Interaction of the Cytoskeletal Framework with Acetylcholine Receptor on the Surface of Embryonic Muscle Cells in Culture

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent a-bungarotoxin and extracted with Triton X-100, using conditions that preserve internal structure. A significant population of the AcCho receptors is retained on the skeletal framework remaining after detergent extraction . The proportion of nonextracted AcCho receptors increases during myotube development. Both photographic images and quantitative fluorescence measurements indicate that AcCho receptors in patched or aggregated areas are retained on the cytoskeleton while the diffuse receptors are partially extracted by detergent. The skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction . Despite an increasing appreciation of the physiological relevance of the spatial arrangement of many integral membrane proteins, the structural basis of the regulation of cell surface topography is poorly understood (for review, see reference 1) . The acetylcholine (AcCho) receptor of skeletal muscle cells is a well characterized integral surface membrane protein that undergoes pronounced changes in distribution on the cell surface during differentiation and innervation of these cells (for review, see references 2-5) . Initially, AcCho receptors on the surface of pre-innervated embryonic muscle cells are distributed randomly or homogeneously (3, 4) . After innervation, the entire AcCho receptor population becomes concentrated into a small patch of membrane adjacent to the nerve ending (3, 6-8) . Studies of muscle innervation in vitro suggest that preexistent surface AcCho receptors are redistributed to the patch at the innervation site (9, 10) . Even in the absence of innervation, AcCho receptors on cultured embryonic muscle cells spontaneously form clusters (11-14) which have an elevated packing density and diminished lateral mobility (15) similar to those ofthejunctional AcCho receptors ofinnervated muscle . Recent findings in other cell types suggest that most plasma membrane proteins are not extracted when lipids are removed with nonionic detergent (16-18) . Rather, the surface proteins form a sheet or lamina that covers the internal skeletal framework remaining after detergent extraction (18) . However, there are a few plasma membrane proteins which are extensively extracted from the surface lamina (16-18) . We report here that in the early development of cultured muscle cells most of the THE JOURNAL OF CELL BIOLOGY " VOLUME 92 JANUARY 1982 231-236 C The Rockefeller University Press " 0021-9525/82/01/0231/06 $1 .00 AcCho receptors are extracted by detergent. With maturation of these cells, a proportion of the receptors patch and become completely bound to the cytoskeletal framework . The association with cytoskeleton of AcCho receptors that are not recognizably clustered also appears to increase . A preliminary report of a portion of these results has appeared (19) . MATERIALS AND METHODS