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Dynamic recruitment of phospholipase C at transiently immobilized GPI-anchored receptor clusters induces IP₃–Ca²⁺ signaling: single-molecule tracking study 2

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

Clusters of CD59, a glycosylphosphatidylinositol-anchored receptor (GPI-AR), with physiological sizes of approximately six CD59 molecules, recruit Gi2 and Lyn via protein–protein and raft interactions. Lyn is activated probably by the Gi2 binding in the same CD59 cluster, inducing the CD59 cluster's binding to F-actin, resulting in its immobilization, termed stimulation-induced temporary arrest of lateral diffusion (STALL; with a 0.57-s lifetime, occurring approximately every 2 s). Simultaneous single-molecule tracking of GFP-PLC2 and CD59 clusters revealed that PLC2 molecules are transiently (median = 0.25 s) recruited from the cytoplasm exclusively at the CD59 clusters undergoing STALL, producing the IP₃–Ca²⁺ signal. Therefore, we propose that the CD59 cluster in STALL may be a key, albeit transient, platform for transducing the extracellular GPI-AR signal to the intracellular IP₃–Ca²⁺ signal, via PLC2 recruitment. The prolonged, analogue, bulk IP₃–Ca²⁺ signal, which lasts for more than several minutes, is likely generated by the sum of the short-lived, digital-like IP₃ bursts, each created by the transient recruitment of PLC2 molecules to STALLed CD59.

Abbreviations used in this paper: DAF, decay accelerating factor; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; GPI-AR, glycosylphosphatidylinositol-anchored receptor; IP₃, inositol-(1,4,5) triphosphate; LDL, low-density lipoprotein; MßCD, methyl-ß-cyclodextrin; PH, pleckstrin homology; PIP₂, phosphatidylinositol-bis(4,5)phosphate; PLAP, placental alkaline phosphatase; PTX, pertussis toxin; SFK, Src-family kinase; STALL, stimulation-induced temporary arrest of lateral diffusion.

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