Updated on 2024/02/15

写真b

 
GOTO Satoshi
 
*Items subject to periodic update by Rikkyo University (The rest are reprinted from information registered on researchmap.)
Affiliation*
College of Science Department of Life Science
Graduate School of Science Doctoral Program in Life Science
Graduate School of Science Master's Program in Life Science
Title*
Professor
Degree
博士(理学) ( 東京大学 )
Research Theme*
  • 私達のような多細胞生物は、多種多様な細胞が協調しあうことによって生存・活動することができる。このように重要な細胞間のやりとりは、細胞表面に提示または細胞外に分泌される蛋白質などを介して行われている。したがって、それらの蛋白質の機能やその制御を研究することは、多細胞生物を理解するうえで非常に重要である。私達は、そのような蛋白質がどのように調節されているかについて、特に翻訳後修飾という観点から研究を行っている。

  • Campus Career*
    • 4 2012 - Present 
      College of Science   Department of Life Science   Professor
    • 4 2012 - Present 
      Graduate School of Science   Master's Program in Life Science   Professor
    • 4 2012 - Present 
      Graduate School of Science   Doctoral Program in Life Science   Professor
     

    Research Areas

    • Life Science / Cell biology

    Research History

    • 4 2012 - Present 
      RIKKYO UNIVERSITY   Graduate School of Science Field of Study: Life Science   Professor

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    • 4 2012 - Present 
      RIKKYO UNIVERSITY   Graduate School of Science Field of Study: Life Science   Professor

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    • 4 2012 - Present 
      RIKKYO UNIVERSITY   College of Science Department of Life Science   Professor

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    Education

    • 4 1987 - 3 1992 
      The University of Tokyo   Graduate School, Division of Science

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      Country: Japan

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    • 4 1983 - 3 1987 
      The University of Tokyo   Faculty of Science

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      Country: Japan

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    Papers

    • SPPL3-dependent downregulation of the synthesis of (neo)lacto-series glycosphingolipid is required for the staining of cell surface CD59. International journal

      Kohei Kawaguchi, Miki Yamamoto-Hino, Satoshi Goto

      Biochemical and biophysical research communications571   81 - 87   24 9 2021

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      Language:English   Publishing type:Research paper (scientific journal)  

      CD59 is a small glycoprotein modified with a glycophosphatidylinositol (GPI) anchor that prevents the formation of the membrane attack complex, thereby protecting host cells from lysis. A previous study identified that cell surface CD59 staining required the intramembrane protease signal peptide peptidase-like 3 (SPPL3). However, the effect of SPPL3 on the staining of CD59 remains unknown. This study shows that SPPL3 is essential for the surface labeling of CD59 but not of major GPI-anchored proteins. Surface CD59 staining requires the intramembrane protease activity of SPPL3 and SPPL3-mediated suppression of the (neo)lacto-series glycosphingolipids (nsGSLs)-but not N-glycan-synthesis pathway. The abundance of nsGSLs may affect complement-dependent cytotoxicity by altering the abundance or accessibility of cell surface CD59.

      DOI: 10.1016/j.bbrc.2021.06.093

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    • Hrd1-dependent Degradation of the Unassembled PIGK Subunit of the GPI Transamidase Complex.

      Kohei Kawaguchi, Miki Yamamoto-Hino, Yoshiko Murakami, Taroh Kinoshita, Satoshi Goto

      Cell structure and function46 ( 2 ) 65 - 71   3 9 2021

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      Language:English   Publishing type:Research paper (scientific journal)  

      Glycosylphosphatidylinositol (GPI)-anchored proteins are post-transcriptionally modified with GPI and anchored to the plasma membrane. GPI is attached to nascent proteins in the endoplasmic reticulum by the GPI transamidase complex, which consists of PIGT, PIGK, GPAA1, PIGU, and PIGS. Of these, PIGK is a catalytic subunit that is unstable without PIGT. This study investigated the pathway by which unassembled PIGK not incorporated into the complex is degraded. We showed that unassembled PIGK was degraded via the proteasome-dependent pathway and that Hrd1 (also known as SYVN1), a ubiquitin ligase involved in the endoplasmic reticulum-associated degradation pathway, was responsible for degradation of unassembled PIGK.Key words: Glycosylphosphatidylinositol, GPI transamidase complex, protein stability, transamidation, ERAD.

      DOI: 10.1247/csf.21019

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    • Subunits of the GPI transamidase complex localize to the endoplasmic reticulum and nuclear envelope in Drosophila. International journal

      Kohei Kawaguchi, Miki Yamamoto-Hino, Nina Matsuyama, Emiko Suzuki, Satoshi Goto

      FEBS letters595 ( 7 ) 960 - 968   4 2021

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      Language:English   Publishing type:Research paper (scientific journal)  

      A total of 10-20% of plasma membrane proteins are anchored by glycosylphosphatidylinositol (GPI). GPI is attached to proteins by GPI transamidase (GPI-T), which contains five subunits named PIGK, PIGS, PIGT, PIGU, and GPAA1. We previously reported that PIGT localizes near the nucleus in Drosophila. However, localizations of the other four subunits remain unknown. Here, we show that a catalytic subunit of GPI-T, PIGK, mainly localizes to the endoplasmic reticulum (ER), while the other four subunits localize to the nuclear envelope (NE) and ER. The NE/ER localization ratio of PIGS differs between cell types and developmental stages. Our results suggest that GPI-T catalyzes GPI attachment in the ER and the other four subunits may have other unknown functions in the NE.

      DOI: 10.1002/1873-3468.14048

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    • Role of the Drosophila YATA protein in the proper subcellular localization of COPI revealed by in vivo analysis.

      Maiko Saito, Minoru Nakayama, Kyota Fujita, Atsuko Uchida, Hiroyuki Yano, Satoshi Goto, Hitoshi Okazawa, Masaki Sone

      Genes & genetic systems95 ( 6 ) 303 - 314   23 3 2021

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      Language:English   Publishing type:Research paper (scientific journal)  

      yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. Here, we reveal a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, our in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI.

      DOI: 10.1266/ggs.20-00027

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    • Lamin is essential for nuclear localization of the GPI synthesis enzyme PIG-B and GPI-anchored protein production in Drosophila. International journal

      Miki Yamamoto-Hino, Kohei Kawaguchi, Masaya Ono, Kazuhiro Furukawa, Satoshi Goto

      Journal of cell science133 ( 6 )   26 3 2020

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      Membrane lipid biosynthesis is a complex process that occurs in various intracellular compartments. In Drosophila, phosphatidylinositol glycan-B (PIG-B), which catalyzes addition of the third mannose in glycosylphosphatidylinositol (GPI), localizes to the nuclear envelope (NE). Although this NE localization is essential for Drosophila development, the underlying molecular mechanism remains unknown. To elucidate this mechanism, we identified PIG-B-interacting proteins by performing immunoprecipitation followed by proteomic analysis. We then examined which of these proteins are required for the NE localization of PIG-B. Knockdown of Lamin Dm0, a B-type lamin, led to mislocalization of PIG-B from the NE to the endoplasmic reticulum. Lamin Dm0 associated with PIG-B at the inner nuclear membrane, a process that required the tail domain of Lamin Dm0. Furthermore, GPI moieties were distributed abnormally in the Lamin Dm0 mutant. These data indicate that Lamin Dm0 is involved in the NE localization of PIG-B and is required for proper GPI-anchor modification of proteins.

      DOI: 10.1242/jcs.238527

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    • Stability of the transamidase complex catalyzing GPI anchoring of proteins. Peer-reviewed International journal

      Kohei Kawaguchi, Tatsuro Sato, Shu Kondo, Miki Yamamoto-Hino, Satoshi Goto

      Biochemical and biophysical research communications512 ( 3 ) 584 - 590   7 5 2019

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      Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors some proteins to the plasma membrane. This anchoring is catalyzed by a transamidase complex (TAC) composed of five subunits: PIG-K, GAA1, PIG-U, PIG-T, and PIG-S (Fig. 1A). PIG-K and GAA1 are predicted to catalyze the first and second steps during attachment of proproteins of GPI-anchored proteins (GPI-APs) to GPI. GPI may be delivered by PIG-U, and PIG-T is required for stability of all TAC subunits when overexpressed in cultured cells. However, protein stability of TAC has not been analyzed using loss-of-function mutants for each subunit. Herein, we analyzed the stability of TAC in knockout and/or knockdown mutants for each subunit. PIG-T and PIG-U, or PIG-T and GAA1, were mutually required for stability, and all three subunits were stable without PIG-S or PIG-K. However, these three subunits were essential for the stability of both PIG-S and PIG-K. By contrast, loss of PIG-S reduced the stability of PIG-K and left the other subunits unaffected. Reduction of PIG-K did not impact any of the other subunits. Thus, PIG-T, PIG-U, and GAA1 may form a core complex associated by PIG-S, and these four subunits may stabilize PIG-K, triggering GPI anchoring reactions. Instability of PIG-K in the absence of the other four subunits may ensure that GPI anchoring is catalyzed only by the completely assembled complex.

      DOI: 10.1016/j.bbrc.2019.03.103

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    • Nuclear envelope localization of PIG-B is essential for GPI-anchor synthesis in Drosophila. Peer-reviewed International journal

      Yamamoto-Hino.M, Katsumata, E, Suzuki, E, Maeda, Y, Kinoshita, T, Goto, S

      J. Cell Sci.131 ( 20 )   2018

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      Membrane lipid biosynthesis is a complex process that takes place in various intracellular compartments. Glycosylphosphatidylinositol (GPI), a lipid involved in membrane anchoring of some proteins, is synthesized by the PIG enzymes. Most PIGs are localized to the endoplasmic reticulum (ER), but Drosophila PIG-B (DmPIG-B) is localized to the nuclear envelope (NE). To determine whether the NE localization of DmPIG-B is functionally important, we defined the determinants of localization and generated an ER-localized form, denoted DmPIG-B[ER]. The enzymatic activity of DmPIG-B[ER] was comparable to that of NE-localized DmPIG-B[NE]. Expression of DmPIG-B[ER] inefficiently rescued the lethality of the PIG-B mutant, whereas DmPIG-B[NE] rescued this lethality fully. DmPIG-B[ER] was preferentially degraded by lysosomes, suggesting that the NE localization is essential for function and stability of the protein. In addition, we found that the region of the ER proximal to the NE is the site of translation of GPI-anchored proteins and addition of GPI. Thus, the NE and proximal ER may provide a platform for efficient GPI anchoring.

      DOI: 10.1242/jcs.218024

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    • Spatzle-Processing Enzyme-independent Activation of the Toll Pathway in Drosophila Innate Immunity Peer-reviewed

      Miki Yamamoto-Hino, Satoshi Goto

      CELL STRUCTURE AND FUNCTION41 ( 1 ) 55 - 60   2016

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:JAPAN SOC CELL BIOLOGY  

      The Toll pathway regulates innate immunity in insects and vertebrates. The Drosophila Toll receptor is activated by a processed form of a ligand, Spatzle. Spatzle-processing enzyme (SPE) is the only enzyme identified to date that functions in converting Spatzle to an active form during the immune response. In the present study, Toll activation induced by immune challenge was almost suppressed in spatzle mutant larvae and adults, whereas it was present in SPE mutant larvae challenged with Micrococcus luteus and adults challenged with Bacillus subtilis. Our data suggest that an unidentified protease besides SPE processes Spatzle under conditions of microbial challenge.

