Efficient gene transfer into primary muscle cells to analyze nerve-independent postsynaptic organization in vitro

Acetylcholine receptor (AChR) clustering on the surface of muscle cells is a hallmark of postsynaptic differentiation at the vertebrate neuromuscular junction (NMJ). Even though the assembly of complex postsynaptic apparatuses is known to rely on both, pre- and postsynaptic signals, the identity of muscle-derived proteins modulating postsynaptic assembly and maintenance is still to be fully elucidated. Efficient gene transfer into muscle cells represents a powerful tool to analyze the contribution of muscle proteins on postsynaptic assembly and maintenance. Here, we describe a protocol that combines efficient electroporation of primary muscle satellite cells with the formation of aneural complex postsynaptic structures on the surface of myotubes. In vitro formed postsynaptic structures share various similarities with in vivo postsynaptic NMJ domains. While primary myotubes express increasing amounts of the ε AChR subunit, associated with NMJ maturation, surface AChR aggregates lack this AChR subunit. Our results also validate the functional expression of a luciferase reporter gene, as well as the response of complex postsynaptic structures to pharmacological treatment. Together, these methods in primary muscle cells are a valuable tool to perform a detailed and accurate analysis of the potential role of muscle-derived proteins on the maintenance of complex postsynaptic structures and to identify nerve-derived signals regulating functional NMJ maturation.