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ABSTRACT: RNA interference (RNAi) using small interfering (siRNA) or short hairpin RNA (shRNA) has become the first choice for gene silencing maneuver in mammalian cells. Because different siRNAs of the same gene have variable silencing efficacy and only limited siRNAs are functional, many candidates are necessary to identify optimal siRNAs. We have previously reported a method named enzymatic production of RNAi library (EPRIL), by which a great variety of shRNA expression constructs (RNAi library) can be produced simultaneously from cDNAs of interest. Recently, we have improved this method and developed a more efficient method. We describe in this chapter detailed protocols for the improved version of EPRIL and high-throughput selection of effective shRNA expression constructs from an RNAi library.
Methods in molecular biology (Clifton, N.J.) 01/2011; 729:123-39.
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Neuroscience Research - NEUROSCI RES. 01/2011; 71.
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ABSTRACT: The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a-induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF-silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a-induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a-induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a- and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.
Molecular biology of the cell 10/2010; 21(20):3590-600. · 5.98 Impact Factor
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Masato Iwabu,
Toshimasa Yamauchi,
Miki Okada-Iwabu,
Koji Sato,
Tatsuro Nakagawa,
Masaaki Funata,
Mamiko Yamaguchi,
Shigeyuki Namiki,
Ryo Nakayama,
Mitsuhisa Tabata, [......],
Masashi Fukayama,
Ichizo Nishino,
Kumpei Tokuyama,
Kohjiro Ueki,
Yuichi Oike,
Satoshi Ishii, Kenzo Hirose,
Takao Shimizu,
Kazushige Touhara,
Takashi Kadowaki
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ABSTRACT: Adiponectin is an anti-diabetic adipokine. Its receptors possess a
seven-transmembrane topology with the amino terminus located
intracellularly, which is the opposite of G-protein-coupled receptors.
Here we provide evidence that adiponectin induces extracellular
Ca2+ influx by adiponectin receptor 1 (AdipoR1), which was
necessary for subsequent activation of
Ca2+/calmodulin-dependent protein kinase kinase β
(CaMKKβ), AMPK and SIRT1, increased expression and decreased
acetylation of peroxisome proliferator-activated receptor γ
coactivator-1α (PGC-1α), and increased mitochondria in
myocytes. Moreover, muscle-specific disruption of AdipoR1 suppressed the
adiponectin-mediated increase in intracellular Ca2+
concentration, and decreased the activation of CaMKK, AMPK and SIRT1 by
adiponectin. Suppression of AdipoR1 also resulted in decreased
PGC-1α expression and deacetylation, decreased mitochondrial
content and enzymes, decreased oxidative type I myofibres, and decreased
oxidative stress-detoxifying enzymes in skeletal muscle, which were
associated with insulin resistance and decreased exercise endurance.
Decreased levels of adiponectin and AdipoR1 in obesity may have causal
roles in mitochondrial dysfunction and insulin resistance seen in
diabetes.
Nature 03/2010; 464:1313-1319. · 36.28 Impact Factor
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ABSTRACT: Glutamate is the major neurotransmitter in the brain, mediating point-to-point transmission across the synaptic cleft in excitatory synapses. Using a glutamate imaging method with fluorescent indicators, we show that synaptic activity generates extrasynaptic glutamate dynamics in the vicinity of active synapses. These glutamate dynamics had magnitudes and durations sufficient to activate extrasynaptic glutamate receptors in brain slices. We also observed crosstalk between synapses--i.e., summation of glutamate released from neighboring synapses. Furthermore, we successfully observed that sensory input from the extremities induced extrasynaptic glutamate dynamics within the appropriate sensory area of the cerebral cortex in vivo. Thus, the present study clarifies the spatiotemporal features of extrasynaptic glutamate dynamics, and opens up an avenue to directly visualizing synaptic activity in live animals.
