Neural Cell Adhesion Between Spinal Cord Molecule Mediates Initial Interactions Neurons and Muscle Cells in Culture

Previous studies in this laboratory have described a cell surface glycoprotein, called neural cell adhesion molecule or N-CAM, that appears to be a ligand in the adhesion between neural membranes. N-CAM antigenic determinants were also shown to be present on embryonic muscle and an N-CAM-dependent adhesion was demonstrated between retinal cell membranes and muscle cells in short-term assays. The present studies indicate that these antigenic determinants are associated with the N-CAM polypeptide, and that rapid adhesion mediated by this molecule occurs between spinal cord membranes and muscle cells. Detailed examination of the effects of anti-(N-CAM) Fab' fragments in cultures of spinal cord with skeletal muscle showed that the Fab' fragments specifically block adhesion of spinal cord neurites and cells to myotubes. The Fab' did not affect binding of neurites to fibroblasts and collagen substrate, and did not alter myotube morphology. These results indicate that N-CAM adhesion is essential for the in vitro establishment of physical associations between nerve and muscle, and suggest that binding involving N-CAM may be an important early step in synaptogenesis. The interaction between nerve and muscle in culture has been extensively studied as a model system for synaptogenesis (13). This work has led to the identification of factors (4, 5) that influence the growth of nerves to muscles, and to a better understanding of the components and structure of the neuromuscular synapse (2-5). Relatively little is known, however, about the substances or mechanisms involved in the initial physical interaction between nerve and muscle. Factors that promote growth of nerves to muscle, such as cell-conditioned media (6) or extracellular matrix components (4), might be involved in the binding of nerve to muscle. In addition, the acetylcholine receptor itself, which is known to exist in aggregates on the muscle before synaptogenesis (1, 3), might influence nerve-muscle adhesion. As yet, however, there is no evidence either that the trophic factors are associated with cell adhesion, or that pre-existing acetylcholine receptor clusters influence the location of synapses (4). Studies in culture suggest that adhesive interactions and transient synapses to muscle can be formed using nerve cells from a variety of tissues including parasympathetic (7) and sympathetic ganglia (8), cerebral cortex (9), and clonal neuroblastoma x glioma hybrid cells (10-12). Furthermore, the neuroblastoma x glioma hybrid, which is derived from mouse, not only binds to muscle from mouse but also to muscle from rat and chick (13). These findings imply that the mechanism of initial binding between neurons and muscle does not possess a stringent tissue or species specificity. Rather, it is likely that a more general adhesive interaction between nerve and muscle establishes an initial physical contact, and that synaptic specificity is determined by, or in combination with, other events. These properties of initial nerve-muscle interactions raised the possibility that they involve the cell surface protein called neural cell adhesion molecule or N-CAM (14-16). The specific affinity of N-CAM for nerve and muscle cells suggests that it is a ligand in the formation of cell-cell bonds (17) and studies using antibodies to N-CAM have demonstrated that the function of this molecule is required for nerve cell sorting (18, 19), and neurite fasciculation (20, 21). N-CAM is present in a variety of species (22, 23) and appears early during embryogenesis in a variety of sites including those that have been determined to differentiate into striated muscle (24). Moreover, antigenic determinants recognized by antibodies against N-CAM have also been shown to be present on the surfaces of embryonic muscle cells, and Fab' fragments prepared from these antibodies inhibit binding of retinal cells, brain membrane vesicles, or reconstituted vesicles containing N-CAM to such muscle cells in short-term assays (25). The purpose of these studies was to identify and characterize further the N-CAM antigens on muscle cells, and to evaluate THE JOURNAL OF CELL BIOLOGY . VOLUME 97 JULY 1983 145-152 © The Rockefeller University Press • 0021-952S/97/07/0145/08 $I . 00 145 on Jne 6, 2017 D ow nladed fom Published July 1, 1983