      DOI: 10.1247/csf.16002

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    • Phenotype-based clustering of glycosylation-related genes by RNAi-mediated gene silencing Peer-reviewed International journal

      Miki Yamamoto-Hino, Hideki Yoshida, Tomomi Ichimiya, Sho Sakamura, Megumi Maeda, Yoshinobu Kimura, Norihiko Sasaki, Kiyoko F. Aoki-Kinoshita, Akiko Kinoshita-Toyoda, Hidenao Toyoda, Ryu Ueda, Shoko Nishihara, Satoshi Goto

      GENES TO CELLS20 ( 6 ) 521 - 542   6 2015

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

      Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, 1,4-galactosyltransferase I, 1,3-galactosyltransferase II, and 1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.

      DOI: 10.1111/gtc.12246

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    • Dynamic regulation of innate immune responses in Drosophila by Senju-mediated glycosylation. Peer-reviewed International journal

      Miki Yamamoto-Hino, Masatoshi Muraoka, Shu Kondo, Ryu Ueda, Hideyuki Okano, Satoshi Goto

      Proceedings of the National Academy of Sciences of the United States of America112 ( 18 ) 5809 - 14   5 5 2015

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      The innate immune system is the first line of defense encountered by invading pathogens. Delayed and/or inadequate innate immune responses can result in failure to combat pathogens, whereas excessive and/or inappropriate responses cause runaway inflammation. Therefore, immune responses are tightly regulated from initiation to resolution and are repressed during the steady state. It is well known that glycans presented on pathogens play important roles in pathogen recognition and the interactions between host molecules and microbes; however, the function of glycans of host organisms in innate immune responses is less well known. Here, we show that innate immune quiescence and strength of the immune response are controlled by host glycosylation involving a novel UDP-galactose transporter called Senju. In senju mutants, reduced expression of galactose-containing glycans resulted in hyperactivation of the Toll signaling pathway in the absence of immune challenges. Genetic epistasis and biochemical analyses revealed that Senju regulates the Toll signaling pathway at a step that converts Toll ligand Spatzle to its active form. Interestingly, Toll activation in immune-challenged wild type (WT) flies reduced the expression of galactose-containing glycans. Suppression of the degalactosylation by senju overexpression resulted in reduced induction of Toll-dependent expression of an antimicrobial peptide, Drosomycin, and increased susceptibility to infection with Gram-positive bacteria. These data suggest that Senju-mediated galactosylation suppresses undesirable Toll signaling activation during the steady state; however, Toll activation in response to infection leads to degalactosylation, which raises the immune response to an adequate level and contributes to the prompt elimination of pathogens.

      DOI: 10.1073/pnas.1424514112

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    • Identification of Proteasome Components Required for Apical Localization of Chaoptin Using Functional Genomics Invited Peer-reviewed International journal

      Hiroyuki Yano, Miki Yamamoto-Hino, Wakae Awano, Kiyoko F. Aoki-Kinoshita, Kayoko Tsuda-Sakurai, Hideyuki Okano, Satoshi Goto

      JOURNAL OF NEUROGENETICS26 ( 1 ) 53 - 63   3 2012

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:INFORMA HEALTHCARE  

      The distinct localization of membrane proteins with regard to cell polarity is crucial for the structure and function of various organs in multicellular organisms. However, the molecules and mechanisms that regulate protein localization to particular subcellular domains are still largely unknown. To identify the genes involved in regulation of protein localization, the authors performed a large-scale screen using a Drosophila RNA interference (RNAi) library, by which Drosophila genes could be knocked down in a tissue-and stage-specific manner. Drosophila photoreceptor cells have a morphologically distinct apicobasal polarity, along which Chaoptin (Chp), a glycosylphosphatidylinositol (GPI)-anchored membrane protein, and the Na+, K+-ATPase are localized to the apical and basolateral domains, respectively. By examining the subcellular localization of these proteins, the authors identified 106 genes whose knockdown resulted in mislocalization of Chp and Na+, K+-ATPase. Gene ontology analysis revealed that the knockdown of proteasome components resulted in mislocalization of Chp to the basolateral plasma membrane. These results suggest that the proteasome is involved, directly or indirectly, in selective localization of Chp to the apical plasma membrane of Drosophila photoreceptor cells.

      DOI: 10.3109/01677063.2012.661497

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    • Structure, function and formation of glycans in Drosophila Peer-reviewed

      Yamamoto-Hino M, Okano H, Kanie O, Goto S

      Glycans: Biochemistry, Characterization and Applications   165 - 188   1 2 2012

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      Glycosylation of proteins shows two striking features: first, there are a large number of glycan structures that are involved in various biological events (diversity) and, second, certain structures are selectively added to specific sites within proteins (specificity) in a context-dependent manner. However, the mechanisms that regulate diversity and specificity are unknown. Over the last few decades, genetic studies in Drosophila have been used to examine the functions mediated by glycosylation in vivo. Recent improvements in the combined use of biochemical/biophysical, genetic, and cell biology approaches has allowed the rapid and detailed analysis of the structure, function and formation of glycans in Drosophila. This chapter aims to provide an overview of the different types of glycans and their biological function(s) in Drosophila. The regulation of glycan synthesis in Drosophila and mammals is also discussed. © 2012 by Nova Science Publishers, Inc. All rights reserved.

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      Other Link: http://orcid.org/0000-0003-4147-6868

    • AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development in Drosophila melanogaster Peer-reviewed International journal

      Satoshi Kametaka, Ai Kametaka, Shinichi Yonekura, Mineyuki Haruta, Seiichi Takenoshita, Satoshi Goto, Satoshi Waguri

      JOURNAL OF CELL SCIENCE125 ( 3 ) 634 - 648   2 2012

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:COMPANY OF BIOLOGISTS LTD  

      Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. In this study, we found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of gamma-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila.

      DOI: 10.1242/jcs.090167

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    • Cisterna-specific Localization of Glycosylation-related Proteins to the Golgi Apparatus Peer-reviewed

      Miki Yamamoto-Hino, Masato Abe, Takako Shibano, Yuka Setoguchi, Wakae Awano, Ryu Ueda, Hideyuki Okano, Satoshi Goto

      CELL STRUCTURE AND FUNCTION37 ( 1 ) 55 - 63   2012

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:JAPAN SOC CELL BIOLOGY  

      The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial- and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial-and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.

      DOI: 10.1247/csf.11037

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    • Multi-stage mass spectrometric information obtained by deconvolution of energy-resolved spectra acquired by triple-quadrupole mass spectrometry Peer-reviewed

      Osamu Kanie, Yoshimi Kanie, Shusaku Daikoku, Yuki Shioiri, Ayako Kurimoto, Sachiko Mutsuga, Satoshi Goto, Yukishige Ito, Katsuhiko Suzuki

      RAPID COMMUNICATIONS IN MASS SPECTROMETRY25 ( 11 ) 1617 - 1624   6 2011

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

      Triple-quadrupole mass spectrometry (TQ-MS) provides the capability to carry out collision-induced dissociation (CID) and it offers advantages in quantification when connected with high-performance liquid chromatography through an electrospray ionization interface. However, although TQ-MS provides information on partial structures through the analysis of product ions obtained by CID experiments, the method only provides single-stage CID experiments, which limits the detailed structural information that can be obtained. Herein, a method of overcoming this limitation of TQ-MS is described. A spectrum obtained by energy-resolved mass spectrometry (ERMS) was used to deconvolute the fragmentation process, with a Galili-antigenic trisaccharide derivative being used as an example. A replot of the ERMS data showing the ratios of the product ions to the precursor ion resulted in a descriptive graph. Analysis of the sum of the ratios of individual product ions to the precursor ion at specific CID energies revealed that the members of a series of product ions were related to each other. The obtained relationships and the m/z values of the product ions provided information on the fragmentation process taking place during the dissociation, indicating that the ERMS spectrum obtained by TQ-MS contained equivalent information to that obtainable by multi-stage MS/MS (MS(n); n >= 2). This method may allow users of triple-quadrupole mass spectrometers to obtain MS(n)-type information by performing a single ERMS experiment, which is even advantageous over quadrupole ion trap (QIT)-MS/MS because CID experiments on individual first-generation product ions are not required. Copyright (C) 2011 John Wiley & Sons, Ltd.

      DOI: 10.1002/rcm.5031

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    • Fluorescence-monitored zero dead-volume nanoLC-microESI-QIT-TOF MS for analysis of fluorescently tagged glycosphingolipids Peer-reviewed International journal

      Shusaku Daikoku, Yasunari Ono, Atsuko Ohtake, Yasuko Hasegawa, Eiichiro Fukusaki, Katsuhiko Suzuki, Yukishige Ito, Satoshi Goto, Osamu Kanie

      ANALYST136 ( 5 ) 1046 - 1050   2011

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROYAL SOC CHEMISTRY  

      An analysis of the glycan processing event is of particular importance to understand the nontemplate dependent synthetic mechanism of the multiple glycosylation reactions taking place in the Golgi apparatus in connection with the post-translational modification of biomolecules. In our efforts to address the issue, we constructed an analysis platform using nano-liquid chromatography (LC), which also worked as a spray tip, with an optical-fiber-based blue (470 nm) light emitting diode (LED)-induced fluorescence (520 nm) detector coupled with a microelectrospray ionization (ESI)-quadrupole ion trap (QIT)-time of flight (TOF) mass spectrometer (MS). This system was designed to enable both quantitative and qualitative analyses of fluorescently tagged molecules such as BODIPY-tagged lactosylceramide. Owing to the zero dead volume after LC separation, an extremely high sensitivity was achieved for the quantitative analysis (260 amol). It was also shown that a simultaneous online structural analysis based on MS could be achieved for the same quantity of analyte. To further demonstrate its potential, an enzymatic reaction of fluorescently tagged lactosylceramide using sialyltransferase was carried out, and the conversion yield was obtained on the basis of fluorescence detection. In addition, the structural details of a product, sialyl lactosylceramide, were obtained by MS and MS/MS analyses.

      DOI: 10.1039/c0an00715c

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    • Identification of Genes Required for Neural-Specific Glycosylation Using Functional Genomics Peer-reviewed International journal

      Miki Yamamoto-Hino, Yoshimi Kanie, Wakae Awano, Kiyoko F. Aoki-Kinoshita, Hiroyuki Yano, Shoko Nishihara, Hideyuki Okano, Ryu Ueda, Osamu Kanie, Satoshi Goto

      PLOS GENETICS6 ( 12 ) e1001254   12 2010

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

      Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, alpha 1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific a1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating alpha 1,3-fucosylation. Further analyses revealed that an RNA-binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells.

