Lipid Domains Detected with of Acetylcholine Receptor Saponin and Filipin Clusters

The acetylcholine receptor (AChR) clusters of cultured rat myotubes contain two distinct, interdigitating, membrane domains, one enriched in AChR, the other poor in AChR but associated with sites of myotube-substrate contact (Bloch, R. J., and B. Geiger, 1980, Cell, 21:25-35). We have used two cholesterol-specific cytochemical probes, saponin and filipin, to investigate the lipid nature of these membrane domains. When studied with freeze-fracture electron microscopy or fluorescence microscopy, these reagents reacted moderately and preferentially with the AChR-rich domains of AChR clusters. Little or no reaction with the membrane in "contact" domains was seen. In contrast, membrane regions surrounding the AChR clusters reacted extensively with filipin. These results suggest that, in rat myotubes, the composition or the state of the lipids differs between the two membrane domains of AChR clusters, and between clusters and surrounding membrane. In chick myotubes, AChR clusters do not appear to react with filipin or saponin, although surrounding membrane reacts extensively with these reagents. The nicotinic acetylcholine receptor (AChR) ~ is distributed heterogeneously within the plasma membrane of vertebrate skeletal muscle. In normal adult muscle fibers, most of the AChR are localized at the postsynaptic membrane of the neuromuscular junction (13). In aneural cultures of vertebrate skeletal muscle cells, AChR also aggregate into limited regions of the plasma membrane (1, 3, 5, 12, 14, 24, 32, 43). Although the presence of nerve may influence the nature or extent of AChR aggregation, a muscle cell can distribute its AChR heterogeneously within its surface membrane even in the absence of nerve. Muscle cells may generate or stabilize these specialized surface structures by both intracellular (6, 16, 1820, 36, 41, 42) and extracellular (2, 6, 9, 11, 21, 34) means. The relative importance of these influences, their relationship to AChR self-aggregation (e.g., reference 10), and the role of the lipid environment in AChR aggregation are still in question. The large clusters of AChR that form in the plasma membranes of cultured rat myotubes are well suited to studies of AChR aggregation. These clusters form almost exclusively where the myotube membrane comes into close contact with the tissue culture substrate (2, 6). As a result, the clusters, t Abbreviations used in this paper: AChR, acetylcholine receptor; abungarotoxin, a-Btx; IMP, intramembrane particle. visualized with a fluorescent derivative of a-bungarotoxin (~Btx), almost always lie in a single plane of focus, allowing the fine structure to be more closely examined. As AChR clusters are localized at the cell-substrate interface, they may also be readily examined by complementary replica freeze-fracture (12, 30, 46) allowing ultrastructural studies of AChR distribution. Largely as a result of light microscopic studies (but see Results, below), the following picture has emerged of the AChR clusters of cultured rat myotubes. (a) Clusters can be 20-30/~m across and as much as 50-100 um long (3, 5). They often assume a rectilinear organization, in which strips of membrane (~1 ~m in width) enriched in AChR alternate with membrane strips of similar size depleted of AChR. (b) Where they are enriched, AChR are organized as apparently regularly spaced smaller aggregates or "speckles" (e.g., Fig. 2 of reference 6). We refer to these areas of the membrane and the linear arrays they generate as "AChR domains." Within AChR domains the intramembrane particles identified with AChR (12) are regularly spaced (37). (c) The membrane strips depleted of AChR are closer to the tissue culture substrate than are the AChR domains and are likely to be involved in cell-substrate contact (6). We refer to these regions as "contact domains." (d) The intracellular cytoskeletal protein, vinculin, is enriched near contact domains, but not near AChR doTHE JOURNAL OF CELL BIOLOGY • VOLUME 97 OCTOBER 1983 1043-1054 © The Rockefeller University Press 0021-9525183/1011043/12 $1.0