Kenzo Hirose

The University of Tokyo, 白山, Tōkyō, Japan

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Publications (73)505.47 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We developed a technique for spatiotemporally precise control of downstream signaling of receptor tyrosine kinase by ultraviolet light irradiation of caged fluorescein-based receptor ligands in cells expressing an antifluorescein antibody-receptor tyrosine kinase chimera. Light-driven ligand uncaging specifically elicited receptor phosphorylation and calcium mobilization in irradiated cells. This technique may be useful for detailed studies of the physiological roles and dynamics of receptor tyrosine kinase signaling.
    Chemistry Letters 02/2015; 44(2):150-151. DOI:10.1246/cl.140901 · 1.30 Impact Factor
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    ABSTRACT: Fluorescent sensors are powerful tools for visualizing cellular molecular dynamics. We present a high-throughput screening system, designated hybrid-type fluorescence indicator development (HyFInD), to identify optimal position-specific fluorophore labeling in hybrid-type sensors consisting of combinations of ligand-binding protein mutants with small molecular fluorophores. We screened sensors for glutamate among hybrid molecules obtained by the reaction of four cysteine-reactive fluorescence probes with a set of cysteine-scanning mutants of the 274 amino acid S1S2 domain of AMPA-type glutamate receptor GluA2 subunit. HyFInD identified a glutamate-responsive probe (enhanced glutamate optical sensor: eEOS) with a dynamic range >2400 %, good photostability, and high selectivity. When eEOS was specifically tethered to neuronal surfaces, it reliably visualized the spatiotemporal dynamics of glutamate release at single synapses, revealing synapse-to-synapse heterogeneity of short-term plasticity.
    Angewandte Chemie 12/2014; 126(49). DOI:10.1002/ange.201407181
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    ABSTRACT: Fluorescent sensors are powerful tools for visualizing cellular molecular dynamics. We present a high-throughput screening system, designated hybrid-type fluorescence indicator development (HyFInD), to identify optimal position-specific fluorophore labeling in hybrid-type sensors consisting of combinations of ligand-binding protein mutants with small molecular fluorophores. We screened sensors for glutamate among hybrid molecules obtained by the reaction of four cysteine-reactive fluorescence probes with a set of cysteine-scanning mutants of the 274 amino acid S1S2 domain of AMPA-type glutamate receptor GluA2 subunit. HyFInD identified a glutamate-responsive probe (enhanced glutamate optical sensor: eEOS) with a dynamic range >2400 %, good photostability, and high selectivity. When eEOS was specifically tethered to neuronal surfaces, it reliably visualized the spatiotemporal dynamics of glutamate release at single synapses, revealing synapse-to-synapse heterogeneity of short-term plasticity.
    Angewandte Chemie International Edition 12/2014; 53(49). DOI:10.1002/anie.201407181 · 11.34 Impact Factor
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    ABSTRACT: Overexpression of growth factor receptors in cancers, e.g. human epidermal growth factor receptor 2 (HER2) in ovarian and breast cancers, is associated with aggressiveness. A possible strategy to treat cancers that overexpress those receptors is blockade of receptor signaling by inducing receptor internalization and degradation. In this study, we developed a cell-based high-throughput screening (HTS) system to identify small molecules that induce HER2 internalization by employing our recently developed acidic-pH-activatable probe in combination with protein labeling technology. Our HTS system enabled facile and reliable quantification of HER2 internalization with a Z' factor of 0.66 and a signal-to-noise ratio of 44.6. As proof of concept, we used the system to screen a ~ 155,000 small-molecule library, and identified three hits that induced HER2 internalization and degradation via at least two distinct mechanisms. This HTS platform should be adaptable to other disease-related receptors in addition to HER2.
    ACS Chemical Biology 08/2014; 9(10). DOI:10.1021/cb500654q · 5.36 Impact Factor
