Figure 3: Tracer mobility is only minimally affected by mGFP-GPI enrichment.

(a) The mean square displacements of Chol-KK114 ON (black) and OFF (grey) mGFP-GPI-enriched areas are plotted as a function of t_lag for one representative cell. Diffusion coefficients were determined by fitting the function MSD=4Dt_lag+4σ²_xy (bold lines). Only the first two data points were considered for fits. We also included an anomalous diffusion fit with MSD=4Dt_lag^α+4σ²_xy (hairlines), yielding α_ON=0.940 and α_OFF=0.922. The first 10 data points were considered for fits. The inset shows a magnification of the first four data points. Only minor deviation from Brownian motion was observed ON as well as OFF mGFP-GPI areas. Relative diffusion coefficients D_ON/D_OFF were determined for 19 individual cells in four independent experiments and plotted as a function of b mGFP-GPI density ρ_mGFP-GPI or (c) average mGFP-GPI spacing d_mGFP-GPI. Data were fitted with D_ON/D_OFF=1—d₀²/d_mGFP-GPI² (c) yielding the characteristic nearest-neighbour distance of mGFP-GPI at the percolation threshold d₀=2.6±0.5 nm. The grey circle indicates the ratio of diffusion coefficients shown in (a). The grey lines represent the s.e. of the fit. (d) Relative diffusion coefficients D_ON/D_OFF of Chol-KK114 as a function of average mGFP-GPI spacing d_mGFP-GPI in T24 cells (open diamonds) and in HeLa cells (closed diamonds, N=9, three independent experiments).