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

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. Vertebrate neuromuscular junctions (NMJs) are synapses between motor neurons and skeletal muscle fibers. The function of this type of synapse is to transmit signals from the central nervous system to muscles and thus stimulate their contraction. The nerve terminal releases the neurotransmitter acetylcholine (ACh), which binds to postsynaptic ACh receptors (AChRs) that are located on the surface of muscle fibers. For efficient synaptic transmission, muscle fibers need to accumulate a high density of AChRs in their postsynaptic membrane 1-4. Rodent muscles cluster components of postsynaptic machinery around day 12 of embryonic development (i.e., before motor neuron axons approach muscle fibers) 5,6. Innervation leads to the dispersion of preexisting AChR clusters 2,7. A single postsynaptic machinery is formed per muscle fiber directly below the nerve terminal. The nerve plays a crucial role in organizing NMJ postsynaptic machinery by secreting signaling molecules, such as agrin and ACh, which regulate the clustering and dispersion of AChRs, respectively 2,8-12. Nerve-derived agrin binds to the surface of low-density lipoprotein receptor-related protein (Lrp4), which activates muscle-specific kinase (MuSK) 13. This triggers an intracellular signaling cascade that activates AChR clustering by the scaffold protein rapsyn 2. 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...

Denervation-Related Neuromuscular Junction Changes: From Degeneration to Regeneration

“…This includes the rearrangement of nerve terminals, subtype shift of AchRs, and so on. Modifications occur inside and outside the cells in NMJ, and peri-synaptic components also play roles during the process such as the Schwann cells (SCs) ( Santosa et al, 2018 ; Chan et al, 2020 ; Pęziński et al, 2020 ).…”

Nanopattern surface improves cultured human myotube maturation

“…At 10 days of differentiation, almost all myotubes displayed clusters of AChR, which reflects a greater maturation of myotubes compared with conventional 2D cultures where only a minority of myotubes have AChR clusters after 2 weeks in culture [ 8 ]. The clusters remained however essentially homogenous, without a sign of lacunarity or pretzel structures, like it was reported in 3D culture [ 8 ] or on surfaces coated with various laminin [ 25 ], morphological changes that reflect the maturation of AChR clusters [ 19 , 26 ]. AChRs were however functional as shown by the Ca 2+ elevations elicited by ACh stimulation.…”

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons

“…Transient transfection was achieved with Jetprime® reagent (Polyplus Transfection) following the manufacturer’s conditions. For culturing, freezing, and thawing of the mouse myoblasts C2C12 (ATCC CRL-1772), we follow the same instructions as described in Pęziński et al, 2020 [ 38 ]. Cell cultures are regularly checked for the absence of mycoplasma contamination by commercially available mycoplasma detection tests (MycoAlert Plus, # LT07-705, Lonza or PlasmoTest, # rep-pt1, Invivogen) according to the manufacturer’s instructions.…”