Single molecules of 594neg-SMs were frequently and transiently recruited to CD59-cluster signaling rafts for 50 ms. (A) Typical superimposed videoframe sequences of a CD59-cluster signaling raft and a single molecule of 594neg-SM, including a colocalization period (frames 4–9), observed at a 33-ms resolution. (B) Trajectories of the CD59-cluster raft and the single molecule of 594neg-SM shown in A. Colocalization within 240 nm (circle) is shown in the orange (594neg-SM) and blue (CD59-cluster raft) trajectories. (C) Duration distributions of colocalization of CD59-cluster-rafts (and CD59TM clusters as a control) by single molecules of 594neg-SM, 594neg-DSPC, and 594neg-DOPC. The exponential colocalization decay time is shown in each box. The numbers of the observed colocalization events were 233, 232, 214, 212, and 226 from left to right. (D) Schematic figure showing four types of transient interactions of SM with SM and CD59 in the PM. (1) Two SM molecules interact by way of a third molecule in a manner dependent on cholesterol and the sphingosine backbone (16-ms lifetime). SM molecules, in addition, transiently associate with CD59 monomers (2; monomer-associated rafts; 12 ms), CD59 transient homodimer rafts (3; whose lifetime is ∼160 ms; 33 ms), and CD59-cluster signaling rafts (4; whose lifetimes are on the order of several to several tens of minutes; 50 ms), in a cholesterol-dependent manner (in a manner dependent on the raft–lipid interaction).