An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machinery

Marcin Pęziński,Tomasz J Prószyński,Bhola Shankar Pradhan,Patrycja Daszczuk,Hanns Lochmüller

doi:10.1038/s41598-020-61347-x

Abstract

Motor neurons form specialized synapses with skeletal muscle fibers, called neuromuscular junctions (NMJs). Cultured myotubes are used as a simplified in vitro system to study the postsynaptic specialization of muscles. The stimulation of myotubes with the glycoprotein agrin or laminin-111 induces the clustering of postsynaptic machinery that contains acetylcholine receptors (AChRs). When myotubes are grown on laminin-coated surfaces, AChR clusters undergo developmental remodeling to form topologically complex structures that resemble mature NMJs. Needing further exploration are the molecular processes that govern AChR cluster assembly and its developmental maturation. Here, we describe an improved protocol for culturing muscle cells to promote the formation of complex AChR clusters. We screened various laminin isoforms and showed that laminin-221 was the most potent for inducing AChR clusters, whereas laminin-121, laminin-211, and laminin-221 afforded the highest percentages of topologically complex assemblies. Human primary myotubes that were formed by myoblasts obtained from patient biopsies also assembled AChR clusters that underwent remodeling in vitro. Collectively, these results demonstrate an advancement of culturing myotubes that can facilitate high-throughput screening for potential therapeutic targets for neuromuscular disorders.

Highlights

Motor neurons form specialized synapses with skeletal muscle fibers, called neuromuscular junctions (NMJs)
Myotubes derived from C2C12 cells or primary myoblasts that were cultured on murine laminin formed numerous large acetylcholine receptors (AChRs) clusters at the bottom of myotubes that often acquired a complex topology that was reminiscent of postsynaptic machinery at the NMJ (Fig. 1d)[34,45,46]
We found that clusters of postsynaptic machinery that were formed in C2C12 myotubes cultured on laminin-121 and laminin-221 were the most developed

Summary

Introduction

Motor neurons form specialized synapses with skeletal muscle fibers, called neuromuscular junctions (NMJs). In mice during the first postnatal weeks, NMJs grow in size and undergo developmental remodeling from simple plaque-shaped structures to topologically complex assemblies[2,14] During this process, postsynaptic machinery becomes perforated with scattered openings that, with time, become more numerous and fuse with each other to form indentations between AChR-rich branches that transform synapses into pretzel-like shapes[15,16]. Recent studies demonstrated that the culturing of muscle cells on mouse laminin-111-coated surfaces leads to the formation of topologically complex clusters of postsynaptic machinery that resemble clusters at the NMJ in vivo[34]. Myotubes cultured on laminin are the only in vitro system where the AChR clusters undergo developmental remodeling, providing the model to study the underlying mechanisms. We found that human primary myotubes that were derived from myoblasts obtained from patient biopsies were able to form AChR clusters with complex topology that contained synaptic podosomes

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Conclusion