論文

Mg2+ is an essential cation to maintain cellular functions, and intracellular Mg2+ concentration ([Mg2+]i) is regulated by Mg2+ channels and transporters. In our previous study, we demonstrated that MPP+ elicits Mg2+ influx across the cell membrane and Mg2+ mobilization from mitochondria, and the resulting [Mg2+]i is an important determinants of the cell viability in MPP+ model of Parkinson's disease (PD). It indicates that cellular Mg2+ transport is one of the important factors to determine the progress of PD. However, whether the expression levels of Mg2+ transport proteins change in the progress of PD has still been obscure. In this study, we estimated the mRNA expression levels of Mg2+ transport proteins upon the exposure to MPP+. In thirteen Mg2+ transport proteins examined, mRNA expression level of SLC41A2 was increased and that of ACDP2, NIPA1 and MMgT2 were decreased. Knockdown of SLC41A2, ACDP2 or NIPA1 accelerated the MPP+-induced cell degeneration, and overexpression attenuated it. The decrease in the mRNA expression levels of NIPA1 and MMgT2 were also elicited by rotenone, H2O2 and FCCP, indicating that mitochondrial dysfunction related to this down-regulation. The increase in that of SLC41A2 was induced by an uncoupler, FCCP, as well as MPP+, suggesting that it is an intrinsic protection mechanism against depolarized mitochondrial membrane potential and/or cellular ATP depletion. Our results shown here indicate that alteration of Mg2+ transport proteins is implicated in the MPP+ model of PD, and it affects cell degeneration.

Cellular energy production processes are composed of many Mg2+ dependent enzymatic reactions. In fact, dysregulation of Mg2+ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg2+ stores. Several biological stimuli alter mitochondrial Mg2+ concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg2+ alteration affect cellular energy metabolism remains unclear. Mg2+ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg2+ uptake system. Here, we comprehensively analyzed intracellular Mg2+ levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg2+ homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg2+ via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg2+ level in response to physiological stimuli.

Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells.

To investigate comprehensive synaptic connectivity, we examined Ca2+ responses with quantitative electric current stimulation by indium-tin-oxide (ITO) glass electrode with transparent and high electro-conductivity. The number of neurons with Ca2+ responses was low during the application of stepwise increase of electric current in short-term cultured neurons (less than 17 days in-vitro (DIV)). The neurons cultured over 17 DIV showed two-type responses: S-shaped (sigmoid) and monotonous saturated responses, and Scatchard plots well illustrated the difference of these two responses. Furthermore, sigmoid like neural network responses over 17 DIV were altered to the monotonous saturated ones by the application of the mixture of AP5 and CNQX, specific blockers of NMDA and AMPA receptors, respectively. This alternation was also characterized by the change of Hill coefficients. These findings indicate that the neural network with sigmoid-like responses has strong synergetic or cooperative synaptic connectivity via excitatory glutamate synapses.

Homeostasis of magnesium ion (Mg2+) plays key roles in healthy neuronal functions, and deficiency of Mg2+ is involved in various neuronal diseases. In neurons, we have reported that excitotoxicity induced by excitatory neurotransmitter glutamate increases intracellular Mg2+ concentration ([Mg2+]i). However, it has not been revealed whether neuronal activity under physiological condition modulates [Mg2+]i. The aim of this study is to explore the direct relationship between neural activity and [Mg2+]i dynamics. In rat primary-dissociated hippocampal neurons, the [Mg2+]i and [Ca2+]i dynamics were simultaneously visualized with a highly selective fluorescent Mg2+ probe, KMG-104, and a fluorescent Ca2+ probe, Fura Red, respectively. [Mg2+]i increase concomitant with neural activity by direct current stimulation was observed in neurons plated on an indium-tin oxide (ITO) glass electrode, which enables fluorescent imaging during neural stimulation. The neural activity-dependent [Mg2+]i increase was also detected in neurons whose excitability was enhanced by the treatment of a voltage-gated K+ channel blocker, tetraethylammonium (TEA) at the timings of spontaneous Ca2+ increase. Furthermore, the [Mg2+]i increase was abolished in Mg2+-free extracellular medium, indicating [Mg2+]i increase is due to Mg2+ influx induced by neural activity. The direct neuronal depolarization by veratridine, a Na+ channel opener, induced [Mg2+]i increase, and this [Mg2+]i increase was suppressed by the pretreatment of a non-specific Mg2+ channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Overall, activity-dependent [Mg2+]i increase results from Mg2+ influx through 2-APB-sensitive channels in rat hippocampal neurons.