Proceedings of the National Academy of Sciences 03/2010; 107(14):6526-31. · 9.68 Impact Factor
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Masato Iwabu,
Toshimasa Yamauchi,
Miki Okada-Iwabu,
Koji Sato,
Tatsuro Nakagawa,
Masaaki Funata,
Mamiko Yamaguchi,
Shigeyuki Namiki,
Ryo Nakayama,
Mitsuhisa Tabata, [......],
Masashi Fukayama,
Ichizo Nishino,
Kumpei Tokuyama,
Kohjiro Ueki,
Yuichi Oike,
Satoshi Ishii, Kenzo Hirose,
Takao Shimizu,
Kazushige Touhara,
Takashi Kadowaki
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ABSTRACT: Adiponectin is an anti-diabetic adipokine. Its receptors possess a seven-transmembrane topology with the amino terminus located intracellularly, which is the opposite of G-protein-coupled receptors. Here we provide evidence that adiponectin induces extracellular Ca(2+) influx by adiponectin receptor 1 (AdipoR1), which was necessary for subsequent activation of Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta), AMPK and SIRT1, increased expression and decreased acetylation of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), and increased mitochondria in myocytes. Moreover, muscle-specific disruption of AdipoR1 suppressed the adiponectin-mediated increase in intracellular Ca(2+) concentration, and decreased the activation of CaMKK, AMPK and SIRT1 by adiponectin. Suppression of AdipoR1 also resulted in decreased PGC-1alpha expression and deacetylation, decreased mitochondrial content and enzymes, decreased oxidative type I myofibres, and decreased oxidative stress-detoxifying enzymes in skeletal muscle, which were associated with insulin resistance and decreased exercise endurance. Decreased levels of adiponectin and AdipoR1 in obesity may have causal roles in mitochondrial dysfunction and insulin resistance seen in diabetes.
Nature 03/2010; 464(7293):1313-9. · 36.28 Impact Factor
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ABSTRACT: FROUNT is a known CCR2-binding protein that facilitates monocyte/macrophage infiltration. Here we report that FROUNT also binds to the C-terminal region of CCR5 and enhances CCR5-mediated cellular chemotaxis. We show that FROUNT overexpression enhances the directionality of chemotaxis, while FROUNT suppression results in impaired responsiveness. Furthermore, we found an increase in consolidated pseudopodium formation in FROUNT-overexpressing cells (FNT cells) on uniform stimulation with CCL4 (MIP1-beta), a specific ligand of CCR5. In most FNT cells, one to two pseudopodia directed toward higher chemokine concentration were found, whereas most FNT-suppressed cells had multiple pseudopodia. The data indicate that FROUNT is involved in sensing and amplifying a shallow extracellular chemokine gradient that leads to a limited number of accurate pseudopodia directed toward the chemokine concentration. In addition to its separate roles in CCR2- and CCR5-mediated chemotaxis, FROUNT, as a common regulator of these receptors, possibly plays a crucial role in the recruitment of immune cells expressing these receptors.
The Journal of Immunology 11/2009; 183(10):6387-94. · 5.79 Impact Factor
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ABSTRACT: Platelet-derived growth factor (PDGF) signaling controls various physiological functions via two receptor subtypes: PDGF receptor (PDGFR) alpha and PDGFRbeta. Nevertheless, our understanding of their roles is limited because of a lack of pharmacological tools to discriminate subtype-specific signaling. We developed a chimeric receptor by combining ligand-binding-domain truncated PDGFRbeta with anti-fluorescein single chain antibody, expecting the control of PDGFRbeta-specific signaling by oligomerized fluorescein as an artificial agonist. Results show that calcium mobilization, Cdc42 activation, and cell migration were elicited specifically by the artificial ligand in cells expressing the chimeric receptor. Our method is expected to be useful to understand the subtype-specific roles of PDGFRs in various cellular functions.
Journal of Pharmacological Sciences 10/2009; 111(3):312-6. · 2.08 Impact Factor
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ABSTRACT: The terrestrial slug Limax has the ability to learn odor associations. This ability depends on the function of the procerebrum, the secondary olfactory center in the brain. Among the various neurotransmitters that are thought to be involved in the function of the procerebrum, glutamate is one of the most important molecules. However, the existence and function of glutamate in this system have been proposed solely on the basis of a few lines of indirect evidence from pharmacological experiments. In the present study, we demonstrated the existence and release of glutamate as a neurotransmitter in the procerebrum of Limax, by using three different techniques: 1) immunohistochemistry of glutamate, 2) in situ hybridization to mRNA of the vesicular glutamate transporter, and 3) real-time imaging of glutamate release within the procerebrum using the glutamate optical sensor EOS2. The release of glutamate within the cell mass layer of the procerebrum was synchronized with oscillation of the local field potential and had the same physiological properties as this oscillation; both were blocked by a serotonin antagonist and were propagated in an apical to basal direction in the procerebrum. Our observations suggest strongly that the oscillation of the local field potential is driven by the glutamate released by bursting neurons in the procerebrum.
