No abstract

Although muscle cell fusion was shown to be an energy-requiring process, release of myoblasts from an EGTA fusion block could be accomplished with Earle's balanced salt solution (containing 1.8 mM Ca ++) free of glucose or any other energy-producing metabolite.The effect of concanavalin A, abrin, and the lectins from wheat germ, soybean, and Lens culinaris on myoblast fusion was examined with synchronized myoblast cultures upon release from fusion block. At a concentration of 15/~g/ml, these lectins were found to inhibit the fusion process to the extent of 62%, 41%, 32%, 8%, and 19%, respectively. Concanavalin A inhibition could be prevented by a-methyl-D-mannoside. The inhibitory effect of all the lectins except abrin could be reversed by changing to the normal, serum-containing medium. The number of binding sites determined for 12q-labeled concanavalin A, wheat germ, and soybean agglutinins was 3.4 • 107, 6.1 • l0 T, and 1.7 • 106, respectively. Although myoblasts were found to have about twice as many binding sites for wheat germ agglutinin as for concanavalin A, concanavalin A was determined to be twice as effective as wheat germ agglutinin as an inhibitor of myoblast fusion. These findings raise the possibility that specific cell surface glycoproteins may be an important factor in this process.Embryonic muscle cells can be grown in culture (11) and, therefore, in vivo myogenesis, which closely corresponds to in vitro myogenesis (9), has become accessible to experimentation by cell culture techniques. Shainberg et al. (33) established an absolute requirement for Ca ++ in the process of myogenesis and they (34), as well as Paterson and Strohman (28), succeeded in synchronizing the process by manipulation of the Ca ++ levels in the culture medium. As Bischoff and Holtzer (3) pointed out, the fusion process, which begins the normal chain of events of myogenesis, must involve at least two separate reactions: (a) cell recognition (or specific adhesion), and (b) subsequent membrane-membrane interaction culminating in cell fusion.Cell recognition and interactions during the process of morphogenesis are believed to be mediated by specific macromolecular components located on the cell membrane and between cells. It 826

The object of these studies is to define the molecular events that occur during synaptogenesis. Our approach is to use single identified Aplysia neurons grown in culture under conditions where chemical synapses are formed. In this report we studied synapses established by R2, a giant cholinergic neuron, onto neurons R15 and L11, and a group of left upper quadrant (LUQ) cells. The detailed electrophysiology of these contacts was described in the preceding paper (Schacher, S., S. G. Rayport, and R. T. Ambron (1985) (J. Neurosci. 5: 2851-2856). Within the animal, R2 synapses on thousands of unicellular mucus glands in the skin. R2 growing in vitro will establish contacts with isolated mucus glands. Although we do not know whether a functional synapse is formed, electron microscopy shows that the membrane in the area of contact is differentiated and that the ending is filled with various types of vesicles. A single R2 regenerating neurites in vitro synthesizes more than 300 polypeptides containing [35S]methionine. Many of these are subsequently transported into the growing neurites. We compared the newly synthesized proteins made by R2 before and after synapse formation and found that the expression of a 68-kilodalton (kd) and 72-kd protein was markedly enhanced after synaptogenesis. The finding that only two proteins were affected implies that many of the proteins required for synapse formation are present in R2 prior to contacting a target cell. Support for this idea was obtained when we compared the proteins present in R2's neurites in vitro with those that are rapidly transported to R2's mature synapses in vivo (Ambron, R. T., S. Schacher, and S. G. Rayport (1985) J. Neurosci. 5: 2866-2873).(ABSTRACT TRUNCATED AT 250 WORDS)

The biosynthetic capabilities of individual neurons of the abdominal ganglion of the marine mollusc Aplysia californica have been analyzed after intrasomatic injection of 3H-monosaccharides. Glycopeptides prepared from the metabolically labeled cells were fractionated using serial lectin affinity and gel filtration chromatography. The fractionation procedure yielded eight populations of glycopeptides, and comparison of two different neurons (R2 and R14) showed that the quantity of the individual species produced is cell-dependent. Structural analysis indicated that the glycoconjugates produced by the Aplysia neuron constitute both O- and N-linked structures as well as an unusual class of oligosaccharide whose linkage to protein is unknown. The O-linked units are small and consist only of N-acetylglucosamine or N-acetylgalactosamine attached to protein. High-mannose-type asparagine-linked units are produced by the neurons, and some of these appear to be processed to biantennary complex-type units that bind to lentil lectin-agarose. Overall, although the Aplysia neurons produce oligosaccharides of a nature similar to that produced by higher eucaryotes, the N- and O-linked structures produced by the neurons do not achieve the complexity of the comparable structures produced by mammalian cells. The results provide a basis for further studies aimed at understanding the role of glycoconjugates in the development of the nervous system.