Parkinson's disease (PD) is a neurodegenerative disorder resulting from mitochondrial dysfunction in dopaminergic neurons. Mitochondria are believed to be responsible for cellular Mg2+ homeostasis. Mg2+ is indispensable for maintaining ordinal cellular functions, hence perturbation of the cellular Mg2+ homeostasis may be responsible for the disorders of physiological functions and diseases including PD. However, the changes in intracellular Mg2+ concentration ([Mg2+]i) and the role of Mg2+ in PD have still been obscure. In this study, we investigated [Mg2+]i and its effect on neurodegeneration in the 1-methyl-4-phenylpyridinium (MPP+) model of PD in differentiated PC12 cells. Application of MPP+ induced an increase in [Mg2+]i immediately via two different pathways: Mg2+ release from mitochondria and Mg2+ influx across cell membrane, and the increased [Mg2+]i sustained for more than 16h after MPP+ application. Suppression of Mg2+ influx decreased the viability of the cells exposed to MPP+. The cell viability correlated highly with [Mg2+]i. In the PC12 cells with suppressed Mg2+ influx, ATP concentration decreased and the amount of reactive oxygen species (ROS) increased after an 8h exposure to MPP+. Our results indicate that the increase in [Mg2+]i inhibited cellular ROS generation and maintained ATP production, which resulted in the protection from MPP+ toxicity.

The quantitative relationship between change in cell shape and ATP consumption is an unsolved problem in cell biology. In this study, a simultaneous imaging and image processing analysis allowed us to observe and quantify these relationships under physiological conditions, for the first time. We focused on two marginal regions of cells: the microtubule-rich € lamella € and the actin-rich € peripheral structure €. Simultaneous imaging and correlation analysis revealed that microtubule dynamics cause lamellar shape change accompanying an increase in ATP level. Also, image processing and spatiotemporal quantification enabled to visualize a chronological change of the relationships between the protrusion length and ATP levels, and it suggested they are influencing each other. Furthermore, inhibition of microtubule dynamics diminished motility in the peripheral structure and the range of fluctuation of ATP level in the lamella. This work clearly demonstrates that cellular motility and morphology are regulated by ATP-related cooperative function between microtubule and actin dynamics.

Background: Indium-tin-oxide (ITO) glass electrodes possess the properties of optical transparency and high electrical conductivity, which enables the electrical stimulation of cultured cells to be performed whilst also measuring the responses with fluorescent imaging techniques. However, the quantitative relationship between the intensity of the stimulating current and the cell response is unclear when using conventional methods that employ a separated configuration of counter and stimulation electrodes. New method: A quantitative electrical current stimulation device without the use of a counter electrode was fabricated. Results: Nerve growth factor (NGF)-induced differentiated PC12 cells were cultured on an ITO single glass electrode, and the Ca2+ response to electrical stimuli was measured using fluorescent Ca2+ imaging. ITO electrode devices with a width less than 0.1mm were found to evoke a Ca2+ response in the PC12 cells. Subsequent variation in the length of the device in the range of 2-10mm was found to have little influence on the efficiency of the electric stimulus. We found that the stimulation of the cells was dependent on the electrical current, when greater than 60μA, rather than on the Joule heat, regardless of the width and length of the conductive area. Comparison with existing method(s): Because of the cells directly in contact with the electrode, our device enables to stimulate the cells specifically, comparing with previous devices with the counter electrode. Conclusions: The ITO device without the use of a counter electrode is a useful tool for evaluating the quantitative neural excitability of cultured neurons.

In vivo toxicity evaluation using model organisms is an important step for the development of new drugs. Here, we report that Ciona intestinalis, a chordate invertebrate, is beneficial to drug toxicity evaluation for the following reasons: rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, and low risk of ethical issues. The dynamic phenotypic change of Ciona larvae during metamorphosis prompted us to examine the effect of cytotoxic drugs on its development by quantifying six toxicity endpoints: degenerated tail size, ampulla length, rotation of body axis, stomach size, heart rate, and body size. As a result, mitochondrial respiratory inhibitors, tubulin polymerization/depolymerization inhibitors, or DNA/RNA synthesis inhibitors showed distinct toxicity profiles against these six endpoints, but drugs with the same targets showed a similar toxicity profile in Ciona. Our results suggest Ciona is an effective animal model for profiling drug toxicity and exploring the mechanisms of drugs with unknown targets.

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.

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.

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS. © 2009 Elsevier Inc. All rights reserved.

Background: The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly. Results: We have prepared a less-fragmented assembly on the basis of scaffold-joining guided by paired-end EST and bacterial artificial chromosome (BAC) sequences, and BAC chromosomal in situ hybridization data. The new assembly (115.2 Mb) is similar in length to the initial assembly (116.7 Mb) but contains 1,272 (approximately 50%) fewer scaffolds. The largest scaffold in the new assembly incorporates 95 initial-assembly scaffolds. In conjunction with the new assembly, we have prepared a greatly improved global gene model set strictly correlated with the extensive currently available EST data. The total gene number (15,254) is similar to that of the initial set (15,582), but the new set includes 3,330 models at genomic sites where none were present in the initial set, and 1,779 models that represent fusions of multiple previously incomplete models. In approximately half, 5′-ends were precisely mapped using 5′-full-length ESTs, an important refinement even in otherwise unchanged models. Conclusion: Using these new resources, we identify a population of non-canonical (non-GT-AG) introns and also find that approximately 20% of Ciona genes reside in operons and that operons contain a high proportion of single-exon genes. Thus, the present dataset provides an opportunity to analyze the Ciona genome much more precisely than ever. © 2008 Satou et al.; licensee BioMed Central Ltd.