      DOI: 10.1371/journal.pgen.1001254

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    • Autophagy-Dependent Rhodopsin Degradation Prevents Retinal Degeneration in Drosophila Peer-reviewed International journal

      Ryosuke Midorikawa, Miki Yamamoto-Hino, Wakae Awano, Yoshimi Hinohara, Emiko Suzuki, Ryu Ueda, Satoshi Goto

      JOURNAL OF NEUROSCIENCE30 ( 32 ) 10703 - 10719   8 2010

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOC NEUROSCIENCE  

      Recent studies have demonstrated protective roles for autophagy in various neurodegenerative disorders, including the polyglutamine diseases; however, the role of autophagy in retinal degeneration has remained unclear. Accumulation of activated rhodopsin in some Drosophila mutants leads to retinal degeneration, and although it is known that activated rhodopsin is degraded in endosomal pathways in normal photoreceptor cells, the contribution of autophagy to rhodopsin regulation has remained elusive. This study reveals that activated rhodopsin is degraded by autophagy in collaboration with endosomal pathways to prevent retinal degeneration. Light-dependent retinal degeneration in the Drosophila visual system is caused by the knockdown or mutation of autophagy-essential components, such as autophagy-related protein 7 and 8 (atg-7/atg-8), or genes essential for PE (phosphatidylethanolamine) biogenesis and autophagosome formation, including Phosphatidylserine decarboxylase (Psd) and CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (Ept). The knockdown of atg-7/8 or Psd/Ept produced an increase in the amount of rhodopsin localized to Rab7, positive late endosomes. This rhodopsin accumulation, followed by retinal degeneration, was suppressed by overexpression of Rab7, which accelerated the endosomal degradation pathway. These results indicate a degree of cross talk between the autophagic and endosomal/lysosomal pathways. Importantly, a reduction in rhodopsin levels rescued Psd knockdown-induced retinal degeneration. Additionally, the Psd knockdown-induced retinal degeneration phenotype was enhanced by Ppt1 inactivation, which causes infantile neuronal ceroid lipofuscinosis, implying that autophagy plays a significant role in its pathogenesis. Collectively, the current data reveal that autophagy suppresses light-dependent retinal degeneration in collaboration with the endosomal degradation pathway and that rhodopsin is a key substrate for autophagic degradation in this context.

      DOI: 10.1523/JNEUROSCI.2061-10.2010

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    • Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt Peer-reviewed International journal

      Akiko Mukai, Miki Yamamoto-Hino, Wakae Awano, Wakako Watanabe, Masayuki Komada, Satoshi Goto

      EMBO JOURNAL29 ( 13 ) 2114 - 2125   7 2010

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      Wingless (Wg)/Wnt has been proposed to exert various functions as a morphogen depending on the levels of its signalling. Therefore, not just the concentration of Wg/Wnt, but also the responsiveness of Wg/Wnt-target cells to the ligand, must have a crucial function in controlling cellular outputs. Here, we show that a balance of ubiquitylation and deubiquitylation of the Wg/Wnt receptor Frizzled determines the cellular responsiveness to Wg/Wnt both in mammalian cells and in Drosophila, and that the cell surface level of Frizzled is regulated by deubiquitylating enzyme UBPY/ubiquitin-specific protease 8 (USP8). Although ubiquitylated Frizzled underwent lysosomal trafficking and degradation, UBPY/USP8-dependent deubiquitylation led to recycling of Frizzled to the plasma membrane, thereby elevating its surface level. Importantly, a gain and loss of UBPY/USP8 function led to up-and down-regulation, respectively, of canonical Wg/Wnt signalling. These results unveil a novel mechanism that regulates the cellular responsiveness to Wg/Wnt by controlling the cell surface level of Frizzled. The EMBO Journal (2010) 29, 2114-2125. doi:10.1038/emboj.2010.100; Published online 21 May 2010

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    • Membrane Protein Location-Dependent Regulation by PI3K (III) and Rabenosyn-5 in Drosophila Wing Cells Peer-reviewed International journal

      Masato Abe, Yuka Setoguchi, Tsubasa Tanaka, Wakae Awano, Kuniaki Takahashi, Ryu Ueda, Akira Nakamura, Satoshi Goto

      PLOS ONE4 ( 10 ) e7306   10 2009

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      The class III phosphatidylinositol-3 kinase (PI3K (III)) regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate (PI(3)P). While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes.

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    • Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry Peer-reviewed International journal

      Yoshimi Kanie, Miki Yamamoto-Hino, Yayoi Karino, Hiroki Yokozawa, Shoko Nishihara, Ryu Ueda, Satoshi Goto, Osamu Kanie

      PLOS ONE4 ( 5 ) e5434   5 2009

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      Background: A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown.
      Methodology/Principal Findings: In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate a-D-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type.
      Conclusions/Significance: These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.

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    • Spatial and temporal regulation of glycosylation during Drosophila eye development Peer-reviewed International journal

      Hiroyuki Yano, Miki Yamamoto-Hino, Satoshi Goto

      CELL AND TISSUE RESEARCH336 ( 1 ) 137 - 147   4 2009

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      Glycosylation plays an essential role during development, in processes such as morphogen distribution, cell-to-cell communication, and extracellular matrix formation. Glycosylation is regulated during development in both a spatial and temporal manner. This study presents a detailed description of glycan distribution from late pupal to adult stages in Drosophila ommatidia by using nine different lectins. The lectins ConA, LCA, and DSA, which recognize high-mannose or complex types of N-linked glycans stain both apical and basolateral membranes of photoreceptor cells, whereas SBA, DBA, and ABA lectins, which bind to mucin-type O-glycans, label the inter-rhabdomeral space. The O-linked GlcNAc moiety is strongly labeled by WGA on the nuclear membrane. The localization of these glycans does not change throughout late pupal development. In contrast, the abundance of O-linked glycans, bisected oligosaccharides, and GlcNAc-containing glycans detected by PNA, PHA-E4, and WGA, respectively, is reduced in rhabdomeres and other subcellular domains during late pupal development. Some of these glycans have also been detected in the Golgi and/or putative secretory vesicles, suggesting their dynamic transport during development. These glycans, whose expression is dynamically regulated in a spatial and temporal manner, may therefore play critical roles in ommatidial development.

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    • [Membrane traffic in Drosophila development].

      Masato Abe, Satoshi Goto

      Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme53 ( 16 Suppl ) 2193 - 9   12 2008

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    • [Regulation of glycosylation in Golgi units].

      Satoshi Goto

      Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme53 ( 12 Suppl ) 1475 - 9   9 2008

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    • N-glycosylation of the Drosophila neural protein Chaoptin is essential for its stability, cell surface transport and adhesive activity Peer-reviewed International journal

      Yu Hirai-Fujita, Miki Yamamoto-Hino, Osamu Kanie, Satoshi Goto

      FEBS LETTERS582 ( 17 ) 2572 - 2576   7 2008

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      Glycosylation of proteins can modulate their function in a striking variety of systems, including immune responses, neuronal activities and development. The Drosophila protein, Chaoptin ( Chp), is essential for the development and maintenance of photoreceptor cells. This protein is heavily glycosylated, but the possible role of this glycosylation is not well- understood. Here we show that mutations introduced into about 1/ 3 of 16 potential N- linked glycosylation sites within Chp impaired its cell adhesive activities when expressed in Drosophila S2 cells. Mutation of 2/ 3 of the glycosylation sites resulted in a marked decrease in Chp protein abundance. These results suggest that Nlinked glycosylation of Chp is essential for its stability and activity. (c) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

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    • Comparative RP-HPLC for rapid identification of glycopeptides and application in off-line LC-MALDI-MS analysis Peer-reviewed International journal

      Yoshimi Kanie, Akiko Enomoto, Satoshi Goto, Osamu Kanie

      CARBOHYDRATE RESEARCH343 ( 4 ) 758 - 768   3 2008

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      Despite the increasing attention being paid to the functions of glycoproteins, their structural analysis is still difficult and hinders functional investigations. Structural analysis of post-translationally modified proteins is thought to be achieved using methods frequently utilized in proteomics research; however, the same methods cannot be used for glycosylated proteins. One of the difficulties associated with the physiochemical properties of glycopeptides and peptides is that the detection of the former is considerably more difficult, because of the existence of glycoforms that increase molecular weight and reduces quantities of individual species. Thus, difficulties are often faced in finding glycopeptide(s) by using MS when analyzing peaks (or fractions) obtained after proteolytic digestion and HPLC. One simple yet difficult solution to this problem would be to develop a purification method that provides better resolution. Our intention has been to address this issue by using a combination of conventional methods. We found that a method consisting of a combination of rough fractionation using a reverse-phase cartridge column under acidic conditions and comparative RP-HPLC, where the two chromatograms obtained using phosphate and borate buffers under basic conditions were compared, is effective for MS-based structural analysis. The applicability of the method in glycoprotein analysis was examined using various samples including ribonuclease B (RNase B), IgG1, ovalbumin (OVA), and asialo fetuin (ASF). The results suggest that the method is useful in the analysis of glycoproteins. (c) 2007 Elsevier Ltd. All rights reserved.

      DOI: 10.1016/j.carres.2007.12.008

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    • Sequential enzymatic glycosyltransfer reactions on a microfluidic device: Synthesis of a glycosaminoglycan linkage region tetrasaccharide Peer-reviewed International journal

      Yasunari Ono, Motohiro Kitajima, Shusaku Daikoku, Toshifumi Shiroya, Shoko Nishihara, Yoshimi Kanie, Katsuhiko Suzuki, Satoshi Goto, Osamu Kanie

      LAB ON A CHIP8 ( 12 ) 2168 - 2173   2008

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      A microfluidic chip carrying three reaction chambers was designed and constructed to examine sequential multiple enzymatic reactions. The synthesis of oligosaccharides in living cells is carried out in the Golgi apparatus where multiple enzymes such as glycosidase and glycosyltransferases act on a variety of substrates to generate glycoconjugates that include glycolipids and glycoproteins. The regulatory mechanism of the process however remains unknown. A microchip-based analysis platform may provide a valuable tool with which to address the issue by mimicking the Golgi function. We thus examined 3 sequential glycosyltransfer reactions on a chip, and succeeded in the synthesis of a tetrasaccharide using immobilized enzymes. Also, the kinetic parameters for a recently identified glycosyltransferase, proteoglycan GalT-I, were obtained for the first time.

      DOI: 10.1039/b809316d

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    • [Golgi units regulating diversity of glycosylation].

      Satoshi Goto

      Seikagaku. The Journal of Japanese Biochemical Society79 ( 1 ) 42 - 5   1 2007

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    • Distinct functional units of the Golgi complex in Drosophila cells Peer-reviewed International journal

      H Yano, M Yamamoto-Hino, M Abe, R Kuwahara, S Haraguchi, Kusaka, I, W Awano, A Kinoshita-Toyoda, H Toyoda, S Goto

      PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA102 ( 38 ) 13467 - 13472   9 2005

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      A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. Here we show that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disk cells are functionally diverse. The UDP-sugar transporter FRINGE-CONNECTION (FRC) is localized to a subset of the Golgi units distinct from those harboring SULFATELESS (SFL), which modifies glucosaminoglycans (GAGs), and from those harboring the protease RHOMBOID (RHO), which processes the glycoprotein SPITZ (SPI). Whereas the glycosylation and function of NOTCH are affected in imaginal disks of frc mutants, those of SPI and of GAG core proteins are not, even though FRC transports a broad range of glycosylation substrates, suggesting that Golgi units containing FRC and those containing SFL or RHO are functionally separable. Distinct Golgi units containing FRC and RHO in embryos could also be separated biochemically by immunoisolation techniques. We also show that Tn-antigen glycan is localized only in a subset of the Golgi units distributed basally in a polarized cell. We propose that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification.

