Neuromuscular junction (NMJ) is a chemical synapse formed between a motor nerve terminal (presynapse) and a cytoplasmic membrane of skeletal muscle (postsynaptic membrane). At the NMJ, acetylcholine acts as neurotransmitter released from the presynapse, and binds to nicotinic acetylcholine receptor (nAChR) on the postsynaptic membrane. On the postsynaptic membrane, nAChRs cluster together with various proteins such as rapsyn, a scaffolding protein for nAChR, and muscle specific receptor tyrosine kinase (MuSK). Agrin, a presynapse-derived factor, signals through the MuSK to induce the clustering of nAChR. It was reported that endocytic recycling of nAChR is strongly concerned with nAChR clustering by light-microscopic observation [ 1]. However, the details of the molecular mechanisms of nAChR clustering remain largely unclear.
Recently, we analyzed the intracellular molecular localization of endocytosed nAChR using correlative light and electron microscopy (CLEM). First, we differentiated C2C12 myoblasts into myotubes, which steadily expressed GFP-tagged rapsyn. Then, we labeled the nAChRs expressed on the cell surface of postsynapse, with particles of colloidal gold-conjugated with anti-nAChR antibodies, and continued to culture the myotubes after adding agrin in the culture medium. By fluorescent microscopy (FM), we observed that the clusters matured approximately 6 hours after agrin addition ( Fig.1. (a)), and began to disassemble into smaller clusters around 18 hours later ( Fig.1. (c)). After that, we made ultra-thin sections of the myotubes, and the local areas of nAChR cluster imaged by FM were further observed by transmission electron microscopy (TEM). As a result, the localization and characterization of the vesicles were obviously different between the maturation ( Fig.1. (b)) and disassembled processes ( Fig.1. (d)).
Fig. 1
CLEM images of clustered and endocytosed nAChR on myotubes
(a) FM image of the matured nAChR cluster. (b) TEM image of the vesicle with endocytosed nAChR on the maturation process. (c) FM image of the collapsed nAChR cluster. (d) TEM image of the vesicle with endocytosed nAChRs on the disassembled process. The white line squares in (a) and (c) represent nAChR clusters visualized with GFP-tagged rapsyn. Black in (b) and white arrows in (d) indicate endocytosed nAChRs labeled with 6nm-gold particles.
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