The long range goal of this proposal is to understand the structural and physiological roles of sarcomere-associated cytoskeletons in the striated muscle cells. Recent studies of two giant structural proteins, titin, and nebulin, suggest that each protein constitutes a set of molecular filaments, forming an elastic matrix in the sarcomere. The sarcomere matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability and the assembly of nascent sarcomeres in developing muscle cells. In the proposed research, we address several questions of central importance and examine them systematically and critically with an integrated experimental approach: (a) What is the conformational basis of titin elasticity? We will test the hypothesis that specific and reversible conformational transition between folded and unfolded states of titin polypeptides underlies its capacity as a molecular spring to generate elasticity. The molecular structure and its conformational transition will be determined at the level of cDNA sequence, amino acid sequence, protein folding, domain organization, contour length, flexibility and extensibility under a variety of experimental conditions. (b) Do titin and nebulin serve as templates or scaffolds for myosin and actin filaments, respectively? We will search for specific interfilament protein interactions and test their effects on actomyosin interaction. Additionally, their potential involvement in the assembly and length regulation of myosin and actin filaments will be examined at the level of self-assembly, nucleated assembly and reconstitution of selectively extracted sarcomeres? (c) How are titin and nebulin organized in sarcomere? We will evaluate a four-filament sarcomere model by high resolution electron microscopy combined with immuno-labeling. Additionally, a systematic search for binding proteins at the Z-and M-lines will be made t elucidate their anchorage in the sarcomere. (d) How does the sarcomere matrix respond to stress? We will evaluate and refine our working hypothesis that the segmental extensibility of titin underlies its capacity as a dual-range molecular spring. A battery of monoclonal antibodies to non-repetitive epitopes will be used to track dynamic translocations and filament strain in sarcomeres of various lengths. Experimental conditions will be designed to facilitate correlation between molecular conformation, sarcomere architecture and muscle mechanics of skeletal and cardiac muscles.
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