      DOI: 10.1073/pnas.0506681102

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    • Approach for functional analysis of glycan using RNA interference Peer-reviewed International journal

      S Nishihara, R Ueda, S Goto, H Toyoda, H Ishida, M Nakamura

      GLYCOCONJUGATE JOURNAL21 ( 1-2 ) 63 - 68   2004

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      The elucidation of the biological role of glycan is one of the most important issues to be resolved following the genome project. RNA interference is becoming an efficient reverse genetic tool for studying gene function in model organisms, including C. elegans and Drosophila melanogaster. Our molecular evolutionary study has shown that a prototype of glycosyltransferases, which synthesize a variety of glycan structures in the Golgi apparatus, was conserved between mammals and Drosophila. For analyses of the basic physiological functions of glycans, we established the Drosophila inducible RNAi knockdown system and applied it to one glycosyltransferase and one transporter, proteoglycan UDP-galactose: beta-xylose 1,4galactosyltransferase I and the PAPS-transporter, respectively. If on the silencing of each gene induced ubiquitously under the control of a cytoplasmic actin promoter, the RNAi knockdown fly died, then the protein was indispensable for life. The expression of the target gene was disrupted specifically and the degree of interference was well correlated with the phenotype. The inducible RNAi knockdown fly obtained using the GAL4-UAS system will pave the way for the functional analysis of glycans.

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    • Molecular cloning and identification of 3 '- phosphoadenosine 5 '-phosphosulfate transporter Peer-reviewed International journal

      S Kamiyama, T Suda, R Ueda, M Suzuki, R Okubo, N Kikuchi, Y Chiba, S Goto, H Toyoda, K Saigo, M Watanabe, H Narimatsu, Y Jigami, S Nishihara

      JOURNAL OF BIOLOGICAL CHEMISTRY278 ( 28 ) 25958 - 25963   7 2003

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      Nucleotide sulfate, namely 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation. Although a specific PAPS transporter is present in Golgi membrane, no study has reported the corresponding gene. We have identified a novel human gene encoding a PAPS transporter, which we have named PAPST1, and the Drosophila melanogaster ortholog, slalom (sll). The amino acid sequence of PAPST1 (432 amino acids) exhibited 48.1% identity with SLL (465 amino acids), and hydropathy analysis predicted the two to be type III transmembrane proteins. The transient expression of PAPST1 in SW480 cells showed a subcellular localization in Golgi membrane. The expression of PAPST1 and SLL in yeast Saccharomyces cerevisiae significantly increased the transport of PAPS into the Golgi membrane fraction. In human tissues, PAPST1 is highly expressed in the placenta and pancreas and present at lower levels in the colon and heart. An RNA interference fly of sll produced with a GAL4-UAS system revealed that the PAPS transporter is essential for viability. It is well known that mutations of some genes related to PAPS synthesis are responsible for human inherited disorders. Our findings provide insights into the significance of PAPS transport and post-translational sulfation.

      DOI: 10.1074/jbc.M302439200

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    • Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons Peer-reviewed International journal

      K Sugimura, M Yamamoto, R Niwa, D Satoh, S Goto, M Taniguchi, S Hayashi, T Uemura

      JOURNAL OF NEUROSCIENCE23 ( 9 ) 3752 - 3760   5 2003

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      Little has been understood about the underlying mechanisms that generate the morphological diversity of dendritic trees. Dendritic arborization neurons in Drosophila provide an excellent model system to tackle this question, and they are classified into classes I-IV in order of increasing arbor complexity. Here we have developed transgenic green fluorescent protein markers for class I or class IV cells, which allowed time-lapse recordings of dendritic birth in the embryo, its maturation processes in the larva, and lesion-induced reactions. The two classes used distinct strategies of dendritic emergence from the cell body and branching, which contributed to differences in their basic arbor patterns. In contrast to the class I cells examined, one cell of class IV, which was a focus in this study, continued to elaborate branches throughout larval stages, and it was much more capable of responding to the severing of branches. We also investigated the cellular basis of field formation between adjacent class IV cells. Our results support the fact that class-specific inhibitory interaction is necessary and sufficient for tiling and confirmed that this intercellular communication was at work at individual dendrodendritic interfaces. Finally, this inhibitory signaling appeared to play a central role when arbors of adjacent cells started meeting midway between the cells and until the body wall became partitioned into abutting, minimal-overlapping territories.

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    • The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila Peer-reviewed International journal

      K Kubota, S Goto, S Hayashi

      DEVELOPMENTAL BIOLOGY257 ( 1 ) 117 - 126   5 2003

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. We describe here essential roles of Wingless in patterning the leg imaginal disc. Firstly, Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it was previously shown that Wingless signal transduction is not active in the proximal leg domain in larvae. Secondly, downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, we provide evidence that Dll restricts expression of a proximal leg-specific gene expression. We propose that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages. (C) 2003 Elsevier Science (USA). All rights reserved.

      DOI: 10.1016/S0012-1606(03)00062-9

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    • GETDB, a database compiling expression patterns and molecular locations of a collection of Gal4 enhancer traps Peer-reviewed

      S Hayashi, K Ito, Y Sado, M Taniguchi, A Akimoto, H Takeuchi, T Aigaki, F Matsuzaki, H Nakagoshi, T Tanimura, R Ueda, T Uemura, M Yoshihara, S Goto

      GENESIS34 ( 1-2 ) 58 - 61   9 2002

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      DOI: 10.1002/gene.10137

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    • UDP-sugar transporter implicated in glycosylation and processing of Notch Peer-reviewed

      S Goto, M Taniguchi, M Muraoka, H Toyoda, Y Sado, M Kawakita, S Hayashi

      NATURE CELL BIOLOGY3 ( 9 ) 816 - 822   9 2001

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      Glycosylation modifies protein activities in various biological processes. Here, we report the functions of a novel UDP-sugar transporter (UST74C, an alternative name for Fringe connection (Frc)) localized to the Golgi apparatus in cellular signalling of Drosophila. Mutants in the frc gene exhibit phenotypes resembling wingless and Notch mutants. Both Fringe-dependent and Fringe-independent Notch pathways are affected, and both glycosylation and proteolytic maturation of Notch are defective in mutant larvae. The results suggest that changes in nucleotide-sugar levels can differently affect Wingless and two distinct aspects of Notch signalling.

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    • EGF receptor attenuates Dpp signaling and helps to distinguish the wing and leg cell fates in Drosophila Peer-reviewed

      K Kubota, S Goto, K Eto, S Hayashi

      DEVELOPMENT127 ( 17 ) 3769 - 3776   9 2000

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      Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. We report here that the receptor tyrosine kinase EGFR antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and EGFR, we show that nuclear transduction of the two signals peaks at different position along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice.

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    • Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning Peer-reviewed

      S Goto, S Hayashi

      DEVELOPMENT126 ( 15 ) 3407 - 3413   8 1999

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      Proximodistal patterning in the Drosophila leg is elaborated from the circular arrangement of the proximal domain expressing escargot and homothorax, and the distal domain expressing Distal-less that are allocated during embryogenesis. The distal domain differentiates multiply segmented distal appendages by activating additional genes such as dachshund. Secreted signaling molecules Wingless and Decapentaplegic, expressed along the anterior-posterior compartment boundary, are required for activation of Distal-less and dachshund and repression of homothorax in the distal domain. However, whether Wingless and Decapentaplegic are sufficient for the circular pattern of gene expression is not known. Here we show that a proximal gene escargot and its activator homothorax ax regulate proximodistal patterning in the distal domain. Clones of cells expressing escargot or homothorax placed in the distal domain induce intercalary expression of dachshund in surrounding cells and reorient planar cell polarity of those cells. Escargot and homothorax-expressing cells also sort out from other cells in the distal domain. We suggest that inductive cell communication between the proximodistal domains, which is maintained in part by a cell-sorting mechanism, is the cellular basis for an intercalary mechanism of the proximodistal axis patterning of the limb.

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    • p38 mitogen-activated protein kinase can be involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis Peer-reviewed

      T Adachi-Yamada, M Nakamura, K Irie, Y Tomoyasu, Y Sano, E Mori, S Goto, N Ueno, Y Nishida, K Matsumoto

      MOLECULAR AND CELLULAR BIOLOGY19 ( 3 ) 2322 - 2329   3 1999

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      p38 mitogen-activated protein kinase (p38) has been extensively studied as a stress-responsive kinase, but its role in development remains unknown. The fruit fly, Drosophila melanogaster, has two p38 genes, D-p38a and D-p38b. To elucidate the developmental function of the Drosophila p38's, we used various genetic and pharmacological manipulations to interfere with their functions: expression of a dominant-negative form of D-p38b, expression of antisense D-p38b RNA, reduction of the D-p38 gene dosage, and treatment with the p38 inhibitor SB203580. Expression of a dominant-negative D-p38b in the wing imaginal disc caused a decapentaplegic (dpp)-like phenotype and enhanced the phenotype of a dpp mutant. Dpp is a secretory ligand belonging to the transforming growth factor beta superfamily which triggers various morphogenetic processes through interaction with the receptor Thick veins (Tkv). Inhibition of D-p38b function also caused the suppression of the wing phenotype induced by constitutively active Tkv (Tkv(CA)). Mosaic analysis revealed that D-p38b regulates the Tkv-dependent transcription of the optomotor-blind (omb) gene in non-Dpp-producing cells, indicating that the site of D-p38b action is downstream of Tkv. Furthermore, forced expression of Tkv(CA) induced an increase in the phosphorylated active form(s) of D-p38(s). These results demonstrate that p38, in addition to its role as a transducer of emergency stress signaling, may function to modulate Dpp signaling.

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    • Cell migration within the embryonic limb primordium of Drosophila as revealed by a novel fluorescence method to visualize mRNA and protein Peer-reviewed International journal

      S Goto, S Hayashi

      DEVELOPMENT GENES AND EVOLUTION207 ( 3 ) 194 - 198   8 1997

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      We report a new technique using fluorescent probes to detect a mRNA and a protein simultaneously in the Drasophila embryo. For in situ hybridization, 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP)/Fast Red TR was used as a fluorescent substrate for alkaline phosphatase. It was possible to compare protein and mRNA expression on a cell by cell basis with a laser scanning confocal microscope. We applied this technique to analyse the dynamics of Distal-less (Dll) enhancer activity in the thoracic limb primordium in the early Drosophila embryo. We stained embryos bearing the Dll early enhancer (Dll-304) fused to the Escherichia coli lacZ gene. LacZ mRNA was delectable in the ventral region of the limb primordium, and beta-galactosidase protein in the dorsal region. In the middle, both mRNA and protein were detectable. These results suggest that the Dll enhancer is activated in the ventral region of the Limb primordium and that Dll-positive cells migrate from a ventral position to a dorsal one within a single limb primordium.

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    • Specification of the embryonic limb primordium by graded activity of Decapentaplegic Peer-reviewed

      S Goto, S Hayashi

      DEVELOPMENT124 ( 1 ) 125 - 132   1 1997

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      Two thoracic limbs of Drosophila, the leg and the wing, originate from a common cluster of cells that include the source of two secreted signaling molecules, Decapentaplegic and Wingless. We show that Wingless, but not Decapentaplegic, is responsible for initial specification of the limb primordia with a distal identity. Limb formation is restricted to the lateral position of the embryo by negative control of the early function of Decapentaplegic and the EGF receptor homolog that determine the global dorsoventral pattern. Late function of Decapentaplegic locally determines two additional cell identities in a dosage dependent manner. Loss of Decapentaplegic activity results in a deletion of the proximal structures of the limb, which is in contrast to the consequence of decapentaplegic mutations in the imaginal disc, which cause a deletion of distal structures. The results indicate that the limb pattern elements are added in a distal to proximal direction in the embryo, which is opposite to what is happening in the growing imaginal disc. We propose that Wingless and Decapentaplegic act sequentially to initiate the proximodistal axis.

