Ab-CTXB-GM1 clusters generated in the PM outer leaflet contained an average of ∼15 GM1 molecules and diffused 2.6 times slower than CD59 clusters. (A) Fluorescence images of non–cross-linked fluorescently labeled CTXB (which could bind up to five GM1 molecules; A633 conjugated with a D/P of 0.80; called CTXB-5-GM1; top) and CTXB clusters induced by the further addition of anti-CTXB Abs (Ab-CTXG-GM1 cluster; bottom), obtained at single-molecule sensitivities. Arrows indicate all of the detected fluorescence spots in each image. (B) Histograms showing the distributions of the signal intensities of individual fluorescent spots of A633-labeled CTXB-5-GM1 (top) and Ab-CTXB-GM1 clusters (bottom). On the basis of these histograms, we concluded that each Ab-CTXB-GM1 cluster contained an average of ∼15 GM1 molecules (see Materials and methods), although the distribution would be quite broad. (C) Typical trajectories of CTXB-5-GM1 (top) and Ab-CTXB-GM1 clusters (bottom) for 0.2 s, obtained at a time resolution of 6.45 ms. (D) Ensemble-averaged MSDs plotted against time, suggesting that in the time scale of 1 s, both CTXB-5-GM1 (top; 154 trajectories) and Ab-CTXB-GM1 clusters (bottom; 91 trajectories) undergo effective simple Brownian diffusion, and the diffusion is slowed by a factor of about 5 after Ab-induced clustering. All error bars represent SEM.