Antibody-induced crosslinking immobilizes acetylcholine receptors. Mobile receptors exhibit anomalous, cholesterol-sensitive diffusion and clustering

Abstract

Synaptic strength depends on the number of cell-surface neurotransmitter receptors in dynamic equilibrium with intracellular pools. Dysregulation of this homeostatic balance occurs e.g. in myasthenia gravis, an autoimmune disease characterized by a decrease in the number of postsynaptic nicotinic acetylcholine receptors. Monoclonal antibody mAb35 mimics this effect. Using STORM nanoscopy we characterize the individual and ensemble dynamics of mAb35-crosslinked receptors. Antibody labeling of live cells results in 80% receptor immobilization. The remaining mobile fraction exhibits a heterogeneous combination of Brownian and anomalous diffusion. Single-molecule trajectories exhibit a two-state switching behavior between free Brownian walks and anticorrelated walks within confinement zones. The latter act as permeable fences (∼34 nm radius, ∼400 ms lifetime). Dynamic clustering, trapping and immobilization also occur in larger areas (∼150 nm radius) with longer lifetimes (11 ± 1 s), in a strongly cholesterol-sensitive manner. Cholesterol depletion increases the size and average duration of the clustering phenomenon; cholesterol enrichment has the opposite effect. The disclosed high proportion of mAb35-crosslinked immobile receptors, together with their anomalous, cholesterol-sensitive diffusion and clustering, provides new insights into the antibody-enhanced antigenic modulation that leads to physiopathological internalization and degradation of receptors in myasthenia.