Single-particle tracking was used to monitor 2 highly expressed molecules in live Xenopus muscle cells: the transmembrane ion channel acetylcholine receptor (AChR) and the outer-leaflet lipid-raft marker ganglioside GM1. AChRs and GM1s were labeled with quantum dots linked to specific toxins, and up to 200 molecules per frame were concurrently tracked at a high sampling frequency (80 Hz) and over a long duration (30 min). In untreated cells, both AChRs and GM1s diffused without marked confinement within a 6-μm range over a long period. However, their instantaneous diffusion coefficient δ was distributed over a broad range and featured an exponential tail. This dynamic heterogeneity was observed in cells under various conditions. Protein networks underneath the membrane, such as...[ Read more ]

Single-particle tracking was used to monitor 2 highly expressed molecules in live Xenopus muscle cells: the transmembrane ion channel acetylcholine receptor (AChR) and the outer-leaflet lipid-raft marker ganglioside GM1. AChRs and GM1s were labeled with quantum dots linked to specific toxins, and up to 200 molecules per frame were concurrently tracked at a high sampling frequency (80 Hz) and over a long duration (30 min). In untreated cells, both AChRs and GM1s diffused without marked confinement within a 6-μm range over a long period. However, their instantaneous diffusion coefficient δ was distributed over a broad range and featured an exponential tail. This dynamic heterogeneity was observed in cells under various conditions. Protein networks underneath the membrane, such as those formed by F-actin and scaffold proteins, were examined for their role in regulating the dynamic heterogeneity; for this analysis, cells were exposed to 5 drugs, and the results showed that whereas ATP depletion homogenized the heterogeneity in δ by eliminating cortical F-actin, reducing the membrane cholesterol content did not exert a major effect. By combining the findings of unconfined diffusion, a broad distribution of δ, and the drastic influence of cortical F-actin disruption, we developed a new hypothesis on membrane organization based on the picket-fence model. Moreover, here we report 2 previously unrecognized behaviors of AChRs: First, AChRs moved linearly toward HB-GAM-coated beads. Our results indicated that AChRs’ linear motion toward the beads is guided by motor proteins, rather than by a vehicle that syncs their step speed. Second, transient coalescence (TC) occurred between AChRs and between GM1s. These TC events indicate that certain immobilization sites exist on the plasma membrane that are likely generated by actin polymerization and cholesterol. The sites were frequently detected between bead-induced immobile AChRs and mobile AChRs, and they might facilitate AChR cluster formation.