara,
Takeshi Kubojima,
Yasunori Okada, Kotaro Oka,
Robin Mange,
Michael Unger,
Akira Funahashi,
Heinz Koeppl
[show abstract]
[hide abstract]
ABSTRACT: In our previous study, we introduced a combination methodology of Fluorescence Correlation Spectroscopy (FCS) and Transmission Electron Microscopy (TEM), which is powerful to investigate the effect of intracellular environment to biochemical reaction processes. Now, we developed a reconstruction method of realistic simulation spaces based on our TEM images. Interactive raytracing visualization of this space allows the perception of the overall 3D structure, which is not directly accessible from 2D TEM images. Simulation results show that the diffusion in such generated structures strongly depends on image post-processing. Frayed structures corresponding to noisy images hinder the diffusion much stronger than smooth surfaces from denoised images. This means that the correct identification of noise or structure is significant to reconstruct appropriate reaction environment in silico in order to estimate realistic behaviors of reactants in vivo. Static structures lead to anomalous diffusion due to the partial confinement. In contrast, mobile crowding agents do not lead to anomalous diffusion at moderate crowding levels. By varying the mobility of these non-reactive obstacles (NRO), we estimated the relationship between NRO diffusion coefficient (Dnro) and the anomaly in the tracer diffusion (α). For Dnro=21.96 to 44.49 μm2/s, the simulation results match the anomaly obtained from FCS measurements. This range of the diffusion coefficient from simulations is compatible with the range of the diffusion coefficient of structural proteins in the cytoplasm. In addition, we investigated the relationship between the radius of NRO and anomalous diffusion coefficient of tracers by the comparison between different simulations. The radius of NRO has to be 58 nm when the polymer moves with the same diffusion speed as a reactant, which is close to the radius of functional protein complexes in a cell.
EURASIP Journal on Bioinformatics and Systems Biology 06/2012; 2012(1):7.
-
Chemistry - An Asian Journal 09/2011; 6(9):2312-5. · 4.50 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report a novel near-infrared fluorescent calcium probe (KFCA), which has good optical properties such as intense NIR fluorescence emission (670 nm, QY: 0.24), excellent ON/OFF ratio (120-fold), and good wavelength-compatibility with visible-light-emissive fluorophores (Fluo-4, DsRed2), and which is applicable for real-time dual-colour intracellular Ca(2+) imaging.
Chemical Communications 08/2011; 47(37):10407-9. · 6.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The phylogenetic position of ascidians as the chordate invertebrates closest to vertebrates suggests that they might possess homologs and/or prototypes of vertebrate peptide hormones and neuropeptides as well as ascidian-specific peptides. However, only a small number of peptides have so far been identified in ascidians. In the present study, we have identified various peptides in the ascidian, Ciona intestinalis. Mass spectrometry-based peptidomic analysis detected 33 peptides, including 26 novel peptides, from C. intestinalis. The ascidian peptides are largely classified into three categories: 1) prototypes and homologs of vertebrate peptides, such as galanin/galanin-like peptide, which have never been identified in any invertebrates; 2) peptides partially homologous with vertebrate peptides, including novel neurotesin-like peptides; 3) novel peptides. These results not only provide evidence that C. intestinalis possesses various homologs and prototypes of vertebrate neuropeptides and peptide hormones but also suggest that several of these peptides might have diverged in the ascidian-specific evolutionary lineage. All Ciona peptide genes were expressed in the neural complex, whereas several peptide gene transcripts were also distributed in peripheral tissues, including the ovary. Furthermore, a Ciona neurotensin-like peptide, C. intestinalis neurotensin-like peptide 6, was shown to down-regulate growth of Ciona vitellogenic oocytes. These results suggest that the Ciona peptides act not only as neuropeptides in the neural tissue but also as hormones in nonneuronal tissues and that ascidians, unlike other invertebrates, such as nematodes, insects, and sea urchins, established an evolutionary origin of the peptidergic neuroendocrine, endocrine, and nervous systems of vertebrates with certain specific molecular diversity.
Endocrinology 04/2011; 152(6):2416-27. · 4.46 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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)).
PLoS ONE 01/2011; 6(8):e23684. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
PLoS ONE 01/2011; 6(4):e18244. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In vivo environments are highly crowded and inhomogeneous, which may affect reaction processes in cells. In this study we examined the effects of intracellular crowding and an inhomogeneity on the behavior of in vivo reactions by calculating the spectral dimension (d(s)), which can be translated into the reaction rate function. We compared estimates of anomaly parameters obtained from fluorescence correlation spectroscopy (FCS) data with fractal dimensions derived from transmission electron microscopy (TEM) image analysis. FCS analysis indicated that the anomalous property was linked to physiological structure. Subsequent TEM analysis provided an in vivo illustration; soluble molecules likely percolate between intracellular clusters, which are constructed in a self-organizing manner. We estimated a cytoplasmic spectral dimension d(s) to be 1.39 ± 0.084. This result suggests that in vivo reactions initially run faster than the same reactions in a homogeneous space; this conclusion is consistent with the anomalous character indicated by FCS analysis. We further showed that these results were compatible with our Monte-Carlo simulation in which the anomalous behavior of mobile molecules correlates with the intracellular environment, leading to description as a percolation cluster, as demonstrated using TEM analysis. We confirmed by the simulation that the above-mentioned in vivo like properties are different from those of homogeneously concentrated environments. Additionally, simulation results indicated that crowding level of an environment might affect diffusion rate of reactant. Such knowledge of the spatial information enables us to construct realistic models for in vivo diffusion and reaction systems.
