The cultivation of muscle cells, whereby myoblasts undergo recognition, cell contact and membrane fusion to form multinucleated myotubes, is a suitable model for in vitro studies of the early stages of fetal muscle development. Despite experimental advantages (a well-defined process in a controlled environment) and decades of research, little is known of the molecular events at the cell surface which lead to or follow apposition of fusion-competent myoblasts. This paucity of knowledge is especially true of muscle cell glycosphingolipids (GSLs), including the gangliosides. This is surprising, since these compounds impart functional specificity to the cell surface by being involved in many cell-cell recognition events, receptor and antigenic functions, the promotion of cellular differentiation and oncogenic transformation. The long-range goal of this proposal is to understand structural and functional changes occurring at the plasma membrane during muscle cell differentiation in vitro, especially in regard to membrane GSLs. We have previously observed, in a comparison of several muscle cell lines, a transient rise in GSL sythesis at the time of myoblast contact and membrane fusion. The proposed work will explore the function of membrane GSLs in muscle cell differentiation, and will lead to a better understanding of human muscle development and provide valuable information which may be useful in exploring the etiology of diseases of muscle development. This will be accomplished using primary cultures of avian myoblasts, and rat L6 muscle cells, by a series of experiments with the following specific aims: 1) Determining if the transient increase in GSL synthesis during myogenesis is due to changes in glycosyltransferase activities; 2) Examining the role of GSLs during myogenesis by (a) examining their synthesis by normal and fusion-defective varients during differentiation; (b) measuring GSL content during myogenesis by HPLC; and (c) incubating normal and fusion-defective myoblasts with exogenous GSLs; 3) Search for myoblast affinities for specific GSLs at different stages of myogenesis using solid-phase assays (TLC overlays and addition of GSL-coated beads to cultures).
