Synapse-Forming Axons and Recombinant Agrin Induce Microprocess Formation on Myotubes

Abstract

We examined cell-surface behavior at nerve-muscle contacts during synaptogenesis in cocultures of rat ventral spinal cord (VSC) neurons and myotubes. Developing synapses in 1-d-old cocultures were identified by the presence of axon-induced acetylcholine receptor (AChR) aggregation. Identified regions were then examined by transmission and scanning electron microscopy. The myotube surface near contacts with axons that induced AChR aggregation typically displayed ruffles, microvilli, and filopodia (microprocesses), indicating motility of the myotube surface. At some of these contact sites microprocesses were wrapped around the axon, resulting in the partial or total "submersion" of the axon within the myotube contours. Sites of myotube contact with somata and dendrites of the same neurons showed much less evidence of motility and surface interaction than sites of contact with axons. Moreover, the distance between opposed membranes of axons and myotubes was smaller than between dendrites or somata and myotubes, suggesting stronger adhesion of axons. These results suggest polarized expression of molecules involved in the induction of microprocess formation and adhesion in developing VSC neurons. We therefore tested the ability of agrin, which is preferentially secreted by axons, to induce microprocess formation in myotubes. Addition of recombinant C-terminal agrin to culture medium resulted in formation of microprocesses within 3 hr. Myotubes transfected with full-length rat agrin constructs displayed numerous filopodia, as revealed by fluorescence microscopy. The results suggest that the induction of muscle cell surface motility may be linked to the signaling processes that trigger the initial formation of the neuromuscular junction.

Modulation of Agrin-induced Acetylcholine Receptor Clustering by Extracellular Signal-regulated Kinases 1 and 2 in Cultured Myotubes

“…(ii) One of the earliest events following nerve-muscle contact, both in vitro and in vivo, is the formation of microprocesses on the muscle fiber surface called myopodia (39,40). However, it is unclear what role myopodia play in NMJ synaptogenesis.…”

Muscle arm development inCaenorhabditis elegans

“…The term 'myopodia' has since been adopted by others to describe the membrane extensions that emanate from embryonic mouse skeletal muscle myotubes and make contact with incoming motor axons (Misgeld et al, 2002). Muscle membrane extension may be a common feature of mammalian skeletal muscle cells, as cultured rat myotubes also extend 'micro-spikes' at sites of motoneuron apposition (Uhm et al, 2001). In all three cases, muscle membrane extensions disappear after the neuromuscular junction has formed, suggesting that these extensions have specific roles in guiding or stabilizing incoming motor axons during development.…”

Increased neuromuscular activity causes axonal defects and muscular degeneration

“…We cannot completely exclude the possibility that presynaptic defects are a consequence of subtle muscle cell damage or of impaired activity-independent processes such as interactions between axons with postsynaptic filapodial projections (Ritzenthaler et al, 2000;Uhm et al, 2001). Interestingly, dynamic behaviors of such myopodia have been proposed to be regulated by neuromuscular transmission (Misgeld et al, 2002).…”