We have described a monoclonat antibody that rounds and detaches chick skeletal myoblasts and myotubes from extracellular substrata. The antibody also inhibits the attachment of myogenic cells to a gelatin-coated substratum but has no detectable effect on myoblast fusion. The cellular response to antibody treatment varies wffh differentiation and cell type. Young myoblasts and myotubes are rapidly rounded and detached by the antibody. Older myotubes require longer incubation times or higher antibody titers for rounding and detachment. Chick embryo fibroblasts, cardiac cells, and neurons are not similarly rounded and remain attached. Since the antibody also detaches cells from embryonic muscle tissue explants, the cell-substratum interaction perturbed by the antibody appears relevant to the in vivo interaction of myogenic cells with their extracellular matrices. Binding studies using iodinated antibody revealed 2-4 x 105 sites per myoblast with an apparent Kd in the range of 2-5 x 10 -9 molar. Embryo fibroblasts bind antibody as well and display approximately twice the number of binding sites per cell. The fluorescence distribution of antigen on myoblasts and myotubes is somewhat punctate and particularly bright along the edge of the myotube. The distribution on fibroblasts was also punctate and was particularly bright along the cell periphery and portions of stress fibers. For both cell types the binding was distinctly different than that reported for collagen, fibronectin, and other extracellular molecules. The antigen, as isolated by antibody affinity chromatography, inhibits antibody-induced rounding. SDS PAGE reveals two unique polypeptides migrating in the region of ~120 and 160 kilodaltons (kd). The most straightforward mechanism for the antibody-induced rounding and detachment is the perturbation of a membrane molecule involved in adhesion. The hypothesized transmembrane link between extracellular macromolecules and the cytoskeleton provides an ovbious candidate. Extracellular macromolecules that comprise the basal lamina and extracellular matrix play a central role in development, tissue maintenance, and regeneration. They are thought to provide the substratum on which cells adhere and migrate, serve as a scaffold for tissue structure, contribute to histospecific cellular adhesions, and provide environmental cues for cellular differentiation (2, 60). In skeletal myo~enesis and regeneration, putative roles for extracellular macromolecules have been identified. Collagen markedly stimulates myogenesis in vitro by providing a substrate conducive to cell attachment (24). Extracellular macromolecules are also implicated in the accumulation of acetylcholine receptors and differentiation of nerve terminals at neuromuscular junctions (7, 9, 5 l). In contrast to this accumulating knowledge and understanding of the cornponents and functions of extracellular macromolecules, membrane components that regulate and participate in the interaction of cells with extracellular matrices remain to be isolated and characterized. One approach to identifying these molecules is through antibodies that perturb adhesion. Gerisch and coworkers have exploited this approach most fully, and have identified molecules participating in Dictyostelium aggregation (23). More recently, this approach has been extended to several different adhesive processes including that of cells to substrata (3, 8, 18, 20, 21, 29, 30, 43, 61). We report a monoclonal antibody CSAT that rounds and detaches skeletal myogenic cells in culture. The antigen with which this antibody reacts is potentially highly significant because it is involved in regulating adhesion and morphology. THE JOURNAL OF CELL BIOLOGY • VOLUME 95 NOVEM6ER 1982 654-666 654 © The Rockefeller U n iversity Press • 0021-9525/82/11/0654/13 $1.00 MATERIALS AND METHODS