Visualization and characterization of GM2-rich lipid raft microdomain

2011 Fiscal Year Final Research Report

• Project/Area Number: 22657029
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Structural biochemistry
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: HIROSE Shigehisa 東京工業大学, 大学院・生命理工学研究科, 教授 (10134199)
• Co-Investigator(Renkei-kenkyūsha): KATO Akira 東京工業大学, 大学院・生命理工学研究科, 助教 (40311336)
• Project Period (FY): 2010 – 2011
• Keywords: 脂質 / タンパク質 / 糖鎖 / 細胞膜 / 脂質ラフト / ガングリオシド / GOTO細胞 / モノクローナル抗体 / フロチリン / エンドサイトーシス / 神経芽腫 / シクロデキストリン

Research Abstract

We raised a monoclonal antibody which enables the visualization of ganglioside GM2-rich microdomains as highly punctate dots on a human neuroblastoma cell, GOTO. Using this antibody, we showed that raft-like microdomains of GM2(GM2rafts) were colocalized with flotillin-2/reggie-1-positive microdomains. Prelabeling of GM2 rafts with a fluorescent probe, Alexa555. Anti-GM2, demonstrated internalization and delivery of the raft. flotillin assembly to the recycling endosomes. Depletion of cholesterol with methyl-β-cyclodextrin(MβCD) resulted in disruption and targeting of GM2 rafts to lysosomes. These results suggest that in GOTO cells, GM2 rafts are actually present in association with flotillin and undergo internalization into distinct pools of endosomal vesicles depending on their cholesterol contents.

Research Products

• [Journal Article] Differential expression of Na+-Clcotransporter and Na+-K+-Clcotransporter2 in the distal nephrons of euryhaline and seawater pufferfishes (2011)
  • Author(s): Kato, A., Muro, T., Kimura, Y., Li, S.-S., Islam, Z., Ogoshi, M., Doi, H., Hirose, S
  • Journal Title: Am. J. Physiol. Regul. Integr. Comp. Physiol Volume: 300 Pages: R284-R297
• [Journal Article] Identification of zebrafish Fxyd11aprotein that is highly expressed inion-transporting epithelium of the gilland skin and its possible role in ionhomeostasis (2010)
  • Author(s): Saito K, Nakamura N, Ito Y, HoshijimaK, Esaki M, Zhao B, Hirose S
  • Journal Title: Front Physiol Volume: 1:129 Pages: 1-14
• [Journal Article] Neuroblastoma GOTO cells are hypersensitive todisruption of lipid rafts (2009)
  • Author(s): Tomioka, R., Minami, N., Kushida, A., Horibe, S., Izumi, I., Kato, A., Fukushima, K., Ideo, H., Yamashita, K., Hirose, S., and Saito, Y.
  • Journal Title: Biochem. Biophys. Res. Commun Volume: 389 Pages: 122-127
• [Presentation] ガングリオシドGM2を主成分とするラフトの挙動 (2012)
  • Author(s): 広瀬茂久
  • Organizer: 第27回動物細胞工学シンポジウムキャンパス・イノベーションセンター
  • Place of Presentation: 東京
  • Year and Date: 20120000
• [Presentation] Methyl-beta-cyclodextrin-mediatedlipid raft disruption causes acuteapoptosis in neuroblastoma GOTO cell (2010)
  • Author(s): Tomioka, R., Kato, A., and Hirose, S
  • Organizer: Keystone Symposia on Molecular and Cellular Biology Molecular Basis for Biological Membrane Organization and Dynamics(A5)
  • Place of Presentation: Snowbird, Utah, USA
  • Year and Date: 20100000
• [Remarks]
  • URL: http://www.hirose.bio.titech.ac.jp/