Journal of Neuroscience Research 06/2009; 87(13):3011-23. · 2.74 Impact Factor
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ABSTRACT: Chromophore-assisted light inactivation is a promising technique to inactivate selected proteins with high spatial and temporal resolution in living cells, but its use has been limited because of the lack of a methodology to prevent nonspecific photodamage in the cell owing to reactive oxygen species generated by the photosensitizer. Here we present a design strategy for photosensitizers with an environment-sensitive off/on switch for singlet oxygen ((1)O(2)) generation, which is switched on by binding to the target, to improve the specificity of protein photoinactivation. (1)O(2) generation in the unbound state is quenched by photoinduced electron transfer, whereas (1)O(2) generation can occur in the hydrophobic environment provided by the target protein, after specific binding. Inositol 1,4,5-trisphosphate receptor, which has been suggested to have a hydrophobic pocket around the ligand binding site, was specifically inactivated by an environment-sensitive photosensitizer-conjugated inositol 1,4,5-trisphosphate receptor ligand without (1)O(2) generation in the cytosol of the target cells, despite light illumination, demonstrating the potential of environment-sensitive photosensitizers to allow high-resolution control of generation of reactive oxygen species in the cell.
Proceedings of the National Academy of Sciences 02/2008; 105(1):28-32. · 9.68 Impact Factor
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ABSTRACT: Astrocytes play a pivotal role in the regulation of neurite growth, but the intracellular signaling mechanism in astrocytes that mediates this regulation remains unclarified. We studied the relationship between spontaneous Ca(2+) oscillations in astrocytes and the astrocyte-mediated neurite growth. We generated Ca(2+) signal-deficient astrocytes in which spontaneous Ca(2+) oscillations were abolished by a chronic inhibition of IP(3) signaling. When hippocampal neurons were cultured on a monolayer of Ca(2+) signal-deficient astrocytes, the growth of dendrites and axons was inhibited. Time-lapse imaging of the advancement of axonal growth cones indicated the involvement of membrane-bound molecules for this inhibition. Among six candidate membrane-bound molecules that may modulate neuronal growth, N-cadherin was downregulated in Ca(2+) signal-deficient astrocytes. Although a blocking antibody to N-cadherin suppressed the axonal growth on control astrocytes, extrinsic N-cadherin expression rescued the suppressed axonal growth on Ca(2+) signal-deficient astrocytes. These findings suggest that spontaneous Ca(2+) oscillations regulate the astrocytic function to promote neurite growth by maintaining the expression of specific growth-enhancing proteins on their surface, and that N-cadherin is one of such molecules.
Journal of Neuroscience 09/2007; 27(33):8957-66. · 7.11 Impact Factor
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ABSTRACT: Imaging neurotransmission is expected to greatly improve our understanding of the mechanisms and regulations of synaptic transmission. Aiming at imaging glutamate, a major excitatory neurotransmitter in the CNS, we developed a novel optical glutamate probe, which consists of a ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor glutamate receptor GluR2 subunit and a small molecule fluorescent dye. We expected that such fluorescent conjugates might report the microenvironmental changes upon protein conformational changes elicited by glutamate binding. After more than 100 conjugates were tested, we finally obtained a conjugate named E (glutamate) optical sensor (EOS), which showed maximally 37% change in fluorescence intensity upon binding of glutamate with a dissociation constant of 148 nm. By immobilizing EOS on the cell surface of hippocampal neuronal culture preparations, we pursued in situ spatial mapping of synaptically released glutamate following presynaptic firing. Results showed that a single firing was sufficient to obtain high-resolution images of glutamate release, indicating the remarkable sensitivity of this technique. Furthermore, we monitored the time course of changes in presynaptic activity induced by phorbol ester and found heterogeneity in presynaptic modulation. These results indicate that EOS can be generally applicable to evaluation of presynaptic modulation and plasticity. This EOS-based glutamate imaging method is useful to address numerous fundamental issues about glutamatergic neurotransmission in the CNS.
European Journal of Neuroscience 05/2007; 25(8):2249-59. · 3.63 Impact Factor
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Neuroscience Research - NEUROSCI RES. 01/2007; 58.
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ABSTRACT: Cells communicate with each other to form organized structures by cell-cell adhesion and cell-cell repulsion, but it remains to be clarified how cell-cell contact information is converted into intracellular signals. Here, we show that cells in contact with neighbouring cells generate local transient intracellular Ca(2+) signals (Ca(2+) lightning). Ca(2+) lightning was observed near cell-cell contact regions and was not observed in the central regions of cells or in solitary cells that were not in contact with other cells. We also show that Ca(2+) lightning is able to regulate cell-cell repulsion by means of PYK2, a Ca(2+)-activated protein tyrosine kinase, which induces focal adhesion disassembly in a Ca(2+)-dependent manner. These results show that cell-cell contact information might be transmitted by Ca(2+) lightning to regulate intracellular events.