Frontiers in physiology. 01/2011; 2:50.
-
[show abstract]
[hide abstract]
ABSTRACT: Mg<sup>2+</sup> plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg<sup>2+</sup> regulation and the Mg<sup>2+</sup> concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg<sup>2+</sup> in mitochondria in intact cells. Here, we have developed a novel Mg<sup>2+</sup>–selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg<sup>2+</sup> concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg<sup>2+</sup>, KMG-104, enabled us to compare the dynamics of Mg<sup>2+</sup> in the cytosol and in mitochondria. With this method, carbonyl cyanide p -(trifluoromethoxy) phenylhydrazone (FCCP)–induced Mg<sup>2+</sup> mobilization from mitochondria to the cytosol was visualized. Although a FCCP–induced decrease in the Mg<sup>2+</sup> 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<sup>2+</sup> and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP<sup>+</sup>). A gradual decrease in the Mg<sup>2+</sup> concentration in mitochondria was observed in response to MPP<sup>+</sup> in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg<sup>2+</sup> 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)).
PLoS ONE 01/2011; 6(8):e23684. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Excess administration of glutamate is known to induce Ca(2+) overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg(2+) in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg(2+) in the excitotoxicity, we simultaneously measured intracellular Ca(2+) and Mg(2+), using fluorescent dyes, Fura red, a fluorescent Ca(2+) probe, and KMG-104, a highly selective fluorescent Mg(2+) 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 Mg(2+) concentration ([Mg(2+)](i)). Extracellular Mg(2+) was not required for this glutamate-induced increase in [Mg(2+)](i), and no increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) or [Mg(2+)](i) was observed in neurons in nominally Ca(2+)-free medium. Application of 5 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca(2+)](i) and [Mg(2+)](i). Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg(2+)](i) but did induce an additive increase in [Ca(2+)](i). Moreover, the glutamate-induced increase in [Mg(2+)](i) was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg(2+) efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca(2+) uniporter, prevented this [Mg(2+)](i) increase. These results indicate that glutamate-induced increases in [Mg(2+)](i) result from the Mg(2+) release from mitochondria and that Ca(2+) accumulation in the mitochondria is required for this Mg(2+) release.
Journal of Neuroscience Research 11/2010; 88(14):3125-32. · 2.74 Impact Factor
-
Developmental Biology 08/2010; 344(1):529. · 4.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Developmental Dynamics 08/2010; 239(8):2278-87. · 2.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Invertebrate Neuroscience 04/2010; 9(3-4):185-93. · 1.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 Ca(2+) 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.
PLoS ONE 01/2010; 5(2):e9164. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mismatch negativity (MMN) is a negative component of event-related brain potentials elicited by stimulus transitions. Stimulus duration transition also elicits MMN (duration MMN), with a magnitude that is related to the degree of duration change and the discrimination ability. The neural substrates of duration MMN have not yet been investigated. We therefore studied how duration transitions in an auditory stimulus train are represented in neurons in the primary auditory cortex of anesthetized guinea pigs. Two types of neuronal responses to the context of changes in stimulus duration were found. One was a reduced response as the duration of the preceding stimulus was increased. Second was an enhancement of the late components of the response including sustained and offset responses at the duration transition. The former may be explained by the previously proposed two-tone suppression, which is dependent on the preceding stimulus duration. The latter is likely to be caused by stimulus-specific adaptation that could be a possible neural generator of duration MMN.
Hearing research 10/2009; 259(1-2):107-16. · 2.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report a method for specifically labelling the surface of cells with two kinds of chemical probes (near-infrared (NIR) fluorescent probes and magnetic resonance (MR) imaging probes) via two genetically expressed tags, and demonstrate the application for in vitro and in vivo dual imaging.
Chemical Communications 08/2009; · 6.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 Ca(2+) 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.
PLoS ONE 02/2009; 4(6):e6036. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Despite the accumulating research on noncoding RNAs (ncRNAs), it is likely that we are seeing only the tip of the iceberg regarding our understanding of the functions and the regulatory roles served by ncRNAs in cellular metabolism, pathogenesis and host-pathogen interactions. Therefore, more powerful computational and experimental tools for analyzing ncRNAs need to be developed. To this end, we propose novel kernel functions, called base-pairing profile local alignment (BPLA) kernels, for analyzing functional ncRNA sequences using support vector machines (SVMs). We extend the local alignment kernels for amino acid sequences in order to handle RNA sequences by using STRAL's; scoring function, which takes into account sequence similarities as well as upstream and downstream base-pairing probabilities, thus enabling us to model secondary structures of RNA sequences. As a test of the performance of BPLA kernels, we applied our kernels to the problem of discriminating members of an RNA family from nonmembers using SVMs. The results indicated that the discrimination ability of our kernels is stronger than that of other existing methods. Furthermore, we demonstrated the applicability of our kernels to the problem of genome-wide search of snoRNA families in the Caenorhabditis elegans genome, and confirmed that the expression is valid in 14 out of 48 of our predicted candidates by using qRT-PCR. Finally, highly expressed six candidates were identified as the original target regions by DNA sequencing.
Nucleic Acids Research 02/2009; 37(3):999-1009. · 8.03 Impact Factor
