Cholesterol-dependent, nanoscale dynamics of single-molecule trajectories dictate the ensemble anomalous diffusion of cell-surface acetylcholine receptors

Single molecule superresolution microscopy was used to follow the translational diffusion of a key neurotransmitter receptor protein, the nicotinic acetylcholine receptor. Individual trajectories and ensemble averages were obtained from single particle tracking of receptors labeled with a monovalent ligand, fluorescent bungarotoxin or a monoclonal antibody. Bayesian inference analysis of the mean square displacements yielded the relative probabilities of the highly heterogeneous mobile populations, a combination of Brownian, anomalous subdiffusive and constrained motions, the proportions of which were differentially modified by cholesterol levels. At the ensemble level, trajectories exhibited weak ergodicity breaking, dominated by anomalous subdiffusion. When the trajectories were separated into subpopulations according to their diffusivity, most of them were found to be ergodic, suggesting that ergodicity breaking stemmed from their heterogeneous nature. The distribution of the trajectories turning angles, markedly anticorrelated in the subdiffusive tracks, appeared to conform to the obstructed diffusion model, characteristically observed in the presence of obstacles. At the single track level, walks were transiently interrupted by intervals of confinement in small ellipsoidal nanodomains with major semi-axis less than 60 nm. In conclusion, a combination of non ergodic and ergodic anomalous mobilities, modulated by cholesterol, coexists with Brownian diffusion. The signature motional behavior at the single-trajectory level, i.e. the nanoscale heterogeneity, underlies the heterogeneous anomalous dynamics at the ensemble level in the complex plasma membrane environment.