Large variations of the long-term (>5 s) diffusion coefficient experienced by single molecules of TfR on the cell surface. (A) A typical trajectory of a TfR molecule tagged by mSiNC-Tf, recorded at video rate (33 ms/frame). Different colors indicate the portions of the trajectory with different diffusivity, as determined by the method described in B. (B) Quantitative analysis results of the trajectory shown in A. (top) The CSD plotted as a function of time. The plot is divided into subintervals, in which a single slope can describe the plot, by eye, and if the portion is longer than 5 s, its slope was obtained, as plotted at the bottom. (C) Distribution of the long-term (>5 s) diffusion coefficients, indicating the presence of long-range spatial variations in the membrane structure, probably reflecting local variations in the actin-based membrane skeleton (open bars; obtained for 30 TfR molecules; 45 separate subintervals exhibiting different diffusion coefficients in time). This should be compared with the histogram of 100-ms-scale diffusion coefficients (black bars, n = 102). The numbers in the box indicate median values (see arrowheads).