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Unraveling of Lipid Raft Organization in Cell Plasma Membranes by Single-Molecule Imaging of Ganglioside Probes

  • Kenichi G. N. SuzukiEmail author
  • Hiromune Ando
  • Naoko Komura
  • Takahiro Fujiwara
  • Makoto Kiso
  • Akihiro Kusumi
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1104)

Abstract

Gangliosides are involved in a variety of physiological roles and particularly in the formation and function of lipid rafts in cell membranes. However, the dynamic behaviors of gangliosides have not been investigated in living cells owing to the lack of fluorescent probes that behave like their parental molecules. This has recently been resolved by developing new fluorescent ganglioside analogues that act similarly to their parental molecules, synthesized by only chemical methods. We performed single fluorescent-molecule imaging and revealed that ganglioside probes dynamically enter and exit rafts containing CD59, a glycosylphosphatidylinositol (GPI)-anchored protein, both before and after stimulation. The residency time of our ganglioside probes in CD59 oligomers was 48 ms after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 and 12 ms, respectively. These results reveal the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they demonstrate that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner and at a much higher frequency than expected. In this chapter, we review methods for the development and single-molecule imaging of new fluorescent ganglioside analogues and discuss how raft domains are formed, both before and after receptor engagement.

Keywords

Fluorescent ganglioside probes Raft domains Single-molecule fluorescence tracking GPI-anchored protein Cell plasma membrane 

Notes

Acknowledgment

This work was supported in part by Grants-in-Aids for Innovative Areas grants to K.G.M.S. [2311001 and 16H01358] and H.A. [24110505 and 26110704], various scientific research grants from the Japan Society for the Promotion of Science (Kiban B awarded to K.G.N.S. [24370055 and 15H04351], H.A. [15H04495], and M.K. [22380067]; Wakate A awarded to H.A. [23688014]; and Kiban S awarded to A.K. [16H06386]), a grant from the Core Research for Evolutional Science and Technology (CREST) project “Creation of Fundamental Technologies for Understanding and Control of Biosystem Dynamics” funded by the Japan Science and Technology Agency (JST) awarded to A.K., a grant from the Naito Foundation and Daiichi-Sankyo Foundation awarded to K.G.N.S., and a grant from the Mizutani Foundation awarded to K.G.N.S. and H.A. The WPI-iCeMS of Kyoto University is supported by the World Premier International Research Center Initiative (WPI) of MEXT.

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Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Kenichi G. N. Suzuki
    • 1
    • 2
    Email author
  • Hiromune Ando
    • 1
    • 2
  • Naoko Komura
    • 1
    • 2
  • Takahiro Fujiwara
    • 2
  • Makoto Kiso
    • 2
  • Akihiro Kusumi
    • 2
    • 3
  1. 1.Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN)Gifu UniversityGifuJapan
  2. 2.Institute for Integrated Cell-Material Sciences (WPI-iCeMS)Kyoto UniversityKyotoJapan
  3. 3.Membrane Cooperativity UnitOkinawa Institute of Science and Technology (OIST)Onna-sonJapan

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