Antibody‐induced crosslinking and cholesterol‐sensitive, anomalous diffusion of nicotinic acetylcholine receptors

Abstract

Synaptic strength depends on the number of cell‐surface neurotransmitter receptors in dynamic equilibrium with intracellular pools. Dysregulation of this homeostatic balance occurs, for example in myasthenia gravis, an autoimmune disease characterized by a decrease in the number of postsynaptic nicotinic acetylcholine receptors (nAChRs). Monoclonal antibody mAb35 mimics this effect. Here we use STORM nanoscopy to characterize the individual and ensemble dynamics of monoclonal antibody‐crosslinked receptors in the clonal cell line CHO‐K1/A5, which robustly expresses adult muscle‐type nAChRs. 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 areas. The latter act as permeable fences (~34 nm radius, ~400 ms lifetime). Dynamic clustering, trapping, and immobilization also occur in larger nanocluster zones (120–180 nm radius) with longer lifetimes (11 ± 1 s), in a strongly cholesterol‐sensitive manner. Cholesterol depletion increases the size of the clustering phenomenon; cholesterol enrichment has the opposite effect. The disclosed high proportion of monoclonal antibody‐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.

Dysregulation of Neuronal Nicotinic Acetylcholine Receptor–Cholesterol Crosstalk in Autism Spectrum Disorder

“…Chronic pharmacological cholesterol depletion using the drug mevinolin on the CHO-K1/A5 cell line (Pediconi et al, 2004) decreases cell-surface nAChRs by inhibition of receptor exocytosis and retention of the protein at the Golgi complex, while acute methyl-β-cyclodextrin mediated cholesterol depletion reduces the amount of cell-surface nAChR by accelerating receptor endocytosis (Borroni et al, 2007). In addition, changes in the distribution of nAChRs have been observed upon cholesterol changes as studied by stimulated emission depletion (STED; Kellner et al, 2007) and singlemolecule localization (SMLM; Mosqueira et al, 2018Mosqueira et al, , 2020 superresolution microscopies. Moreover, changes in cholesterol levels alter the translational mobility of the receptor in the plane of the plasma membrane, as measured by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy (Baier et al, 2010) and SMLM (Mosqueira et al, 2018(Mosqueira et al, , 2020.…”

“…In addition, changes in the distribution of nAChRs have been observed upon cholesterol changes as studied by stimulated emission depletion (STED; Kellner et al, 2007) and singlemolecule localization (SMLM; Mosqueira et al, 2018Mosqueira et al, , 2020 superresolution microscopies. Moreover, changes in cholesterol levels alter the translational mobility of the receptor in the plane of the plasma membrane, as measured by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy (Baier et al, 2010) and SMLM (Mosqueira et al, 2018(Mosqueira et al, , 2020. Pharmacologically induced cholesterol depletion leads to changes in nAChR stability, topographical distribution and long-range dynamic association with other nAChR molecules, and transiently affects the channel kinetics of those receptors remaining at the surface as a compensatory mechanism for the temporary loss of cell-surface receptors (Borroni et al, 2007).…”

Physiological Considerations for Modeling in vivo Antibody-Target Interactions

“…The diffusion rate of bound antibodies on lymphocytes has been estimated to be approximately 10 –10 cm 2 /s ( Elson et al, 1976 ), nearly four orders of magnitude lower than the diffusion constant in solution. These hindrances influence the avidity between two species by reducing molecular dispersion on the cell surface after dissociation events, thereby promoting rebinding ( Mosquera et al, 2020 ). Rebinding can contribute to apparent increases in k on , or decreases in k off , between molecules in vivo ( Vauquelin and Charlton, 2013 ).…”

The Plasma Membrane as the Arena for Receptor-Receptor Crosstalk

“…Lowering cholesterol levels causes rapid nAChR (muscle type) internalization, and compensatory gain-offunction of the nAChRs remaining at the plasma membrane [110][111][112]. Studies using stimulated emission depletion (STED) [113] and single-molecule localization (SMLM) [114,115] superresolution microscopies have further shown contrasting changes in the distribution of nAChR nanoclusters and individual molecules upon cholesterol depletion or enrichment. In addition, changes in the cholesterol concentration of the membrane followed using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy [108] and SMLM [114,115] have also been shown to modify the translational mobility of the receptor in the plane of the plasma membrane.…”