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    • Enhancer-trap detection of expression patterns corresponding to the polar coordinate system in the imaginal discs of Drosophila melanogaster Peer-reviewed International journal

      Satoshi Goto, Teiichi Tanimura, Yoshiki Hotta

      Roux's Archives of Developmental Biology204 ( 6 ) 378 - 391   6 1995

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      We have isolated three classes of "enhancertrap" lines of Drosophila in which lacZ expression patterns in the imaginal discs are consistent with the idea of a polar (radial and angular) coordinate system of positional information. In the first class (HZ76), a circular pattern was expressed transiently during the mid-third instar larval stage when the radial components of the coordinate are probably generated. The expression patterns of the second class (HZ84) were sector-shaped and circular in the leg disc, suggesting a correlation with both radial and angular coordinate values. The expression patterns found in the third class (PZ63 and PZ22) were circular and appeared to reflect radial positional values. Expression in the latter two classes always began in the presumptive dorsal region of the leg disc and gradually spread to the ventral region. These developmental profiles of expression suggested the existence of a centre that initiates patterned gene expression in the presumptive dorsal region of the leg disc. The PZ22 line showed transient expression during tarsal segmentation, suggesting its involvement in tarsal segment formation. We have cloned the PZ22 gene and partially determined its sequence. The deduced amino acid sequence contained a zinc finger motif found in DNA/RNA binding proteins. By in situ hybridization, we determined that the PZ22 gene was transcribed in the leg disc in a pattern identical to that of the lacZ expression. In addition, it was expressed transiently in the embryonic mesoderm during mesoderm segmentation. The PZ22 gene, therefore, may function both in mesodermal segmentation in the embryo and in tarsal segmentation in the imaginal disc. © 1995 Springer-Verlag.

      DOI: 10.1007/BF00360483

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    • PURIFICATION, CHARACTERIZATION, AND AMINO-ACID-SEQUENCES OF PEPSINOGENS AND PEPSINS FROM THE ESOPHAGEAL MUCOSA OF BULLFROG (RANA-CATESBEIANA) Peer-reviewed

      E YAKABE, M TANJI, M ICHINOSE, S GOTO, K MIKI, K KUROKAWA, H ITO, T KAGEYAMA, K TAKAHASHI

      JOURNAL OF BIOLOGICAL CHEMISTRY266 ( 33 ) 22436 - 22443   11 1991

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      Two pepsinogens (pepsinogens 1 and 2) were purified from the esophageal mucosa of the bullfrog (Rana catesbeiana), and their molecular weights were determined to be 40,100 and 39,200, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 70-residue sequences of both pepsinogens are the same, including the 36-residue activation segment. Furthermore, a cDNA clone encoding frog pepsinogen was obtained and sequenced, which permitted deduction of the complete amino acid sequence (368 residues) of one of the pepsinogen isozymogens. The calculated molecular weight of the protein (40,034) coincided well with the values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results are incompatible with the previous report (Shugerman R. P., Hirschowitz, B. I., Bhown, A. S., Schrohenloher, R. E., and Spenney, J. G. (1982) J. Biol. Chem. 257, 795-798) that the major pepsinogen isolated from the bullfrog esophageal gland is a unique "mini" pepsinogen with a molecular weight of approximately 12,000-34,000.
      The two pepsinogens were immunologically indistinguishable from each other and related to human pepsinogen C. The deduced amino acid sequence was also more homologous with those of pepsinogens C than those of pepsinogens A and prochymosin. These results indicate that the frog pepsinogens belong to the pepsinogen C group. They were both glycoproteins, and therefore, this is the first finding of carbohydrate-containing pepsinogens C. Both pepsinogens were activated to pepsins in the same manner by an apparent one-step mechanism. The resulting pepsins were enzymatically indistinguishable from each other, and their properties resembled those of tuna pepsins.

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    • ISOLATION OF DROSOPHILA GENOMIC CLONES HOMOLOGOUS TO THE EEL SODIUM-CHANNEL GENE Peer-reviewed

      H OKAMOTO, K SAKAI, S GOTO, E TAKASUISHIKAWA, Y HOTTA

      PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES63 ( 7 ) 284 - 288   9 1987

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    Misc.

    • 翻訳後修飾と選別輸送をつかさどる小胞体・ゴルジ体の“オルガネラ・ゾーン” Invited Peer-reviewed

      後藤聡, 山本(日野)美紀

      生体の科学69   551 - 555   12 2018

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    • In Vivo RNAi-Based Screens: Studies in Model Organisms Invited Peer-reviewed International journal

      Miki Yamamoto-Hino, Satoshi Goto

      GENES4 ( 4 ) 646 - 665   12 2013

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      Language:English   Publisher:MDPI AG  

      RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.

      DOI: 10.3390/genes4040646

      PubMed

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    • ユビキチン化によるFrizzledのリソソーム分解を介したWntシグナル強度の制御 Peer-reviewed

      Daocharad Burana, 後藤聡, 駒田雅之

      細胞工学32   396 - 400   2013

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    • Balanced ubiquitination determines cellular responsiveness to extracellular stimuli Invited Peer-reviewed International journal

      Akiko Mukai, Miki Yamamoto-Hino, Masayuki Komada, Hideyuki Okano, Satoshi Goto

      CELLULAR AND MOLECULAR LIFE SCIENCES69 ( 23 ) 4007 - 4016   12 2012

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      Language:English   Publisher:SPRINGER BASEL AG  

      Signal strength evoked by ligand stimulation is crucial for cellular responses such as fate decision, cell survival/death, secretion, and migration. For example, morphogens are secreted signaling molecules that form concentration gradients within tissues and induce distinct cell fates in a signal strength-dependent manner. In addition to extracellular ligand abundance, the sensitivity of signal-receiving cells to ligands also influences signal strength. Cell sensitivity to ligands is controlled at various levels: receptor presentation at the cell surface, positive/negative regulation of signal transduction, and target gene activation/repression. While the regulation of signal transduction and gene transcription is well studied, receptor presentation is still not fully understood. Recently, it was reported that cellular sensitivity to the Wingless (Wg)/Wnt morphogen is regulated by balanced ubiquitination and deubiquitination of its receptor Frizzled (Fz). In this review, we review how ubiquitination regulates receptor presentation at the cell surface for the detection of extracellular signal strength.

      DOI: 10.1007/s00018-012-1084-4

      PubMed

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    • 糖鎖関連遺伝子の大規模スクリーニング

      山本(日野)美紀, 山本(日野)美紀, 蟹江善美, 粟野若枝, 木下聖子, 西原祥子, 岡野栄之, 上田龍, 蟹江治, 蟹江治, 後藤聡, 後藤聡

      生化学   2011

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    • 受容体のユビキチン化と脱ユビキチン化を介した細胞のWnt応答性の制御 Peer-reviewed

      駒田雅之, 後藤聡

      細胞工学29   1237 - 1243   2010

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    • マススペクトロメトリー法によるショウジョウバエの糖タンパク質chaoptinの構造解析

      蟹江善美, 日野美紀, 後藤聡, 蟹江治

      日本糖質学会年会要旨集28th   2008

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    • 糖鎖修飾の場—ゴルジ体ユニットによる糖鎖修飾の制御 Invited Peer-reviewed

      後藤聡

      蛋白質核酸酵素53   1475 - 1479   2008

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    • ショウジョウバエにおけるメンブレントラフィック Invited Peer-reviewed

      阿部将人, 後藤聡

      蛋白質核酸酵素53   2193 - 2199   2008

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    • 糖ペプチドのMS解析のためのアルカリ条件逆相HPLC精製法

      蟹江善美, 榎本明珠子, 蟹江治, 後藤聡

      質量分析総合討論会講演要旨集55th   2007

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    • ショウジョウバエを用いた糖鎖修飾制御因子のゲノムワイドスクリーニング

      山本(日野)美紀, 矢野弘之, 蟹江善美, 粟野若枝, 平井ゆう, 桑原玲子, 木下(青木)聖子, 上田龍, 西原祥子, 西原祥子, 蟹江治, 後藤聡, 後藤聡

      生化学   2007

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    • Regulation of glycosylation by golgi units Invited Peer-reviewed

      Satoshi Goto

      Trends in Glycoscience and Glycotechnology18 ( 104 ) 347 - 351   2006

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      Language:English   Publishing type:Book review, literature introduction, etc.   Publisher:Gakushin Publishing Company  

      Most secretory and transmembrane proteins are post-translationally modified with various glycans. The structures of glycans attached to the proteins depend on proteins, cells, tissues, ages, species and individuals. However, the mechanism of regulation to generate such diverse glycans in a cell has not been revealed. In this review, we would like to discuss our finding, "Golgi units" that would regulate the variety of glycosylation. ©2006 FCCA (Forum: Carbohydrates Coming of Age).

      DOI: 10.4052/tigg.18.347

      Scopus

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    • RNAi変異体ハエバンクを用いたショウジョウバエ遺伝子機能解析

      藤谷和子, 佐渡由希子, 高橋邦明, 内藤雄樹, 山田智之, 森下真一, 西郷薫, 後藤聡, 西原祥子, 上田龍

      日本分子生物学会年会講演要旨集28th   543   25 11 2005

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      Language:Japanese  

      J-GLOBAL

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    • マススペクトロメトリー法によるショウジョウバエの糖タンパク質の構造解析

      蟹江善美, 日野美紀, 後藤聡, 蟹江治

      日本糖質学会年会要旨集25th   2005

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    • Functional glycomics using Drosophila RNAi system

      S Nishihara, M Hino, H Yoshida, N Sasaki, S Goto, H Toyoda, R Ueda

      GLYCOBIOLOGY14 ( 11 ) 1065 - 1065   11 2004

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      Language:English   Publishing type:Research paper, summary (international conference)   Publisher:OXFORD UNIV PRESS INC  

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    • Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter

      S Kamiyama, T Suda, R Ueda, H Yoshida, N Kikuchi, Y Chiba, S Goto, H Toyoda, H Narimatsu, Y Jigami, S Nishihara

      GLYCOBIOLOGY13 ( 11 ) 857 - 857   11 2003

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      Language:English   Publishing type:Research paper, summary (international conference)   Publisher:OXFORD UNIV PRESS INC  

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    • PAPSトランスポーターのクローニングと機能解析~PAPSトランスポーターは生命維持に必須である

      神山伸, 須田健, 上田龍, 浜本洋, 鈴木正之, 菊池紀広, 千葉靖典, 後藤聡, 西原祥子

      日本糖質学会年会要旨集24th   52   3 7 2003

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      Language:Japanese  

      J-GLOBAL

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    • 発生のおける小胞輸送の役割 Invited Peer-reviewed

      後藤聡

      実験医学21   136 - 141   2003

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      Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (other)  

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    • ショウジョウバエ脚のパターン形成、近遠軸形成機構 Invited Peer-reviewed

      後藤聡, 林茂生

      実験医学17   306 - 312   1999

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      Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (other)  

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    • 発生研究のための蛍光プローブによるmRNAとタンパク質の二重染色法 Invited Peer-reviewed

      後藤聡, 林茂生

      実験医学16   523 - 527   1998

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      Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (other)  

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    • Dpp, Wgによるショウジョウバエの翅・肢誘導と近遠軸形成 Invited Peer-reviewed

      後藤聡, 林茂生

      実験医学15   342 - 349   1997

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      Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (other)  

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    Books and Other Publications

    • Localization of glycosyl enzymes and nucleotide-sugar transporters in the endoplasmic reticulum and the Golgi apparatus

      Yamamoto-Hino,M, Goto,S( Role: Joint author)

      SpringerReference, Heidelberg  2014 

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      Language:English Book type:Other

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    • 「ゴルジ体(糖修飾の場として)」 生化学辞典

      後藤聡( Role: Joint author)

      朝倉書店  2012 

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      Language:Japanese Book type:Dictionary, encyclopedia

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    • 「EGF経路」「ERK/MAPK経路」「Hedgehogシグナル」「AKT」 岩波生物学辞典

      後藤聡( Role: Joint author)

      岩波書店  2012 

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      Language:Japanese Book type:Dictionary, encyclopedia

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    • Structure, Function and Formation of Glycans in Drosophila.

