Myofibrillogenesis is a highly complex process that depends on the coordinated assembly and integration of a number of contractile, cytoskeletal and signaling proteins into regular arrays, the sarcomeres. Myosin Binding Protein C (MyBP-C), a protein associated with thick filaments, is believed to have both a structural and a regulatory role within the muscle cell, by contributing to the normal assembly and stabilization of thick filaments and modulating the number of myosin heads available for involvement in the contractile cycle. Obscurin, a giant myofibrillar protein, that closely surrounds Z-disks and M-lines, interacts specifically and directly with a novel isoform of MyBP-C slow, MyBP-C(+) slow, that appears to selectively concentrate at the M-line. Consequently, we hypothesize that the binding of obscurin with MyBP-C(+) slow contributes to the assembly, stabilization and maintenance of sarcomeric myosin into regular A-bands, through formation of primitive M-lines that may serve as scaffolding structures. We propose to test this hypothesis through three specific aims: (I) to characterize the novel MyBP-C(+) slow isoform in differentiating and adult skeletal muscle fibers, using a wide array of molecular, cellular and immunological approaches;(II) to study the interaction between obscurin and MyBP-C(+) slow qualitatively, by in vitro and in vivo binding assays, and quantitatively, by surface plasmon resonance technology and (III) to investigate the physiological significance of the binding of obscurin to MyBP-C(+) slow in the assembly and organization of myosin thick filaments into periodic A-bands, using adenovirally-mediated gene transfer and small inhibitory RNA technology. Knowledge gained from the proposed studies will provide new insights into the molecular mechanisms that integrate thick filaments into sarcomeres of normal skeletal fibers and how they are compromised in human muscle disease.
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