The Role of Nebulin Isoforms in Thin Filament Assembly

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DESCRIPTION (provided by applicant): Crucial to our understanding of cardiac and skeletal muscle myofilament function or dysfunction during disease is an understanding of the events and interactions involved in myofilament assembly. The long term goal of this project is to elucidate these interactions. Essential to this work is defining key proteins and assessing their role in the structure. As an initial step in this research, the roles of nebulin family members in the regulation and function of the actin based cytoskeleton will be analyzed. Nebulin and nebulette are large modular proteins associated with the thin filaments of skeletal muscle and cardiac muscle, respectively. The defining element of the nebulin family members is the presence of a 35 residue repeat that serves as the basic actin binding motif for the family members. The organization of the repeat domain has led to the postulation that these proteins serve as blueprints for the architecture of the thin filaments during development. These proteins also contain 3 other domains, an acidic N-terminal domain, a linker domain and a Src homology 3 (SH3) domain. It is hypothesized that these other domains, many of which are unique to each family member, serve to regulate actin binding functions and target interactions of the nebulin modules to distinct subcellular locations. To test these hypotheses: 1) the linker domains of three nebulin family members (nebulin, nebulette and Lasp 1) will be analyzed both in vivo and in vitro for actin binding activity and targeting functions dependent and independent of the nebulin modules; 2) the orientation of nebulette and the position of the linker and N-terminal domains will be mapped by immuno-electron microscopy; 3) the N-terminal domains of nebulette and nebulin will be examined for targeting and regulatory functions by examining the ability of this domain to interact with thin filaments in vivo and in vitro; and 4) chimeric nebulin-nebulette genes will be constructed and expressed in cardiomyocytes to evaluate the thin filament ruler hypothesis. This work will involve cellular and molecular biological techniques to alter gene expression in primary cultures of cardiac and skeletal muscle. Immunofluorescence and electron microscopy will be used to evaluate ultrastructural organization both in vitro and in vivo and biochemical and biophysical techniques to define molecular interactions. The results of this analysis will aid in our understanding of the roles of nebulin family members in the organization and functions of the thin filaments of striated muscle and to their roles in cytoskeletal dynamics. In addition, information gained here will add to our knowledge of the roles of these proteins in normal and diseased states.