      Yamamoto-Hino,M, Okano,H, Kanie,O, Goto,S( Role: Joint author)

      Nova Science publishers, Inc., NY  2012 

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      Language:English Book type:Scholarly book

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    • 5. 「成虫原基」新生化学実験講座「発生・分化・老化」

      後藤聡, 谷村禎一( Role: Joint author)

      東京化学同人  1992 

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      Language:Japanese Book type:Dictionary, encyclopedia

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    Research Projects

    • Analysis of the mechanism of exacerbation of neurodegeneration by innate immunity

      Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research 

      Goto Satoshi

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      4 2019 - 3 2022

      Grant number:19K06445

      Grant amount:\4420000 ( Direct Cost: \3400000 、 Indirect Cost:\1020000 )

      Living organisms have an innate defense response called innate immunity to protect themselves from various pathogens. Recently, it has been shown that innate immunity is activated not only during infection but also in neurodegenerative diseases, but it is not well understood whether the activation of innate immunity is involved in the progression of neurodegeneration. Using a fly model of neurodegenerative disease, we have shown that a molecule induced during innate immune activation acts in a pro-neurodegenerative manner, and we have named this molecule D-knif. In this study, we investigated whether the same phenomenon is observed in mammals and the effect of the human D-knif family on human neurons, and were able to show that the D-knif family molecules also act to promote neurodegeneration in humans.

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    • 糖鎖およびリン酸修飾の基盤となる選別輸送ゾーンの分子機構と生理機能の解析

      文部科学省  科学研究費助成事業 

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      7 2017 - 3 2022

      Grant type:Competitive

      Grant amount:\17900000 ( Direct Cost: \12530000 、 Indirect Cost:\5370000 )

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    • Toward an integrative understanding of functional zones in organelles

      Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research 

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      6 2017 - 3 2022

      Grant number:17H06413

      Grant amount:\120640000 ( Direct Cost: \92800000 、 Indirect Cost:\27840000 )

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    • 核膜と小胞体の機能的連携

      日本学術振興会  科学研究費助成事業 

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      4 2016 - 3 2017

      Grant type:Competitive

      Grant amount:\2800000 ( Direct Cost: \1960000 、 Indirect Cost:\840000 )

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    • Mechanism of glycan change involved in the activation of innate immunity

      Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research 

      YAMAMOTO(HINO) MIKI, GOTO Satoshi, KASE Sawako

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      4 2014 - 3 2017

      Grant number:26440066

      Grant amount:\5070000 ( Direct Cost: \3900000 、 Indirect Cost:\1170000 )

      The innate immune system is the first line of defense encountered by invading pathogens. We have found that immune challenge leads to change host glycosylation, which involved in full activation of immune response. Our aim in this study was to reveal the mechanism of the host glycan change by immune challenge.
      In this study, we found that glucosaminoglycan (GAG) regulates immune activity. GAG suppresses hyper-activation of the Toll pathway in the steady state. Interestingly, pathogen infection reduces the expression of a GAG synthesizing enzyme to enhance immune responses. These results indicate that GAG plays an important role in both suppression of runaway inflammation and enhancement of immune responses.

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    • GPI修飾の新規メカニズムとその破綻によるシグナル伝達異常の解明

      文部科学省  科学研究費助成事業 

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      4 2013 - 3 2015

      Grant type:Competitive

      Grant amount:\8200000 ( Direct Cost: \5740000 、 Indirect Cost:\2460000 )

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    • 自然免疫をダイナミックに制御する糖鎖修飾の解析

      日本学術振興会  科学研究費助成事業 

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      4 2012 - 4 2014

      Grant type:Competitive

      Grant amount:\14100000 ( Direct Cost: \9870000 、 Indirect Cost:\4230000 )

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    • mRNAの局在による翻訳後修飾の制御

      文部科学省  科学研究費助成事業 

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      4 2011 - 3 2013

      Grant type:Competitive

      Grant amount:\7200000 ( Direct Cost: \5040000 、 Indirect Cost:\2160000 )

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    • 糖鎖修飾による自然免疫システムの多様な制御機構

      住友財団  住友財団、基礎科学研究助成 

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      4 2011 - 3 2013

      Grant type:Competitive

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    • Regulatory mechanism of glycosylation

      Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research 

      GOTO Satoshi

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      2009 - 2011

      Grant number:21570157

      Grant amount:\5330000 ( Direct Cost: \4100000 、 Indirect Cost:\1230000 )

      Glycosylation plays an important role in various physiological processes such as development, immunity and neural functions. However, mechanisms regulating glycosylation still remain largely unknown. To elucidate it, we performed a large scale screen using Drosophila RNAi library and identified novel genes. We analyzed the functions of the genes and found novel mechanisms of mRNA of glycosylation related genes.

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    • mRNA局在による翻訳多様性の制御

      日本学術振興会  科学研究費助成事業 

      後藤 聡

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      2009 - 2010

      Grant number:21112525

      Grant amount:\7800000 ( Direct Cost: \6000000 、 Indirect Cost:\1800000 )

      私達は、ショウジョウバエの初期胚においてGPIアンカーを生合成する膜結合型酵素Pig-BのmRNAが一部の粗面小胞体領域に局在していることを見出していた。そこで、本研究ではmRNAの局在がPig-B蛋白質の活性に重要な役割を果たしているかを明らかにことを目指した。具体的には、Pig-BmRNAの局在を変化させるために、その3'UTRを改変したコンストラクトを発現するショウジョウバエ系統を作成した。実際に、その局在が変化したかを調べたところ、本来の領域とは異なる小胞体領域に局在することを確認した。現在、本来とは異なる領域で翻訳されたPig-B蛋白質が正しい活性を有しているかを確認中である。そのために、本来とは異なる領域で翻訳されるPig-Bをpig-B変異体で発現させ、pig-B変異体の表現型を抑圧できるかを調べている。
      また、Pig-B蛋白質に対する抗体を作成したところ、endogenous Pig-B蛋白質を検出する良い抗体が得られた。それを用いて細胞染色したところ、驚いたことに、いままで言われてきた小胞体ではなく、核膜に局在していた。このことは、GPIが、いままでいわれてきたような小胞体ではなく、核膜で生合成されていることを示している。
      さらに、GPIを蛋白質に付加させるTransamidaseに対する抗体の作成に取り掛かった。その抗体を用いることによって、実際にGPIが付加されるのが小胞体なのか、核膜なのかが明らかになると思われる。

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    • 糖修飾による核膜孔複合体と核形態の維持メカニズム

      日本学術振興会  科学研究費助成事業 

      後藤 聡

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      2007 - 2008

      Grant number:19038029

      Grant amount:\7800000 ( Direct Cost: \7800000 )

      核膜孔は、細胞内でO-GlcNAcにより糖鎖修飾される蛋白質複合体としてもよく知られている。しかし、その糖鎖修飾の意義については必ずしも明らかになっていない。我々は、先行実験よりショウジョウバエの精子形成過程では核膜孔上の糖修飾がダイナミックに制御されていることを見出した。すなわち、体細胞では、常に糖修飾を受けている核膜孔が、精子形成過程の減数分裂直後に一過的に糖修飾が失われることを見出した。さらに興味深いことに、糖修飾を失った核膜孔は徐々に消失することも見出した。これらの観察結果は、O-GlcNAcによる糖修飾が核膜孔の安定性に寄与している可能性を示唆している。そこで、我々は核膜孔上の糖鎖の役割を解析するために、O-GlcNAcを付加する酵素O-GlcNAc Transferase(OGT)およびO-GlcNAcを脱離させる酵素O-GlcNAc'aseのノックダウンや強制発現できる系を構築した。その系を用いてショウジョウバエの精巣でOGTをノックダウンしたところ、正常よりも早い段階から核膜孔が核膜から一部消失することを見出した。このことは、糖修飾が核膜孔の安定性に寄与していることを強く示唆している。そこで、O-GlcNAcによって糖修飾を受ける主要な核膜孔蛋白質であるNup62をノックダウンしたところ、一部の核膜孔が核膜から消失することも見出した。この結果は、Nup62が核膜孔の形成維持に重要な役割を果たしていることを示している。また、Nup62をノックダウンすると器官が正常に発生せず、結果として致死になる。このことは、Nup62が生体にとって必須であることを示している。

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    • 分泌と分解による細胞外因子の制御メカニズム

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 矢野 弘之

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      2006 - 2007

      Grant number:18060044

      Grant amount:\7000000 ( Direct Cost: \7000000 )

      細胞間の情報伝達や接着は,分泌蛋白質や細胞膜蛋白質によって制御されている。そのような蛋白質は,極性のある上皮細胞や神経細胞上では,その極性にそって異なる局在をしているものが多い。さらに,それらの蛋白質は糖鎖や脂質による翻訳後修飾を受けることによって,正しい機能を獲得する。例えば,ショウジョウバエの分泌性シグナル分子であるWingless(Wg)は,脂質修飾および糖修飾を受けた後,上皮細胞のアピカル側へと分泌される。また,ショウジョウバエの眼の形成に必須であるChaoptinという細胞接着因子も,糖修飾を受けた後,光受容細胞のアピカル側の細胞膜へと輸送される。しかし,これらの蛋白質の分泌や極性輸送の制御メカニズムについてはまだまだわからないことが数多く残されている。さらに,翻訳後修飾の制御メカニズムについてはほとんどわかっていないといっても過言ではない。そこで,我々は,ショウジョウバエを用いて蛋白質の分泌や極性輸送の制御メカニズムを解析したところ,分泌制御についてはTSG101と呼ばれる分子がWinglessの分泌に重要な役割を果たしていることを見出した。さらに解析を進めたところ,TSG101が関わる分泌過程はERからゴルジ体への輸送過程であることが明らかとなった。また,これとは独立に進めたChaoptin の極性輸送に関与する分子のゲノムワイドスクリーニングの結果,非常に興味深い分子を同定することにも成功した。

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    • 糖ー核酸トランスポーターの細胞内局在と機能

      日本学術振興会  科学研究費助成事業 

      後藤 聡

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      2006 - 2007

      Grant number:18059036

      Grant amount:\4600000 ( Direct Cost: \4600000 )

