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GPI-anchored receptor clusters transiently recruit Lyn and G for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1

¹ Membrane Mechanisms Project, International Cooperative Research Project, Japan Science and Technology Agency (ICORP-JST), The Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan
² Department of Biology, The Johns Hopkins University, Baltimore, MD 21218

Correspondence to Akihiro Kusumi: akusumi{at}frontier.kyoto-u.ac.jp

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold–induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Gi2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein–protein and lipid–lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP₃ production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Gi2. Because both Gi2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Gi2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.

Abbreviations used in this paper: CCD, charge-coupled device; DAF, decay accelerating factor; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DRM, detergent-resistant membrane; GPI-AR, glycosylphosphatidylinositol-anchored receptor; IP₃, inositol-(1,4,5) triphosphate; LDL, low-density lipoprotein; MßCD, methyl-ß-cyclodextrin; PLAP, placental alkaline phosphatase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PTX, pertussis toxin; SFK, Src-family kinase; STALL, stimulation-induced temporary arrest of lateral diffusion; TCZ, transient confinement zone; TIRF, total internal reflection fluorescence.

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