EMBO Reports 12/2006; 7(11):1117-23. · 7.36 Impact Factor
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ABSTRACT: Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.
EMBO Reports 05/2006; 7(4):390-6. · 7.36 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 09/2005; 50(10 Suppl):1388-94.
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ABSTRACT: Nitric oxide (NO) is an intercellular messenger regulating neuronal functions. To visualize NO signalling in the brain, we generated a novel fluorescent NO indicator, which consists of the heme-binding region (HBR) of soluble guanylyl cyclase and the green fluorescent protein. The indicator (HBR-GFP) was expressed in the Purkinje cells of the mouse cerebellum and we imaged NO signals in acute cerebellar slices upon parallel fibre (PF) activation with a train of burst stimulations (BS, each BS consisting of five pulses at 50 Hz). Our results showed that the intensity of synaptic NO signal decays steeply with the distance from the synaptic input near PF-Purkinje cell synapses and generates synapse-specific long-term potentiation (LTP). Furthermore, the NO release level has a bell-shaped dependence on the frequency of PF activity. At an optimal frequency (1 Hz), but not at a low frequency (0.25 Hz) of a train of 60 BS, NO release as well as LTP was induced. However, both NO release and LTP were significantly reduced at higher frequencies (2-4 Hz) of BS train due to cannabinoid receptor-mediated retrograde inhibition of NO generation at the PF terminals. These results suggest that synaptic NO signalling decodes the frequency of neuronal activity to mediate synaptic plasticity at the PF-Purkinje cell synapse.
The Journal of Physiology 09/2005; 566(Pt 3):849-63. · 4.72 Impact Factor
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ABSTRACT: BAD, a member of the Bcl-2 protein family, promotes mitochondria-dependent apoptosis. Here, we report that BAD dissociates from 14-3-3zeta at each G2/M phase of proliferating lymphoid cells. The cell cycle-dependent dissociation of BAD was associated with phosphorylation at Ser-128, whereas mutant S128A-BAD, in which Ser-128 was converted to alanine, remained associated with 14-3-3zeta throughout the cell cycle. Although the cell cycle-dependent dissociation of BAD per se did not induce apoptosis, growth factor deprivation induced prompt apoptosis at the G2/M phase but not at the G1 phase. In cells expressing S128A-BAD, growth factor deprivation-induced apoptosis was markedly delayed and was accompanied by a delayed dephosphorylation of growth factor-dependent regulatory serine residues. These results indicate that BAD induces apoptosis upon detecting the coincidence of G2/M phase and growth factor deprivation.
Journal of Biological Chemistry 08/2005; 280(28):26225-32. · 4.77 Impact Factor
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ABSTRACT: Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for beta-galactosidase, which is the most widely used reporter enzyme.
Journal of the American Chemical Society 05/2005; 127(13):4888-94. · 9.91 Impact Factor
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ABSTRACT: Phosphorylation of the RMLC (regulatory myosin light chain) regulates the activity of myosin II, which is critically involved in the motility of both muscle and non-muscle cells. There are both Ca2+-dependent and -independent pathways for RMLC phosphorylation in smooth-muscle cells, and the latter pathway is often involved in an abnormal contractility in pathological states such as asthma and hypertension. Therefore pharmacological interventions of RMLC phosphorylation may have a therapeutic value. In the present study, we developed a new genetically encoded biosensor, termed CRCit (ECFP-RMLC-Citrine, where ECFP is enhanced cyan fluorescent protein), that detects RMLC phosphorylation using fluorescence resonance energy transfer between two variants of the green fluorescent protein fused to both the N- and C-termini of RMLC. When expressed in primary cultured vascular smooth-muscle cells, CRCit detected the Ca2+-dependent RMLC phosphorylation with a high spatiotemporal resolution. Furthermore, we could specifically assay the agonist-induced Ca2+-independent phosphorylation of RMLC when Ca2+ signalling in cells expressing CRCit was suppressed. Thus CRCit may also be used for the high throughput screening of compounds that inhibit abnormal smooth-muscle contraction.
Biochemical Journal 02/2005; 385(Pt 2):589-94. · 4.90 Impact Factor
