Abstract
Translational motion of neurotransmitter receptors is key for determining receptor number at the synapse and hence, synaptic efficacy. We combine live-cell STORM superresolution microscopy of nicotinic acetylcholine receptor (nAChR) with single-particle tracking, mean-squared displacement (MSD), turning angle, ergodicity, and clustering analyses to characterize the lateral motion of individual molecules and their collective behaviour. nAChR diffusion is highly heterogeneous: subdiffusive, Brownian and, less frequently, superdiffusive. At the single-track level, free walks are transiently interrupted by ms-long confinement sojourns occurring in nanodomains of ~36 nm radius. Cholesterol modulates the time and the area spent in confinement. Turning angle analysis reveals anticorrelated steps with time-lag dependence, in good agreement with the permeable fence model. At the ensemble level, nanocluster assembly occurs in second-long bursts separated by periods of cluster disassembly. Thus, millisecond-long confinement sojourns and second-long reversible nanoclustering with similar cholesterol sensitivities affect all trajectories; the proportion of the two regimes determines the resulting macroscopic motional mode and breadth of heterogeneity in the ensemble population.
Highlights
Transmission of chemical signals in the synapse is regulated by the crosstalk between neurotransmitter receptors and scaffolding proteins, lipids, and the cytoskeleton
Cholesterol is an abundant component in the post-synaptic membrane, and there is a plethora of evidence on the variety of modulatory roles exerted by this lipid on the nAChR1
The physical models tested to reveal the source of the anomalous diffusion suggest that coexisting macromolecular self-crowding of the nicotinic acetylcholine receptor (nAChR) and obstacles in the form of percolating barriers operate in the time scale of tens of milliseconds to seconds, hampering free nAChR diffusion
Summary
Transmission of chemical signals in the synapse is regulated by the crosstalk between neurotransmitter receptors and scaffolding proteins, lipids, and the cytoskeleton. Comprehension of the various phenomena that determine this spatio-temporal homeostatic balance is key to understanding the function of the synapse in health and disease One such phenomenon is the motion of receptors in the plasma membrane. The members of the pLGIC superfamily are descendants of an ancestral “proto-channel” which appeared early in phylogenetic evolution, before the prokaryote-eukaryote dichotomy, and almost all prokaryotes lack cholesterol, the pLGIC exhibit the same sterol-recognition motifs as their eukaryotic counterparts[2,3] This remarkable degree of conservation at the molecular scale points to the importance of cholesterol in pLGIC function. We know that cholesterol exhibits preference over other lipid species for the superficial region surrounding the surface of the receptor protein; about 15 cholesterol molecules appear to be located at this region[4] These cholesterol molecules are in constant exchange with the bulk bilayer cholesterol[5], which is a key regulator of physical membrane properties at large. A correlation could be established between the behaviour at the individual trajectory level and the heterogeneous motional behaviour at the ensemble level: we found that the nanoscale dynamics of the single-trajectories dictate the population dynamics at the mesoscopic level, and that cholesterol fine tunes free walk diffusion and the stability of confinement zones
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