One of the first events in NMJ formation is aggregation of AChR (muscular nicotinic acetylcholine receptor) in clusters. Clustering is induced by deposition of neural agrin on muscle fibres. Agrin is the master gene controlling development and maturation of the neuromuscular junction (NMJ). At the NMJ and in myotubes AChRs undergo turnover with half life that varies from 24h (in denervated muscle and in myotubes) to 10 days (innervated NMJ). In vivo studies on rats whose muscles were transfected with agrin cDNA had shown that agrin causes metabolic stabilization of clustered AChRs in a dose-dependent manner. Thus agrin mimics the effects of the nerve on AChR turnover. Nevertheless, agrin’s effects could be indirect since this neuronal protein also causes co aggregation of receptors for other factors released by the nerve. To specifically study the role of agrin on AChR turnover we used aneurally cultured myotubes exposed to chromatographically purified recombinant neural agrin to induce clusters formation. Turnover data were collected selectively from clustered AChRs by means of a fluorescence imaging approach. We found that agrin had no effects on AchR turnover. Our imaging study also showed that AChR clusters are stable units: new synthetized receptors slip in these high density zones maintaining inalterated the morfological aspect and the receptor density in the cluster. At difference with turnover, the area of AChR aggergates was found to be exquisitely sensitive to agrin’s dose and incubation time. In details, low doses of agrin (0.1-0.5 nM) frequently induced the formation of large clusters, whereas higher doses (4-8 nM) generated abundant clusters of small size. The data indicate that clusters are dynamic units, forming, expanding and shrinking during time and in relation to agrin’s dose. The data also suggest that agrin dosage is fundamental for appropriate growth of the postsynaptic membrane during NMJ maturation in vivo.

Sinaptogenesi e controllo della stabilità del recettore per l'acetilcolina nel muscolo scheletrico

FUMAGALLI, Guido Francesco
2003-01-01

Abstract

One of the first events in NMJ formation is aggregation of AChR (muscular nicotinic acetylcholine receptor) in clusters. Clustering is induced by deposition of neural agrin on muscle fibres. Agrin is the master gene controlling development and maturation of the neuromuscular junction (NMJ). At the NMJ and in myotubes AChRs undergo turnover with half life that varies from 24h (in denervated muscle and in myotubes) to 10 days (innervated NMJ). In vivo studies on rats whose muscles were transfected with agrin cDNA had shown that agrin causes metabolic stabilization of clustered AChRs in a dose-dependent manner. Thus agrin mimics the effects of the nerve on AChR turnover. Nevertheless, agrin’s effects could be indirect since this neuronal protein also causes co aggregation of receptors for other factors released by the nerve. To specifically study the role of agrin on AChR turnover we used aneurally cultured myotubes exposed to chromatographically purified recombinant neural agrin to induce clusters formation. Turnover data were collected selectively from clustered AChRs by means of a fluorescence imaging approach. We found that agrin had no effects on AchR turnover. Our imaging study also showed that AChR clusters are stable units: new synthetized receptors slip in these high density zones maintaining inalterated the morfological aspect and the receptor density in the cluster. At difference with turnover, the area of AChR aggergates was found to be exquisitely sensitive to agrin’s dose and incubation time. In details, low doses of agrin (0.1-0.5 nM) frequently induced the formation of large clusters, whereas higher doses (4-8 nM) generated abundant clusters of small size. The data indicate that clusters are dynamic units, forming, expanding and shrinking during time and in relation to agrin’s dose. The data also suggest that agrin dosage is fundamental for appropriate growth of the postsynaptic membrane during NMJ maturation in vivo.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/242127
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