Acetylcholine Receptor Organization in Membrane Domains in Muscle Cells

Abstract

Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction formation; however, it has not been demonstrated whether rapsyn is the only cause of receptor immobilization. Here, we used single-molecule tracking methods to investigate nAChR mobility in plasma membranes of myoblast cells during their differentiation to myotubes in the presence and absence of rapsyn. We found that in myoblasts the majority of nAChR were immobile and that ϳ20% of the receptors showed restricted diffusion in small domains of ϳ50 nm. In myoblasts devoid of rapsyn, the fraction of mobile nAChR was considerably increased, accompanied by a 3-fold decrease in the immobile population of nAChR with respect to rapsynexpressing cells. Half of the mobile receptors were confined to domains of ϳ120 nm. Measurements performed in heterologously transfected HEK cells confirmed the direct immobilization of nAChR by rapsyn. However, irrespective of the presence of rapsyn, about one-third of nAChR were confined in 300-nm domains. Our results show (i) that rapsyn efficiently immobilizes nAChR independently of other postsynaptic scaffold components; (ii) nAChR is constrained in confined membrane domains independently of rapsyn; and (iii) in the presence of rapsyn, the size of these domains is strongly reduced.

Cholesterol modulates acetylcholine receptor diffusion by tuning confinement sojourns and nanocluster stability

“…36 nm. Other SPT studies reported that 20% of nAChRs in muscle myoblasts showed restricted diffusion in small domains having similar dimensions 26 . We found that the less diffusive subpopulations spent the longest stopovers in the confined state; the confined sojourns of Brownian trajectories had shorter durations, with superdiffusive trajectories displaying the shortest confined lifetimes (Fig.…”

Ligand-Induced Dynamics of Neurotrophin Receptors Investigated by Single-Molecule Imaging Approaches

“…Proteins endogenously present at the plasma membrane can be detected using specific antibodies (or their Fab fragments), which can in turn be detected using secondary antibodies (or their Fab fragments): the primary or (if needed) the secondary antibody is usually conjugated to small organic dyes, gold nanoparticles, or fluorescent quantum dots (Qdots) [ 88 , 89 , 90 , 91 ]. If the proteins are membrane receptors, a possible alternative to antibodies is using their specific ligands conjugated to the same kind of probes [ 92 , 93 , 94 ]. In the second approach, the proteins of interest are fused to so called “chemical tags”.…”

Cell-surface translational dynamics of nicotinic acetylcholine receptors

“…It has long since been known that rapsyn (formerly called 43K protein) affects nAChR distribution at the cell surface ( Barrantes et al, 1980 ; Burden et al, 1983 ; Ramarao and Cohen, 1998 ). This effect varies along development, as illustrated in a recent study on the effect of rapsyn on nAChR mobility followed along myoblast development in culture ( Piguet et al, 2011 ). The myristoylated N-terminus of rapsyn molecules anchors nAChRs to the plasma membrane in a 1:1 stoichiometry, playing a major role during myoblast differentiation and neuromuscular junction development.…”

“…The myristoylated N-terminus of rapsyn molecules anchors nAChRs to the plasma membrane in a 1:1 stoichiometry, playing a major role during myoblast differentiation and neuromuscular junction development. In myoblasts the majority of the receptors were found to be immobile, with 20% of the receptors exhibiting restricted diffusion in small domains of about 50 nm ( Piguet et al, 2011 ). Before differentiation, only 2% of the nAChRs showed Brownian diffusion, 24% diffused in confined regions, and 74% were immobile.…”

“…About 50% of the mobile receptors were confined to domains of about 120 nm. Irrespective of the presence of the nAChR-anchoring protein rapsyn, nAChR was confined to domains : when rapsyn was present, the size of the domains diminished ( Piguet et al, 2011 ). This study is in agreement with our study using direct imaging of nAChR nanoclusters using superresolution microscopy in cells devoid of rapsyn ( Kellner et al, 2007 ).…”