The lipid bilayer of acetylcholine receptor clusters of cultured rat myotubes is organized into morphologically distinct domains.
We have studied the composition and organization of the lipid bilayer at the large, substrate-associated clusters of acetylcholine receptors (AChR) that form in cultured rat myotubes. These clusters have a characteristic morphology consisting of alternating linear domains of AChR-rich and AChR-poor membrane, the latter involved in attaching the myotube to the substrate. We partially purified AChR clusters by extracting cultured rat myotubes with the cholesterol-specific detergent, saponin. The lipid bilayer of the cluster preparation was analyzed biochemically and the substructure of the bilayers was studied morphologically using the fluorescent probes, dansyl polymyxin B, and 3,3'-di(C12H25 and C18H37) indocarbocyanine iodide (C12- and C18-diI). Our results demonstrate that preparations of AChR clusters have a lipid composition biochemically similar to that of the surrounding plasma membrane. Morphologically, however, the lipid bilayer appears to be arranged into domains that resemble the interdigitating pattern seen for the AChR. This distinctive lipid organization is not due to the use of saponin to purify clusters, as we obtained similar results with clusters isolated by physically shearing myotube cultures. The domain-like organization of the bilayer at clusters is disrupted by treatments that disperse AChR clusters in intact myotubes or that remove peripheral membrane proteins from isolated clusters. This suggests that such proteins may contribute to the organization of the bilayer. Two additional factors may also contribute to the organization of the bilayer: physical constraints imposed by sites of substrate attachment and, to a lesser extent, "boundary" lipid associated with AChR.
Scher
M G
MG
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201.
Bloch
R J
RJ
D000199:Actins / Q000502:physiology; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000648:ultrastructure*; D003599:Cytoskeleton / Q000502:physiology; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000737:chemistry*; D009132:Muscles / Q000648:ultrastructure*; D009469:Neuromuscular Junction / Q000648:ultrastructure; D010743:Phospholipids / Q000737:chemistry; D051381:Rats; D011940:Receptor Aggregation; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013049:Spectrin / Q000502:physiology
D000199:Actins; D001386:Azides; D008563:Membrane Lipids; D010743:Phospholipids; D011978:Receptors, Nicotinic; D013049:Spectrin; D002118:Calcium