Junctional folds are unique membrane specializations developed progressively during the postnatal maturation of vertebrate neuromuscular junctions (NMJs), but how they are formed remains elusive. Previous studies suggested that topologically complex acetylcholine receptor (AChR) clusters in muscle cultures undergo a series of transformations, resembling the postnatal maturation of NMJs in vivo. We first demonstrated the presence of membrane infoldings at AChR clusters in cultured muscles. Live-cell super-resolution imaging further revealed that AChRs are gradually redistributed to the crest regions and spatially segregated from acetylcholinesterase along the elongating membrane infoldings over time. Mechanistically, lipid raft disruption or caveolin-3 knockdown not only inhibits membrane infolding formation at aneural AChR clusters and delays agrin-induced AChR clustering in vitro but also affects junctional fold development at NMJs in vivo. Collectively, this study demonstrated the progressive development of membrane infoldings via nerve-independent, caveolin-3–dependent mechanisms and identified their roles in AChR trafficking and redistribution during the structural maturation of NMJs.
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