In physiological and pathological events, extracellular ATP plays an important role by controlling several types of purinergic receptors and changing cytoskeleton dynamics. To know the process of ATP-dependent cytoskeleton remodeling, we focused on cofilin, a key regulator of actin cytoskeleton, and investigated the dynamics of cofilin in PC12 cells through fluorescent protein-labeled cofilin and actin, Ca2+ imaging, and fluorescence resonance energy transfer (FRET) techniques. As a result, ATP induced intracellular Ca2+ increase, following cofilin rods' formation. ATP-induced cofilin rods' formation was not observed in cells expressing unphosphorylatable variant of cofilin. A P2X receptor agonist, but not P2Y, induced the formation of cofilin rods, whereas calmodulin and calcineurin inhibitors suppressed it. These results indicate that Ca2+ influx through P2X receptors induces the formation of cofilin rods via calcineurin-dependent dephosphorylation of cofilin. This pathway might be one candidate to explain the effects of ATP on neuronal development and injury. © 2007 Elsevier Inc. All rights reserved.

In vertebrates, Brachyury, a T-box transcription factor gene, seems to have a dual role in the differentiation of axial midline mesoderm cells into notochord and gastrulation cell movements regulated by non-canonical Wnt/planar cell polarity (Wnt/PCP) signaling. To understand the function of Brachyury-downstream genes in chordate embryos, from a series of our survey on differential expression, including subtractive hybridization, dot-blot assays, EST sequences and the expression patterns in whole-mount in situ hybridization at embryonic stages, we developed a knowledge database called "CINOBI: Ciona Notochord and Brachyury-downstream gene Index" to create comprehensive catalogues of Brachyury-downstream gene sets in Ciona intestinalis. Combining genome and large-scale cDNA data, we were able to characterize 450 non-redundant Brachyury-downstream genes: Twenty-four genes were newly annotated as notochord-expressed genes. Several genes are components of signaling pathways such as Wnt/PCP, NfκB and TGF-β signaling. We propose that Brachyury is linked to these pathways regulating the expression of each component, and such a regulatory mechanism might be conserved among chordates. © 2008 The Author(s).

Formation of the chordate body is accomplished by a complex set of morphogenetic movements including convergent extension of notochord cells. In the ascidian Ciona intestinalis, Brachyury plays a key role in the formation of the notochord, and more than 30 Bra-downstream notochord genes have been identified. In the present study, we examined the effects of functional suppression of nine Bra-downstream notochord genes, which include Ci-PTP, Ci-ACL, Ci-prickle, Ci-netrin, Ci-trop, Ci-Noto3, Ci-ASAK, Ci-ERM and Ci-pellino. When the function of the first two genes (Ci-PTP and Ci-ACL) was suppressed with specific morpholinos, the notochord cells failed to converge, while functional suppression of Ci-prickle resulted in a failure of intercalation, and therefore the cells in these three types of embryo remained in the mid-dorsal region of the embryo. Functional suppression of the next four genes (Ci-netrin, Ci-trop, Ci-Noto3 and Ci-ASAK) resulted in the partial defect of intercalation, and the notochord did not consist of a single row. In addition, when the function of the last two genes (Ci-ERM and Ci-pellino) was suppressed, notochord cells failed to elongate in the embryo, even though convergence/extension took place normally. These results indicate that many Bra-downstream notochord genes are involved in convergence/extension of the embryo. © 2007 The Authors.

Background: Ascidian larval epidermal sensory neurons (ESNs) each extend a single cilium into the outer body structure, or tunic, to form a unique external sensory network, the ASNET (ascidian dendritic network in tunic). The functions of ESNs and the ASNET are unknown, though it has been suggested that they play important roles in swimming larvae. In this study, we used transgenic larva expressing the photoconvertible protein Kaede pan-neuronally to identify the orientation and morphology of single ESN external processes within the ASNET. Results: When individual ESN cell bodies were UV-irradiated, the photoconverted Kaede protein diffused into the external processes that projected to the edge of the tunic, indicating that these processes are cytoplasmically connected to the cell bodies of ESNs. We were, therefore, able to comprehensively catalog the morphology and orientation of each ESN external process. Most trunk ESNs appeared to extend neurites to the palp, but no processes were observed to emanate from palp neurons. ESN processes differed systematically in their morphology and orientation, suggesting that the ASNET is formed in a regulated, non-random fashion. Conclusions: This study reveals the organization of ESN sensory fields in the ascidian larval tunic.