Acetylcholine Receptors Direct Rapsyn Clusters to the Neuromuscular Synapse in Zebrafish

Abstract

Clustering of nicotinic muscle acetylcholine receptors (AChRs) requires association with intracellular rapsyn, a protein with an intrinsic ability to self-cluster. Previous studies on sofa potato (sop), an AChR null line of zebrafish, have suggested that AChRs may play an active role in subsynaptic localization of rapsyn clusters. To test this proposal directly, we identified and cloned the gene responsible for the sop phenotype and then attempted to rescue subsynaptic localization of the receptor-rapsyn complex in mutant fish. sop contains a leucine to proline mutation at position 28, near the N terminus of the zebrafish AChR ␦ subunit. Transient expression of mutant ␦ subunit in sop fish was unable to restore surface expression of muscle AChRs. In contrast, expression of wild-type ␦ subunit restored the ability of muscle to assemble surface receptors along with the ability of fish to swim. Most importantly, the ability of rapsyn clusters to localize effectively to subsynaptic sites also was rescued in large part. Our results point to direct involvement of the AChR molecule in restricting receptor-rapsyn clusters to the synapse.

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms

“…Although AChRs are required for the postsynaptic clustering of several of the proteins observed in the NMJ such as acetylcholinesterase (AChE), syntrophin isoforms, and rapsyn (36-38), we found that AChE and MuSK are, whereas rapsyn is not, clustered at the postsynaptic membrane (Fig. S3), consistent with previous findings (35,(39)(40)(41). ACh Inhibits Pre-and Postsynaptic Differentiation Through Nonpostsynaptic AChR.…”

“…These results indicate that although agrin-MuSK signaling is necessary for both expression of AChR in postsynaptic clusters and specialization of presynaptic nerve terminals, expression of AChR in clusters per se is not an essential intermediary in this process. Interestingly, synaptic vesicles are also clustered in zebrafish AChR cluster mutants nic-1 (29) and sop, which are deficient in the AChRδ subunit (34,35). Although AChRs are required for the postsynaptic clustering of several of the proteins observed in the NMJ such as acetylcholinesterase (AChE), syntrophin isoforms, and rapsyn (36-38), we found that AChE and MuSK are, whereas rapsyn is not, clustered at the postsynaptic membrane (Fig.…”

Essential roles of the acetylcholine receptor γ-subunit in neuromuscular synaptic patterning

“…In addition, rapsyn appears to associate with AChRs in an intracellular compartment prior to its insertion into the plasma membrane (Marchand et al, 2002;Moransard et al, 2003), suggesting that rapsyn and AChRs may arrive at the plasma membrane together. Furthermore, rapsyn clusters are absent from the NMJ in AChR δ-subunit mutants of zebrafish, demonstrating that AChRs are required for synaptic localization of rapsyn (Ono et al, 2004;Ono et al, 2002). Together, these observations support the hypothesis that the full complement of AChR subunits is required for targeting rapsyn to synaptic sites.…”

“…Synaptic localization of rapsyn occurs at the initial stages of neuromuscular synaptogenesis (Noakes et al, 1993), and deletion of rapsyn leads to a total absence of AChR clustering (Gautam et al, 1995). Furthermore, rapsyn is absent from synaptic sites in AChR mutants of zebrafish (Ono et al, 2004;Ono et al, 2002). Because of its essential role in AChR clustering, we asked whether the localization of rapsyn was affected in γ -/-muscles.…”

accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1

“…Mutants carrying reduced striations in somatic muscles, such as fibrils unbundeled and sapje, were shown to have abnormal myofiber organization (Felsenfeld et al, 1990;Bassett et al, 2003). The genes mutated in the mutants nic/twister, sofa potato, ache/zieharmonika, twitch once and sapje were, respectively, identified as the α-subunit of the nicotinic acetylcholine receptor, the δ-subunit of the nicotinic acetylcholine receptor, acetylcholinesterase, rapsyn and the Duchenne muscular dystrophy gene (Sepich et al, 1998;Lefebvre et al, 2004;Ono et al, 2004;Behra et al, 2002;Downes and Granato, 2004;Ono et al, 2002;Bassett et al, 2003).…”