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Establishment of a cellular system for visualizing intracellular lipid rafts using a new cholesterol-binding probe

Research Project

Project/Area Number 14580660
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionTokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare

Principal Investigator

HAYASHI Masami  Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare, Tokyo Metropolitan Institute of Gerontology, Biomembrane Research Group, Research Associate, 東京都老人総合研究所・生体膜機能研究グループ, 研究助手 (00192279)

Co-Investigator(Kenkyū-buntansha) IWASHITA Yoshiko  Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare, Tokyo Metropolitan Institute of Gerontology, Biomembrane Research Group, Senior Research Scientist, 福祉振興財団・東京都老人総合研究所・生体膜機能研究グループ, 副参事研究員 (50111498)
Project Period (FY) 2002 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥2,900,000 (Direct Cost: ¥2,900,000)
KeywordsCHOLESTEROL / FIBROBLAST / LIPID RAFTS / PERERINGOLYSIN O / VISUALIZATION / GREEN FLUORESCENT PROTEIN / MICRODOMAIN / ラフト / 線維芽細胞 / 脂質マイクロドメイン / 脂質ミクロドメイン / MDCK細胞
Research Abstract

We prepared a non-cytolytic derivative of perfringolysin O (θ-toxin), a cholesterol-binding cytolysin, as a probe for cholesterol. It is already shown that the probe (BCθ; protease-nicked and biotinylated θ-toxin) binds selectively to cholesterol in cholesterol-rich membrane domains (lipid rafts) of intact cells, and is useful for visualizing cholesterol-rich membrane domains on the cell surface. To visualize further intracellular cholesterol-rich membrane domains of living cells, we established a cellular system. We have shown that the specificity and high affinity of BCθ for cholesterol and its property of targeting to cholesterol-rich membrane domains can all be ascribed to the C-terminal domain (D4) of θ-toxin. Based on this finding, we constructed a fused protein of D4 and enhanced green fluorescent protein (EGFP). First, the fused protein, EGFP-D4,was overexpressed in E. coli and purified for characterization. We confirmed that EGFP-D4 is a fluorescent protein targeting to cholesterol-rich membrane domains. Then, an EGFP-D4 DNA fragment was inserted into a vector for mammalian gene expression and introduced into mouse fibroblasts. We isolated several stable cell lines expressing EGFP-D4 at variable levels and showed that the expressed EGFP-D4 was partially associated with intracellular vesicular structures by fluorescence microscopy. Characterization of the vesicles is now under way.

Report

(3 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] Ohno-Iwashita, Y.et al.: "Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts"Anaerobe. 10(2). 125-134 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Reid, P.C.et al.: "A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain"J.Lipid Res.. 45(3). 582-591 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Aoki, T.et al.: "Sequestration of cross-linked membrane molecules to caveolae in two different pathways"Acta Histochemica et Cytochemica. 36(2). 165-171 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sugii, S.et al.: "Biotinylated theta toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells"J.Lipid Res.. 44(5). 1033-1041 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Moebius, W.et al.: "Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway"Traffic. 4. 222-231 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Shimada, Y.et al.: "The C-terminal domain of perfringolysin O is an essential cholestereol-binding unit targeting to cholesterol-rich microdomains"Eur.J.Biochem.. 269(24). 6195-6203 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ohno-Jwashita, Y. et al.: "Perfringolysin O, a cholesterol-binding Cytolysin, as a probe for lipid rafts."Anaerobe. 10(2). 125-134 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Reid, P.C.et al.: "A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain."J. Lipid Res.. 45(3). 582-591 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Aoki, T. et al.: "Sequestration of cross-linked membrane molecules to caveolae in two different pathways."Acta Histochemica et Cytochemica. 36(2). 165-171 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sugii, S. et al.: "Biotinylated theta toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells."J. Lipid Res.. 44(5). 1033-1041 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Moebius, W. et al.: "Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway."Traffic. 4. 222-231 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Shimada, Y. et al.: "The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains."Eur.J.Biochem.. 269(24). 6195-6203 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ohno-Iwashita, Y. et al.: "Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts"Anaerobe. 10(2). 125-134 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Reid, P.C.et al.: "A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain"J.Lipid Res.. 45(3). 582-591 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Aoki, T. et al.: "Sequestration of cross-linked membrane molecules to caveolae in two different pathways"Acta Histochemica et Cytochemica. 36(2). 165-171 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Sugii, S. et al.: "Biotinylated theta toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells"J.Lipid Res.. 44(5). 1033-1041 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Heijnen, H.F.G.et al.: "Concentration of rafts in platelet filopodia correlates with recruitment of c-Src and CD63 to these domains"J.Thrombosis and Haemostasis. 1(6). 1161-1173 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Moebius, W. et al.: "Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway"Traffic. 4. 222-231 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Shimada, Y. et al.: "The C-terminal domain of perfringolysin O is an essential cholestereol-binding unit targeting to cholesterol-rich microdomains"Eur. J. Biochem.. 269(24). 6195-6203 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Terashita, A. et al.: "Lipid binding activity of a neuron-specific protein NAP-22 studied in vivo and in vitro"J. Neurosci. Res.. 70(2). 172-179 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Kokubo, H. et al.: "Ultrastructural localization of flotillin-1 to cholesterol-rich membrane microdomains, rafts, in rat brain tissue"Brain Res.. 965(1-2). 83-90 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] 嶋田有紀子, 岩下淑子: "コレステロールから視た膜機能ドメイン-コレステロールを見分けるプローブの開発"生化学. 74(7). 547-551 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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