Single-Molecule Imaging of Signal Transduction via GPI-Anchored Receptors

  • Kenichi G. N. SuzukiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)


Lipid rafts have been drawing extensive attention as a signaling platform. To investigate molecular interactions in lipid rafts, we often need to observe molecules in the plasma membranes of living cells because chemical fixation and subsequent immunostaining with divalent or multivalent antibodies may change the location of the target molecules. In this chapter, we describe how to examine dynamics of raft-associated glycosylphosphatidylinositol (GPI)-anchored receptors and interactions of the receptors with downstream signaling molecules by single-particle tracking or single-molecule imaging techniques.

Key words

Single-particle tracking Single-molecule imaging GPI-anchored protein Rafts Colocalization Temporal confinement Src family kinase PLCγ IP3 Calcium 



This work was supported in part by Grants-in-Aid for Specific Research (B) (No. 24370055) and by Innovative Areas (No. 2311002) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


  1. 1.
    Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572PubMedCrossRefGoogle Scholar
  2. 2.
    Brown DA, Rose JK (1992) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68:533–544PubMedCrossRefGoogle Scholar
  3. 3.
    Heerklotz H (2002) Triton promotes domain formation in lipid raft mixtures. Biophys J 83:2693–2701PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Casadei BR, Domingues CC, de Paula E, Riske KA (2014) Direct visualization of the action of Triton X-100 on giant vesicles of erythrocyte membrane lipids. Biophys J 106:2417–2425PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Tanaka KAK, Suzuki KGN, Shirai YM, Shibutani ST, Miyahara MSH, Tsuboi H et al (2010) Membrane molecules mobile even after chemical fixation. Nat Methods 7:865–866PubMedCrossRefGoogle Scholar
  6. 6.
    Suzuki KGN, Kasai RS, Hirosawa KM, Nemoto YL, Ishibashi M, Miwa Y et al (2012) Transient GPI-anchored homodimer rafts are units for raft organization and function. Nat Chem Biol 8:774–783PubMedCrossRefGoogle Scholar
  7. 7.
    Suzuki KGN, Fujiwara TK, Sanematsu F, Iino R, Edidin M, Kusumi A (2007) GPI-anchored receptor clusters transiently recruit Lyn and Gα for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1. J Cell Biol 177:717–730PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Suzuki KGN, Fujiwara TK, Edidin M, Kusumi A (2007) Dynamic recruitment of phospholipase Cγ at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2. J Cell Biol 177:731–742PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Suzuki K, Sterba RE, Sheetz MP (2000) Outer membrane monolayer domains from two-dimensional surface scanning resistance measurements. Biophys J 79:448–459PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Suzuki K, Sheetz MP (2001) Binding of cross-linking glycosylphosphatidylinositol-anchored proteins to discrete actin-associated sites and cholesterol-dependent domains. Biophys J 81:2181–2189PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Suzuki KGN (2012) Lipid rafts generate digital-like signal transduction in cell plasma membranes. Biotechnol J 7:753–761PubMedCrossRefGoogle Scholar
  12. 12.
    Suzuki KGN, Kasai RS, Fujiwara TK, Kusumi A (2013) Single-molecule imaging of receptor-receptor interactions. Methods Cell Biol 117:373–390PubMedCrossRefGoogle Scholar
  13. 13.
    Murray EW, Robbins SM (1998) Antibody cross-linking of the glycosylphosphatidylinositol-linked protein CD59 on hematopoietic cells induces signaling pathways resembling activation by complement. J Biol Chem 273:25279–25284PubMedCrossRefGoogle Scholar
  14. 14.
    Suzuki K, Ritchie K, Kajikawa E, Fujiwara T, Kusumi A (2005) Rapid hop diffusion of a G-protein-coupled receptor in the plasma membrane as revealed by single-molecule techniques. Biophys J 88:3659–3680PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Fujiwara T, Ritchie K, Murakoshi H, Jacobson K, Kusumi A (2002) Phospholipids undergo hop diffusion in compartmentalized cell membrane. J Cell Biol 157:1071–1081PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Simson R, Sheets ED, Jacobson K (1995) Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis. Biophys J 69:989–993PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Hirose K, Kadowaki S, Tanabe M, Takeshima H, Iino M (1999) Spatiotemporal dynamics of inositol 1,4,5-triphosphate that underlies complex Ca2+ mobilization patterns. Science 284:1527–1530PubMedCrossRefGoogle Scholar
  18. 18.
    Raucher D, Sheetz MP (2001) Phospholipase C activation by anesthetics decreases membrane-cytoskeleton adhesion. J Cell Sci 114:3759–3766PubMedGoogle Scholar
  19. 19.
    Sahl SJ, Leutenegger M, Hilbert M, Hell SW, Eggeling C (2010) Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids. Proc Natl Acad Sci U S A 107:6829–6834PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Institute for Frontier Medical SciencesKyoto UniversityKyotoJapan
  2. 2.National Centre for Biological Sciences (NCBS)/Institute for Stem Cell Biology and Regenerative Medicine (inStem)BangaloreIndia

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