Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes.
The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced prominently through the agrin/MuSK signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes both AChR clustering and cluster stability were dependent on cholesterol since depletion by methyl-ß-cyclodextrin inhibited cluster formation or dispersed established ones. Importantly, AChR clusters resided in ordered membrane domains - a biophysical property of rafts - as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, which all generate membranes with similar cholesterol/GM1 ganglioside contents and were enriched in several postsynaptic components, notably AChR, syntrophin and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintain clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.