Abstract
BackgroundThe formation of acetylcholine receptor (AChR) cluster is a key event during the development of the neuromuscular junction. It is induced through the activation of muscle-specific kinase (MuSK) by the heparan-sulfate proteoglycan agrin released from the motor axon. On the other hand, DC electric field, a non-neuronal stimulus, is also highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells.Methodology/Principal FindingsTo understand its molecular mechanism, quantum dots (QDs) were used to follow the movement of AChRs as they became clustered under the influence of electric field. From analyses of trajectories of AChR movement in the membrane, it was concluded that diffuse receptors underwent Brownian motion until they were immobilized at sites of cluster formation. This supports the diffusion-mediated trapping model in explaining AChR clustering under the influence of this stimulus. Disrupting F-actin cytoskeleton assembly and interfering with rapsyn-AChR interaction suppressed this phenomenon, suggesting that these are integral components of the trapping mechanism induced by the electric field. Consistent with the idea that signaling pathways are activated by this stimulus, the localization of tyrosine-phosphorylated forms of AChR β-subunit and Src was observed at cathodal AChR clusters. Furthermore, disrupting MuSK activity through the expression of a kinase-dead form of this enzyme abolished electric field-induced AChR clustering.ConclusionsThese results suggest that DC electric field as a physical stimulus elicits molecular reactions in muscle cells in the form of cathodal MuSK activation in a ligand-free manner to trigger a signaling pathway that leads to cytoskeletal assembly and AChR clustering.
Highlights
The formation of the neuromuscular junction (NMJ) has been extensively studied as a model for understanding synapse development
These results suggest that DC electric field as a physical stimulus elicits molecular reactions in muscle cells in the form of cathodal muscle-specific kinase (MuSK) activation in a ligand-free manner to trigger a signaling pathway that leads to cytoskeletal assembly and acetylcholine receptor (AChR) clustering
In response to DC electric field, AChRs became clustered along the cathode-facing edge of cultured Xenopus muscle cells as shown by R-BTX labeling (Fig. 1, C and D)
Summary
The formation of the neuromuscular junction (NMJ) has been extensively studied as a model for understanding synapse development. During the assembly of vertebrate NMJ, acetylcholine receptors (AChRs) are clustered to a very high density of ,10,000 per mm in the postsynaptic membrane of the skeletal muscle, about 1,000-fold higher than that at extrajunctional membrane [1] This key event in NMJ development is mediated by the activation of muscle-specific tyrosine kinase receptor MuSK during motor innervation [2]. The formation of acetylcholine receptor (AChR) cluster is a key event during the development of the neuromuscular junction It is induced through the activation of muscle-specific kinase (MuSK) by the heparan-sulfate proteoglycan agrin released from the motor axon. DC electric field, a non-neuronal stimulus, is highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells
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