Regulation of acetylcholine receptor clustering by ADF/cofilin-directed vesicular trafficking

Postsynaptic receptor localization is crucial for synapse development and function, but the underlying cytoskeletal mechanisms remain elusive. Using Xenopus neuromuscular junctions as a model, we found that actin depolymerizing factor (ADF)/cofilin regulated actin-dependent vesicular trafficking of acetylcholine receptors (AChRs) to the postsynaptic membrane. Active ADF/ cofilin was concentrated in small puncta adjacent to AChR clusters and was spatiotemporally correlated with the formation and maintenance of surface AChR clusters. Notably, increased actin dynamics, vesicular markers and intracellular AChRs were all enriched at the sites of ADF/cofilin localization. Furthermore, a substantial amount of new AChRs was detected at these ADF/ cofilin-enriched sites. Manipulation of either ADF/cofilin activity through its serine-3 phosphorylation or ADF/cofilin localization via 14-3-3 proteins markedly attenuated AChR insertion and clustering. These results suggest that spatiotemporally restricted ADF/cofilin-mediated actin dynamics regulate AChR trafficking during the development of neuromuscular synapses. Chemical synapses represent a major form of neuronal connections in the vertebrate nervous system that underlie a wide spectrum of neural functions. A prominent feature of chemical synapses is the presence of a postsynaptic apparatus containing highly concentrated receptors for effective reception of neurotransmitters released from the presynaptic nerve terminal. Regulation of postsynaptic receptor localization is therefore crucial for synapse formation, function and modulation 1–3 . At present, the cellular mechanisms underlying the spatiotemporal control of receptor trafficking and clustering at the postsynaptic site remain poorly understood. Because of its size, accessibility and simplicity 4 , the neuromuscular junction (NMJ) is a good model for studying the spatial distribution and trafficking of postsynaptic AChRs. Previous studies have shown that aneural AChR clusters can spontaneously form in the absence of innervation and nerve-secreted factors in vivo 5 . Nerve innervation, however, induces site-directed clustering of AChRs through redistribution from aneural clusters, recruitment of diffuse receptors and new synthesis from the subsynaptic nuclei 4,6 . Two counteracting nerve-derived factors, agrin and acetylcholine, regulate the redistribution of AChRs on the muscle membrane 7 . Agrin activates muscle-specific tyrosine kinase (MuSK) for inducing AChR clustering on the postsynaptic membrane 8 ,w hereas acetylcholine disperses extrasynaptic AChR clusters 9,10 . It remains unclear how AChRs are spatiotemporally delivered to the synaptic site during synapse formation. Passive diffusion trap and/or active trafficking mechanisms may be involved in AChR redistribution 11,12 . Clustered AChRs are believed to be immobilized via scaffolding connections to the actin cytoskeleton 13,14 , thus their redistribution probably requires dynamic changes in the cortical actin network. We used a combination of live-cell imaging and molecular and pharmacological manipulations to investigate the cytoskeletal control of AChR trafficking during synapse formation. We found that ADF/ cofilin is important for synaptic targeting of AChRs. ADF/cofilin accumulated at the nascent synaptic site before the clustering of surface AChRs. Furthermore, the disassembly of the spontaneous AChR clusters was preceded by the disappearance of active ADF/cofilin aggregates. Localized ADF/cofilin was associated with increased dynamic actin turnover and spatially correlated with the surface delivery of intracellular AChRs through vesicular trafficking. We identified that 14-3-3 molecules are essential for the spatial localization of ADF/cofilin for the regulation of AChR trafficking. Finally, alteration of ADF/cofilin activity or disruption of its localization prevented the formation of new AChR clusters induced by synaptogenic stimuli. These findings indicate that spatiotemporally restricted ADF/cofilincontrolled actin dynamics regulate the surface targeting of postsynaptic receptors at synaptic sites.