In culture, single skeletal myoblasts fuse together to form contractile, multinucleated myotubes. This is a striking process and a good model for studying muscle differentiation and related diseases of muscle development, including the muscular dystrophies. The long-range goal of this project is to understand the roles of membrane glycosphingolipids (GSLs) in muscle cell recognition and fusion. In a previous comparative study changes were found in the synthesis of specific GSL structures during myoblast membrane fusion, but the exact nature of these changes varied with each cell line examined. The one consistent observation was a transient increase in the synthesis of total GSLs at the time membrane fusion. Recent work has focussed on two clonal muscle cell lines, one which fuses to form myotubes in vitro (E63) and another which is fusion-defective (fu-1). E63 cells demonstrated transient increased GSL synthesis at fusion, while fu-1 cells did not. Direct assay of several glycosyltransferases revealed that two were maximally activated at the time of myoblast contact and membrane fusion: GM3 synthase and LacCer synthase (absent in fu-1 cells). By using whole cell homogenates for these studies the involvement of activation of synthetic enzymes in the Golgi apparatus versus increased expression of surface glycosyltransferases was not determined. A direct involvement of membrane GSLs in myogenesis has recently been documented by examining the effect of a specific inhibitor of glycosylceramide synthase (D-threo-PDMP). PDMP blocked E63 myotube formation and this inhibition could be overcome by concurrent exposure of the cells to exogenous GSLs. To further delineate the involvement of membrane GSls in muscle cell differentiation experiments with the following specific aims are proposed: 1) Determining the effects of added agents (oligosaccharides, glycohydrolases and exogenous GSLs) on normal and fusion-defective myoblasts; 2) Analyzing the effects of GSL metabolic inhibitors; 3) Examining surface carbohydrate specificities by co-culturing normal and fusion-defective myoblasts after various biochemical manipulations; and 4) Exploring the possible role of surface glycosyltransferases in myoblast interactions during myogenesis.
