We plan to study the structure and functions of the outer membrane of developing skeletal myoblasts using monoclonal antibodies. A """"""""library"""""""" of these antibodies has been prepared and used to distinguish quantitative and topographic changes on the myoblast surface that accompany differentitation. We shall use these antibodies to isolate and characterize the molecular species on the myoblast membrane and to identify those which are germane to myoblast development. Immunochemical mapping of the distribution and densitites of deteminants on the membrane will be achieved using computer-assisted immunofluorescence and photometry. Immunoelectron microscopy, will be used to confirm and expand these maps. Finer resolution of the topography of the myoblast membrane will be achieved by the combined application of monoclonal antibodies and resonance energy transfer. This method will also be used to identify molecular interactions between membrane antigens on single cells and those interactions which mediate the association and fusion of myoblasts. The monoclonal antibodies will also be used to select and isolate cellsurface mutants lacking specific membrane components. These mutants will be used to determine the effects of loss of individual membrane components on the molecular morphology and functions of the differentiating myoblast membrane. This combined genetic and immunochemical approach to defining the myoblast membrane will lead to the identification of membrane components with their respective functions and to an understanding of how membrane structure and function relate to cell differentiation. These studies will provide the basis for future genetic analysis of the skeletal myoblast membrane, for the isolation of myogenic stem cells and for the examination of earlier stages of myogenesis both in vitro and in vivo.
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