Effect of actin perturbation on the mobility of mouse FcγR, determined by single-particle tracking (SPT). (A) Representative tracks of FcγRII/III, labeled under control conditions using primary Fab fragments and detected with quantum dots, as described in Materials and methods. All tracks were obtained from ≥20-s movies with an image acquisition rate of 33 Hz. The motion type was determined by moment scaling spectrum analysis and only tracks of at least 20 frames were analyzed (see Materials and methods). The cyan lines in A indicate tracks of FcγR with unrestricted diffusion, whereas the pink lines indicate tracks classified as restricted by the SPT software. The black lines in A indicate the closing of gaps as performed by the SPT software. Tracking of receptors in jasplakinolide-treated cells was limited to areas of the plasma membrane devoid of blebs. (B and C) Percentage of FcγRII/III (B) and FcγRI (C) tracked receptors undergoing either restricted or unrestricted diffusion under control or drug-treated conditions. (D) Summary of the calculated diffusion coefficients for FcγRI and FcγRII/III tracked in macrophages pretreated with vehicle (DMSO) or actin-perturbing agents. Data in B–D are means ± SEM of three independent experiments, with at least 10 cells analyzed per condition per experiment.