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Journal of Lipid Research, Vol. 47, 2121-2133, October 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

Françoise Stetzkowski-Marden^*, Katharina Gaus^1,, Michel Recouvreur^*, Annie Cartaud^* and Jean Cartaud^2,*

^* Biologie Cellulaire des Membranes, Institut Jacques Monod, Unité Mixte de Recherche 7592, Centre National de la Recherche Scientifique, Université Paris 6, Université Paris 7, F-75251 Paris Cedex 05, France
Max Planck Institute for Cell Biology and Genetics, Dresden D-01307, Germany

Published, JLR Papers in Press, July 1, 2006.

¹ Present address of K. Gaus: Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Sydney 2052 NSW, Australia.

² To whom correspondence should be addressed. e-mail: cartaud{at}ijm.jussieu.fr

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 predominantly through the agrin/muscle-specific kinase (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, because depletion by methyl-ß-cyclodextrin inhibited cluster formation or dispersed established clusters. 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, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these 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 maintains 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.

Supplementary key words lipid rafts • nicotinic acetylcholine receptor • neuromuscular junction

Abbreviations: AChR, nicotinic acetylcholine receptor; -Bgtx, -bungarotoxin; CTX, cholera toxin B subunit; DRM, detergent-resistant membrane; ESA, epidermal surface antigen; GP, global polarization; MßCD, methyl-ß-cyclodextrin; MuSK, muscle-specific kinase

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