The applicant proposes to increase our understanding of the regulatory circuitry that controls the proliferation, fusion and differentiation of skeletal myoblasts to form muscle fibers during development and aging. In an effort to extend the understanding of the regulation of myoblast fusion, an early step in muscle tissue formation and regeneration the applicant proposes to determine rigorously the effects of certain cell surface molecules, substrates, and growth factors on the proliferation and differentiation of myoblasts in tissue culture using novel assays. The applicant also proposes to study the heterodimerization of members of the helix-loop helix family of myogenic regulators in live proliferating and differentiating muscle cells. The test system will involve chimeric proteins composed of these regulators and complementing DATE - galactosidase (DATE -gal) deletion mutants, and will permit analysis of heterodimerization and nuclear translocation in the intact cell. This approach will serve as a powerful adjunct to the in vitro methods routinely used for studying dimerization by examining these interactions in a biologically relevant context, the intact cell. The applicant will assess in mice which of the expressed myogenic activators and inhibitors actually interact to form heterodimers during development. A few key regulators will be selected for study in vivo using a knock in of a construct containing a myogenic regulator-DATE -gal chimera, and heterodimerization will be assayed by DATA -gal complementation. This will determine, for example, whether and when twist and Myf5 which are co- expressed in the developing mouse embryo, heterodimerize to prevent myogenesis. The experiments outlined in this proposal are possible by the recent adaptation of DATE -gal complementation in mammalian cells. The fusion of two myoblasts expressing strongly complementing deletion mutant peptides of DATE -gal results in DATE - gal enzyme activity that provides a rapid, reproducible, and quantitative measure of cell fusion. Similarly, DATE -gal activity resulting from the interaction of two chimeric proteins containing weakly complementing DATE -gal peptides measures the extent of interaction of the non-DATE -gal portions of the chimeras in an intact cell. These assays provide critical new tools for use in the proposed studies that will contribute to our understanding of the regulatory mechanisms underlying muscle development and aging.
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