      糖核酸トランスポーターは,小胞体やゴルジ体の膜上にあり,細胞質で生成された糖-核酸を小胞体やゴルジ体の内腔に輸送する蛋白質である。輸送された糖-核酸は,様々な蛋白質や脂質の糖修飾に用いられる。申請者らは,Fringe-Connections(FRC)と呼ばれる新規の糖-核酸トランスポーターを同定し,それがすべてのゴルジ体にはなく,ごく一部のゴルジ体に局在し,ごく一部の蛋白質の糖修飾にだけ関与することを見出した。しかし,FRC以外のトランスポーターについては,ほとんど解析がなされていない。そこで,本研究では,FRC以外のトランスポーターについても,一部のゴルジ体にしか局在しないものがあるのか,さらに,その局在によって糖修飾の特異性が制御されているのかを明らかにする予定である。本年度,我々は,データベースから抽出したトランスポーターについて,タグを付加し,ショウジョウバエ個体で発現させて,細胞内の局在を詳細に解析した。特に,すべてのゴルジ体に局在するのか,一部のゴルジ体だけに局在するのかに着目して解析し,一部のゴルジ体だけに局在するトランスポーターを同定した。さらに,トランスポーター同士の局在を比較して,同じゴルジ体に局在するトランスポーターのグループと異なるゴルジ体に局在するトランスポーターグループなどに分類し,トランスポーターの種類と局在についての詳細なリストを作成している。さらに,トランスポーターをノックアウトしたところ,思いもかけない表現型が得られた。

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    • 極性輸送の分子機構

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 阿部 人

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      2006 - 2007

      Grant number:18050045

      Grant amount:\6600000 ( Direct Cost: \6600000 )

      発生の過程で組織がダイナミックな形態形成運動を行う場合,それに伴って様々な膜蛋白質の局在もダイナミックに変化する。しかし,そのような膜蛋白質の移動を制御するメカニズムはよくわかっていない。私達は,様々な膜蛋白質がダイナミックに輸送されるショウジョウバエの翅形成過程に着目し,その分子メカニズムを解析している。特に,apical側の接着構造の形成に関わるE-cadherinやplanar cell polarityの形成に関わるFlamingo,そして,basal側の接着構造の形成に関わるIntegrinに着目し,様々な小胞輸送関連分子をスクリーニングしたところ,PI3Kinase classIIIと,それによって生成されるPI(3)Pに結合するFYVEdomainを持つ遺伝子が同定された。まず,PI3KinaseのノックダウンでE-cadherin,Flamingo,Integrinの局在を調べたところ,細胞内に異常な蓄積が観察された。ところが,FYVEdomainをもつRbsn-5のノックダウンでは,Integrinの局在に異常は観察できたが,E-cadherinとFlamingoの局在には異常は観察できなかった。この結果は,PI3Kinaseは膜蛋白質一般の輸送に関与しているが,その下流のFYVEdomainをもつ遺伝子において,輸送の選択性が制御されていることを示唆している。さらに,Rbsn-5の機能を解析するために,細胞内の輸送コンパートメントについて調べたところ,興味深いことに,Rbsn-5が局在するbasal側のエンドソームはapical側のエンドソームと比較して著しく大きいことがわかった。また,そのbasal側のエンドソームの形成にRbsn-5が必要であることもわかった。以上の結果より,極性細胞において,apical側とbasal側ではエンドソームが異なることによって,違う制御が可能になっており,それに関わる分子のひとつがRbsn-5であることがわかった。

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    • ゴルジ体抽出画分による糖修飾の再構築

      日本学術振興会  科学研究費助成事業 

      佐藤 利行, 後藤 聡, 蟹江 治

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      2005 - 2006

      Grant number:17657048

      Grant amount:\3600000 ( Direct Cost: \3600000 )

      糖タンパク質の性質や活性を試験管内で測定するには、合成した蛋白質に正しい糖鎖を付加する必要がある。小胞体で翻訳されたタンパク質はゴルジ体へと小胞輸送され、順序よく糖が付加される。しかし、試験管内で糖鎖を制御しながら付加する技術は発展途上であり、本研究では一連の反応を試験管内で再現し、「試験管内で任意の糖鎖を付加する技術」を開発する。
      昨年度は、出芽酵母Saccharomyces cerevisiaeの生体より小胞体膜画分やゴルジ体膜画分、可溶性画分を抽出する方法を確立した。さらにそれらを用いて、試験管内での標的タンパク質の糖修飾系を確立した。本年度は、試験管内での糖修飾反応系の検証をさらに行った。
      試験管内で反応を起こし、糖修飾による標的タンパク質の高分子化を明確にするために、S.cerevisiaeのN型糖鎖合成の初期反応に関与する糖転移酵素複合体M-PolIのサブユニットの1つ、Vanlpをコードする遺伝子(van1)を破壊したS.cerevisiaeを形質転換し、相同組換えにより標的タンパク質を高発現するvan1破壊株を作製した。その破壊株から小胞体膜画分を抽出した。その小胞体中には、N型糖鎖が成熟型より短く付加された標的タンパク質が存在した。
      この小胞体画分と、野生株から別々に抽出したゴルジ体画分および可溶性画分を試験管内で混合し小胞輸送を起こした。結果、標的タンパク質が高分子化されたことをSDS-PAGE上で確認し、試験管内で糖鎖修飾されたと示唆された。しかし、SDS-PAGE上で検出された高分子化は、野生株における成熟型標的タンパク質の見かけ分子量よりも大きいものであった。
      反応により糖修飾された標的タンパク質の分子量は、野生株における成熟型標的タンパク質の分子量と等しくなるとの予想とは反するものであり、試験管内において糖鎖修飾を制御するためには、細胞におけるさらなる糖鎖伸長決定機構の解明が必要であると示唆された。

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    • 精子形成過程における核膜孔複合体の脱糖鎖と崩壊について

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 矢野 弘之

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      2005 - 2006

      Grant number:17050031

      Grant amount:\6400000 ( Direct Cost: \6400000 )

      核膜孔は、細胞内でO-GlcNAcにより糖鎖修飾される蛋白質複合体としてもよく知られている。しかし、その糖鎖修飾の意義については必ずしも明らかになっていない。我々は、先行実験よりショウジョウバエの精子形成過程では核膜孔上の糖修飾がダイナミックに制御されていることを見出した。すなわち、体細胞では、常に糖修飾を受けている核膜孔が、精子形成過程の減数分裂直後に一過的に糖修飾が失われることを見出した。さらに興味深いことに、糖修飾を失った核膜孔は徐々に消失することも見出した。これらの観察結果は、O-GlcNAcによる糖修飾が核膜孔の安定性に寄与している可能性を示唆している。そこで、我々は核膜孔上の糖鎖の役割を解析するために、O-GlcNAcを付加する酵素O-GlcNAc Transferase(OGT)およびO-GlcNAcを脱離させる酵素O-GlcNAc'aseのノックダウンや強制発現できる系を構築した。その系を用いてショウジョウバエの精巣でOGTをノックダウンしたところ、正常よりも早い段階から核膜孔が核膜から一部消失することを見出した。このことは、糖修飾が核膜孔の安定性に寄与していることを強く示唆している。そこで、O-GlcNAcによって糖修飾を受ける主要な核膜孔蛋白質であるNup62をノックダウンしたところ、一部の核膜孔が核膜から消失することも見出した。この結果は、Nup62が核膜孔の形成維持に重要な役割を果たしていることを示している。

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    • 多細胞個体における小胞輸送機能についての網羅的解析

      日本学術振興会  科学研究費助成事業 

      後藤 聡

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      2004 - 2005

      Grant number:16044251

      Grant amount:\6100000 ( Direct Cost: \6100000 )

      小胞輸送の多細胞個体レベルでの役割を明らかにするために、ショウジョウバエをモデル系に用いて解析をおこなった。まず、酵母や培養細胞を用いた先行実験より明らかにされてきた「小胞輸送に関与する分子」のショウジョウバエホモログをデータベースから検索しリスト化した。そのリストにある遺伝子について、ショウジョウバエ個体でRNAiによるノックダウンを誘導できる系統を作成した。それらの系統を用いて、ショウジョウバエの翅、眼、背中で各遺伝子をノックダウンし、どのような表現型が見られるかを詳細に観察した。特に翅には、翅脈・感覚器官・剛毛・微毛などの構造があり、それらが形成されるのに必要な情報伝達系などがわかっているので、翅の構造異常を詳細に観察することによって、小胞輸送がどのような情報伝達系の制御に関わっているかを大まかに推測することができる。そこで翅の表現型をもとに遺伝子を分類したところ、大きく5種類に分類することができた。その中で特に我々が注目しているのは、細胞の大きさが変化する表現型であった。というのも、細胞の大きさの制御には、Insulin-PI3K情報伝達系が関与していることがわかっているが、その情報伝達経路の制御に小胞輸送がどのように関与しているは明らかではなかったからである。
      我々は、同定した新規遺伝子が本当にInsulin-PI3K情報伝達経路の制御に関与しているのか、もし関与しているとするならば、どのようなメカニズムかということを今後明らかにしていく予定である。

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    • 発生における分子機構のイメージング

      日本学術振興会  科学研究費助成事業 

      川崎 能彦, 後藤 聡, 平田 たつみ, 後藤 聡

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      1999 - 2003

      Grant number:11242207

      Grant amount:\55900000 ( Direct Cost: \55900000 )

      細胞の形態変化や移動は、発生において重要な役割を果たしている。しかし、その制御機構の生体における解析は遅れている。その主な理由として、生体内で細胞の形態変化・運動を経時的に観察し、そのメカニズムを解析する系が整っていないことが挙げられる。そこで本研究では、ショウジョウバエや哺乳動物の脳組織片を用いて、細胞形態・運動の制御機構を解析を進めた。
      後藤らは、翻訳後修飾に注目して解析を行った。発生において糖修飾やプロセッシング等の翻訳後修飾は重要な役割を果たしている。しかし、そういった修飾がどのように制御されているのかについては、ほとんどわかっていない。翻訳後修飾が行われる場は、主としてゴルジ体である。そこで、様々なゴルジ体マーカーを用いて詳細な細胞内イメージングをおこなったところ、驚いたことに、ゴルジ体には多様な種類があることが明らかとなった。この結果が示すところは、我々が見いだした多様なゴルジ体によって、多様な翻訳後修飾が制御されている可能性である。そこで、遺伝学的、生化学的解析に加え、実際の糖修飾が行われている過程をイメージングしたところ、それぞれ異なるゴルジ体では異なる糖修飾がなされていることがわかった。
      川崎、平田らは、神経細胞の移動に注目して解析を行った。マウス胚の終脳において腹側接線方向への新規の細胞移動を明らかにした後、その細胞移動の制御機構について解析を進めた。その結果、終脳背側の新皮質領域には、この細胞の腹側接線方向への移動を促進する何らかの作用が勾配を持って存在すること。終脳腹側の線状体原基の領域には、この細胞の侵入を阻害する何らかの機構が存在することなどを見いだし、これらの細胞が移動する具体的な様子をイメージング技術を用いて明らかにすることに成功した。

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    • Signaling systems that respond to cell-cell contact and regulate epithelial polarity

      Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research 

      HAYASHI Shigeo, NAGAFUCHI Akira

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      1999 - 2002

      Grant number:11235202

      Grant amount:\100200000 ( Direct Cost: \100200000 )

      (Hayashi) To understand the molecular basis for cell motility control during organogenesisi of tubular organs, we have developed an in vivo imaging system of cytosletetal and membrane components and made the following findings.
      1)In vivo function of the small GTPase RAC was examined. Activation of RAC caused down-regulation of E-cadherin expression and inhibited epithelial cell adhesion and a loss of apical-basal polarity. The results suggests that Rac is primarily involved in maintaining plasticity of epithelia to assure smooth remodeling during morphogenesis.
      2)Extension of cytoplasmic processes was studied using tracheal terminal branch as a model, and was found that the stereotyped pattem of terminal branch extension correlates with expression of signaling molecules defining positional information in the epidermis. We found that extension of terminal branch is guided by Hedgehog and Dpp that are secreted from the epidermis, thereby correct matching of respiratory system with its target organ.
      3)A large scale screening for genes regulating epithelial morphogenesis was performed using a gain of function system. Several novel gene activity controlling cell adhesion, cell motility and guidance were identified.
      (Nagafuchi) To understand the precise roles of cell-cell adhesion in the establishment of epithelia, the function of cadherin-catenin system was assessed by gene target approach in F9 teratocarcinome cell line.
      1)F9 cell deficient in alpha-catenin still able to form tight junction. The result suggets that tight junction can form in the absence of cadherin-catenin dependent strong cell-cell adhesion.
      2)Functions of plakoglobin and beta-catenin were assessed by the gene knockout approach. F9 cells deficient of beta catenin are able to establish nearly complete epithelia. Knockout of plakoglobin caused defects in desmosome formation. Double knockout cells failed to express many of epithelia-specific genes, suggesting essential requirement for those two genes in epithelia formation. The results also suggest that the function of beta catenin can be compensated by plakoglobin.

