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Medientyp:
E-Artikel
Titel:
An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machinery
Beteiligte:
Pęziński, Marcin;
Daszczuk, Patrycja;
Pradhan, Bhola Shankar;
Lochmüller, Hanns;
Prószyński, Tomasz J.
Erschienen:
Springer Science and Business Media LLC, 2020
Erschienen in:Scientific Reports
Sprache:
Englisch
DOI:
10.1038/s41598-020-61347-x
ISSN:
2045-2322
Entstehung:
Anmerkungen:
Beschreibung:
<jats:title>Abstract</jats:title><jats:p>Motor neurons form specialized synapses with skeletal muscle fibers, called neuromuscular junctions (NMJs). Cultured myotubes are used as a simplified <jats:italic>in vitro</jats:italic> 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 <jats:italic>in vitro</jats:italic>. Collectively, these results demonstrate an advancement of culturing myotubes that can facilitate high-throughput screening for potential therapeutic targets for neuromuscular disorders.</jats:p>