SELECTIVE-INHIBITION OF A STEP OF MYOTUBE FORMATION WITH WHEAT-GERM-AGGLUTININ IN A MURINE MYOBLAST CELL-LINE, C2C12

Myoblast cells, C2C12, which is an established cell line from satellit e cells of skeletal muscle of C3H mouse, start to fuse and form multin ucleated cells (myotubes) and begin to express creatine phosphokinase and myosin, when culture medium is changed from the growth medium to t he differentiation medium. Among the 12 lectins that we tested, wheat germ agglutinin apparently suppressed the myotube development judged b y phase-contrast microscopy, but did not affect the induction of creat ine phosphokinase activity. The addition of N-acetylglucosamine or N,N ',N''-triacetylchitotriose, which is a specific ligand for wheat germ agglutinin, to the differentiation medium, recovered this apparently s uppressive effect of wheat germ agglutinin on the myotube development. Tachypleus tridentatus (Japanese horseshoe crab) lectin that specific ally recognizes N-acetylneuraminic acid, one of the sialic acids, show ed no effect on the myotube development. It was suggested that wheat g erm agglutinin suppressed the process through recognizing N-acetylgluc osamine containing sugar. Surprisingly, even in the presence of wheat germ agglutinin, the ratio of mononucleated cell numbers to the genomi c DNA content, which represents the fusion level, decreased after incu bation in the differentiation medium, indicating that even when wheat germ agglutinin was present in the medium, cell fusion, which is the i nitial step of the myotube formation, occurred. Immunostaining with an ti-skeletal muscle myosin antiserum confirmed that the myosin expressi ng cells actually fused and formed multinucleated cells. Their shape, however, was thin compared to that in the absence of wheat germ agglut inin. We propose that the membrane fusion step to form myotubes is com posed of two distinct steps in C2C12; one fusion step is to form long and thin myotubes from mononucleated cells and the other one is to dev elop fat myotubes. Wheat germ agglutinin specifically inhibits the lat ter fusion step.