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    • 1分子イメージングによる生体分子の時間的・空間的動態の解析

      日本学術振興会  科学研究費助成事業 

      後藤 聡

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      1999 - 2000

      Grant number:11780542

      Grant amount:\2200000 ( Direct Cost: \2200000 )

      分泌蛋白や膜蛋白の多くは、Golgi体において多様な糖鎖修飾を受けている。近年のショウジョウバエを用いた遺伝学的な解析から、糖鎖修飾が発生において重要な役割果たしていることが明らかとなってきた。申請者は、Golgi体に局在する新規の糖核酸輸送体(UST74C)を同定し、その変異体の解析を行った。変異体の表現型は、多様な細胞間相互作用に用いられているNotch受容体の変異形質によく似ていた。糖核酸は糖鎖修飾の基質であるので、この変異体においては糖修飾に異常が生じていると考えられた。そこで、変異体の蛋白質の糖鎖構造を幅広く調べたところ、Notch受容体上の糖鎖構造に特に異常が生じていた。なぜ、特定の蛋白質の糖鎖構造に異常が生じるかについて解析するために、この分子の細胞における分布を調べたところ、すべてのGolgi体ではなく一部のGolgi体にのみ局在していることがわかった。UST74Cが一部のGolgi体に局在する過程を観察するために、UST74CにGFP(Green Fluorescent Protein)を融合した遺伝子をもつトランスジェニックショウジョウバエを作成し、Golgi体の細胞内での動態を観察する計画を進めている。このことにより、Golgi体の多様性を生み出すメカニズムを解くことができると考えている。この研究は、細胞内小器官の動態を遺伝学的にアプローチできる系として、非常にユニークなものである。

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    • 複数の細胞外シグナル因子による遺伝子発現制御の解析

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 林 茂生

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      1999 - 1999

      Grant number:11154228

      Grant amount:\2100000 ( Direct Cost: \2100000 )

      発生や再生といった生命現象において、細胞の増殖や運命決定に細胞外からシグナルによる制御は重要な役割を果たしている。我々は、細胞運命に関わる遺伝子の発現が複数の細胞外シグナルによって制御されている系として、ショウジョウバエの翅・脚が形成される過程に着目して研究を進めている。
      ショウジョウバエの翅・脚は、TGF-beta superfamilyに属するDPPとWNT familyに属するWG、Drosophila EGF ReceptorのリガンドであるSPITZといった複数の細胞外シグナルによって胚期に誘導される。その際、それぞれのシグナル因子の発現が時間的空間的に異なったパターンで正しく制御されている。我々は、それぞれのシグナル因子の役割を調べるために、突然変異体や強制発現系を用いて解析し、さらにそれぞれのシグナル伝達経路においてクロストークの可能性を検討した。
      また、翅・脚形成過程において時間的空間的に発現が制御されている遺伝子を網羅的に検索するために、Gal4を用いたエンハンサートラップ系統を作成し、胚期と幼虫期に翅・脚原基で発現している系統、および成虫の脚の形態の異常を示す系統をスクリーニングしている。

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    • ショウジョウバエ脚のパターン形成機構の解析

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 林 茂生

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      1999 - 1999

      Grant number:11152236

      Grant amount:\2800000 ( Direct Cost: \2800000 )

      生物の形態は種によって多様であるが、共通性も多く見いだされる。このことは、形態形成のメカニズムには先祖型があり、それに修正を加えつつ多様な形態が生み出された可能性を示唆している。そこで、我々はショウジョウバエの脚をモデル系に用いて、形作りの基本メカニズムを明らかにし、形の多様性を生み出す機構を解析するための基本的情報を得たいと考えている。ショウジョウバエの脚は、胚期に誘導された原基が幼虫期に増殖・パターン形成を行い、蛹期に形態形成運動をすることによって形成される。我々は、胚期に翅・脚原基が誘導されるメカニズムについて、さらに幼虫期における近遠軸パターンの形成機構について解析し、新たなモデルを提出するに至った。しかし、脚形成機構の全貌を明らかにするにはほど遠い。というのも、脚の形成に関わる遺伝子のかなりのものが未知だからである。そこで、我々は脚形成に関わる遺伝子を網羅的に検索するために、Ga14を用いたエンハンサートラップ系統を作成・スクリーニングし、胚期と幼虫期に脚原基で発現している系統、および成虫の脚の形態に異常を示す系統を同定した。さらに、上述のスクリーニングで同定された系統のすべてについて、挿入箇所の同定を進めている。ショウジョウバエでは、全染色体の塩基配列を決定するゲノムプロジェクトが修了しており、その情報は本年度末あたりに公開される予定である。その情報をもとに、各系統で同定された発現パターンを示す遺伝子の候補を得る予定である。その結果、脚原基において、どのような遺伝子が、どのような発現パターンを示しているかを網羅的にリストアップすることができ、脚の形成機構メカニズムの解明に大きな進歩をもたらすことができるのではないかと考えている。

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    • 細胞外シグナル因子による遺伝子発現制御の解析

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 林 茂生

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      1998 - 1998

      Grant number:10173228

      Grant amount:\2000000 ( Direct Cost: \2000000 )

      本年度は、ショウジョウバエの翅・脚の原基が胚期に形成されるときに、EGFレセプターを介したシグナルが重要な役割を果たしていることを明らかにした。細胞外シグナル因子Wingless(Wg)によって翅・脚共通の前駆細胞が誘導されたのち、違う種類の細胞外シグナル因子Decapentaplegic(Dpp)の濃度の差によって翅または脚の運命に決定される。EGFレセプターの突然変異体では、この運命決定の過程に異常が生じて、脚原基が正常に形成されなくなった。また、EGFレセプターのリガンドであるSpitzの変異体や、レセプターの下流で働いていると考えられているMAPKカスケードの変異体でも同様の表現型を示したので、Spitz-EGFR-MAPKカスケードというセットが、脚の運命決定に必要であることがわかった。

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    • ショウジョウバエ肢の近遠軸形成の解析

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 林 茂生

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      1998 - 1998

      Grant number:10171235

      Grant amount:\2300000 ( Direct Cost: \2300000 )

      本年度は、ショウジョウバエの脚の近遠軸にそったパターン形成において、遠位部と近位部の細胞間相互作用が重要な役割を果たしていることを新たに見い出した。ショウジョウバエの脚の原基は胚期に誘導形成されるが、そのとき同時に、遠位部と近位部の二つの領域からなる最も単純な近遠軸も形成される。遠位部では、Distalless(Dll)というhomeobox typeの転写制御因子が、近位部では、escargot(esg)というzinc finger typeの転写制御因子が発現している。幼虫期に、それらの発現領域の中間でdachshund(dac)という転写制御因子が発現誘導される。dacの発現誘導には、遠位部で発現しているDllが必要であることがわかっていたが、今回我々は、近位部に発現しているesgも必要であることを新たに見い出し、遠位・近位部の相互作用こそが近遠軸形成に必須であることを示した。この結果は、パターン形成における新しいメカニズムを分子レベルで解く手がかりになると考えられる。

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    • Dppシグナルの強度による遺伝子発現制御の解析

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 林 茂生

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      1997 - 1997

      Grant number:09277223

      Grant amount:\2200000 ( Direct Cost: \2200000 )

      細胞外のシグナル因子によって転写が制御されているメカニズムについては、近年のシグナル伝達の研究によって多くの知見が得られている。ところが、発生・形態形成の過程におけるモルフォジェンと呼ばれるシグナル因子の場合、シグナルの強度に応じて標的遺伝子の発現が制御されていることが知られているが、そのメカニズムについてはほとんどわかっていない。我々は、ショウジョウバエの肢原基形成過程において、モルフォジェンと考えられているDecapentaplegic(Dpp)のシグナル強度が、細胞の運命決定に重要な役割を果たしていることを見い出した。つまり細胞にDppシグナルが強く入ったときには翅の運命に決定され、弱く入ったときには脚の運命に決定される。我々は、この過程に働く他のシグナル分子について解析したところ、ショウジョウバエのEGF receptor(EGFR)を介したシグナルが、Dppと逆の作用を示すことを見い出した。つまり、EGFRのmutantでは、脚原基を構成する細胞の数が減少し、そのかわり翅原基の細胞数が増加していた。このことは、翅または脚への運命決定にDppシグナルに加えてEGFRを介したシグナルも重要な役割を果たしていることを示している。我々はさらに、DppシグナルとEGFRを介したシグナルの関係について研究を進めている。

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    • Dpp,Wgによるショウジョウバエ翅・肢形成の解析

      日本学術振興会  科学研究費助成事業 

      後藤 聡, 林 茂生

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      1997 - 1997

      Grant number:09275230

      Grant amount:\2400000 ( Direct Cost: \2400000 )

      我々は、ショウジョウバエの肢を用いて器官形成とそのパターン形成について研究を行っている。ショウジョウバエの肢は、胚発生期に分泌性シグナル因子であるWgによって誘導され、その後Dppによって翅・脚への運命決定がおこなわれる。同時に脚の近遠軸の原型も形成される。本年度は、胚期に誘導された近遠軸の原型をもとに、どのようにして成虫の複雑な近遠軸が形成されるのかについて研究をおこなった。胚期の脚原基の近位部では転写制御因子であるescargot(esg)が発現し、遠位部では転写制御因子であるDistalless(Dll)が発現している。そこで、esgまたはDllを、本来発現していない場所の少数の細胞で強制発現させ、その効果を調べた。その結果、Dllを発現した細胞は、それ自身遠位部の構造を作るとともに周囲の細胞に働きかけて、より近位部の構造を誘導した。esgを発現した細胞は、周囲の細胞に、より遠位部の遺伝子発現を誘導した。以上の結果より、胚期に誘導された脚原基においてDllを発現している遠位部の細胞からのシグナルとesgを発現している近位部の細胞群からのシグナルにより、その間に中間部の運命が誘導されると考えられた。このことは、移植実験等の結果から提出されたintercalation modelと良い対応をみせており、我々の結果はintercalation modelの分子的な基盤をあたえるものと考えられる。

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