Molecular and functional characterization of membrane infoldings in the development of neuromuscular synapses

The vertebrate neuromuscular junction (NMJ) has been widely used as a model synapse for studying the formation, maintenance, and plasticity of chemical synapses. In the past few decades, extensive research progress has been made to understand how acetylcholine receptors (AChR) are spatially clustered at the postsynaptic membrane in response to nerve-secreted agrin at developing NMJs. As revealed by the ultrastructural studies using electron microscopy, and more recently using superresolution microscopy, we now know that the postsynaptic junctional folds at mature NMJs exhibit spatially segregated distribution of different postsynaptic proteins throughout the crests and troughs regions. However, the molecular mechanisms underlying the formation of membrane specializations are largely unknown. Our preliminary data convincingly demonstrated that membrane infoldings are frequently observed in spontaneously formed aneural AChR clusters in Xenopus muscle cells cultured on extracellular matrix (ECM)-coated substratum, further suggesting that nerve-independent aneural AChR clusters exhibit the resemblance to synaptic AChR clusters in vivo. In this project, we will primarily use Xenopus tissue cultures in combination with different molecular manipulations and super-resolution imaging to answer a series of cell biology questions concerning the molecular and functional characterizations of membrane infoldings in association with aneural and synaptic AChR clusters. Since abnormalities of the junctional fold architecture are the most common phenotypes associated with several neuromuscular disorders, the results of this project will provide a better understanding of not only the molecular mechanisms underlying membrane infolding formation in NMJ development but also the pathogenic mechanisms underlying the structural changes of junctional folds in patients with muscular dystrophies.