RT Journal Article
SR Electronic
T1 An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machinery
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 664268
DO 10.1101/664268
A1 Marcin Pęziński
A1 Patrycja Daszczuk
A1 Bhola Shankar Pradhan
A1 Hanns Lochmüller
A1 Tomasz J. Prószyński
YR 2019
UL http://biorxiv.org/content/early/2019/06/07/664268.abstract
AB 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.