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    ABSTRACT: Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.
    Angewandte Chemie International Edition 06/2014; 53(24). DOI:10.1002/anie.201402030 · 11.34 Impact Factor
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    ABSTRACT: Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.
    Angewandte Chemie 06/2014; 126(24). DOI:10.1002/ange.201402030
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    ABSTRACT: Brain injury induces phenotypic changes in astrocytes, known as reactive astrogliosis, which may influence neuronal survival. Here we show that brain injury induces inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) signaling in astrocytes, and that the Ca(2+) signaling is required for astrogliosis. We found that type 2 IP3 receptor knockout (IP3R2KO) mice deficient in astrocytic Ca(2+) signaling have impaired reactive astrogliosis and increased injury-associated neuronal death. We identified N-cadherin and pumilio 2 (Pum2) as downstream signaling molecules, and found that brain injury induces up-regulation of N-cadherin around the injured site. This effect is mediated by Ca(2+)-dependent down-regulation of Pum2, which in turn attenuates Pum2-dependent translational repression of N-cadherin. Furthermore, we show that astrocyte-specific knockout of N-cadherin results in impairment of astrogliosis and neuroprotection. Thus, astrocytic Ca(2+) signaling and the downstream function of N-cadherin play indispensable roles in the cellular responses to brain injury. These findings define a previously unreported signaling axis required for reactive astrogliosis and neuroprotection following brain injury.
    Proceedings of the National Academy of Sciences 06/2013; DOI:10.1073/pnas.1300378110 · 9.81 Impact Factor
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    ABSTRACT: A fluorescent aptamer sensor was applied to the analysis of extracellular chemical transmitter dynamics. We utilized a tocopherol-labeled aptamer, which allowed the direct anchoring of the fluorescent aptamer on the cell surface while retaining its performance as a fluorescent sensor. The fast-responsive fluorescent DNA aptamer sensor, which targets adenine compounds, was anchored on the surface of brain astrocytes. Fluorescence imaging of the aptamer-anchored astrocytes enabled the real-time monitoring of release of adenine compounds as a gliotransmitter, which was synchronized with calcium wave propagation in neighboring cells.
    Journal of the American Chemical Society 06/2012; 134(23):9561-4. DOI:10.1021/ja302551p · 11.44 Impact Factor
  • Neuroscience Research 09/2011; 71. DOI:10.1016/j.neures.2011.07.403 · 2.15 Impact Factor
  • Neuroscience Research 09/2011; 71. DOI:10.1016/j.neures.2011.07.1426 · 2.15 Impact Factor
  • Kohtaroh Sugao, Kenzo Hirose
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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. DOI:10.1007/978-1-61779-065-2_8 · 1.29 Impact Factor
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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. DOI:10.1091/mbc.E10-02-0095 · 5.98 Impact Factor
  • ChemInform 08/2010; 33(31). DOI:10.1002/chin.200231215
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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. DOI:10.1038/nature08991 · 42.35 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. DOI:10.1073/pnas.0913154107 · 9.81 Impact Factor
  • Neuroscience Research 01/2010; 68:e220. DOI:10.1016/j.neures.2010.07.2543 · 2.15 Impact Factor
  • Neuroscience Research 01/2010; 68:e235. DOI:10.1016/j.neures.2010.07.1040 · 2.15 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. DOI:10.4049/jimmunol.0803469 · 5.36 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 10/2009; 87(13):3011-23. DOI:10.1002/jnr.22130 · 2.73 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. DOI:10.1254/jphs.09136SC · 2.11 Impact Factor

Publication Stats

3k Citations
505.47 Total Impact Points

Institutions

  • 2000–2015
    • The University of Tokyo
      • • Department of Neurology
      • • Faculty & Graduate School of Medicine
      • • Department of Pharmaceutical Sciences
      白山, Tōkyō, Japan
  • 2006–2007
    • Nagoya University
      Nagoya, Aichi, Japan
  • 1999
    • Kansai Medical University
      Moriguchi, Ōsaka, Japan