The cytoplasm of striated muscle cells contains, besides actin and myosin filaments, contains at least two interconnected lattices. An intermediate filament lattice envelops and links all sarcomeres to the membrane skeleton, mitochondria, nuclei, and sarcoplasmic reticulum. Inside the sarcomere, a cytoskeletal matrix consisted of a set of elastic titin filaments and a set of inextensible nebulin filaments provides structural continuity. Both lattices generate restoring force. Active force and elastic force are transmitted through specialized anchor structures of the sarcomere. One important stress-bearing structure is the Z line, a dense and narrow structure that anchors and organizes four major filaments: actin, titin, nebulin and desmin filaments. The Z lines are now known to play important roles in the structural organization of sarcomere, the transmission of mechanical forces as well as the stress signaling pathways.? Its dense structure however poses technical challenges and the variability of protein composition made it difficult to generalize findings from one muscle to the next. Our projects address the Z line structure and function from several prospectives. 1. What are the roles of titin, nebulin (skeletal muscles), nebulette (a nebulin-ike protein in the heart) in the assembly and integrity of the Z line in vertebrate muscle? 2. What are the composition and structure of the unusually broad Z line of sonic muscle of Midshipman fish? 3. What are the roles of protein kinases, nonmuscle myosin and other signaling proteins in the function of the Z lines? 4. What is its relationship to the anomalous nemaline rod Z bodies found in aging heart muscle, in diseased skeletal muscle known as nemaline myopathy? ? The distribution of titin, nebulin, nebulette nonmuscle myosin IIB and into the myofibrils and the Z lines are being studied with fluorescence techniques with either monoclonal antibodies to these proteins, or by the use of fluorescent fusion proteins synthesized within the muscle cells. To identify protein composition, especially the proteins that interact with titin, nebulin and nebulette in the Z line, we are applying both molecular biological methods (yeast two hybrid screening), as well as biochemical techniques to search for interacting proteins. We have succeeded in resolving the high-resolution structure of the unusually broad Z band (1 micron, roughly 20 times the wide of vertebrate Z lines) in the sonic muscle of Midshipman fish is being studied by electron microscopy, X-ray diffraction and biochemical methods. Interestingly, the Z band are also attachment sites of a very elaborate intermediate filaments lattice and junctional complexes at the Z band that provide the necessary radial force to assemble and maintain the tubular shape of the sonic muscle fiber. We also discovered novel assembly/disassembly intermediates (myosin flares and leptomeres) that suggest a new mechanism of muscle growth by splitting longitudinally along the myotubes. The myofibrillogenesis is being approached by establishing a culture system for the sonic muscle. To visualize the contraction of the sonic muscle, we have also succeeded in visualizing the detailed organization of muscle fibers in intact sonic muscle by magnetic resonance imaging (MRI) and analyzed the sound producing characterized by laser vibrometry. These studies are important in the understanding of how contractile machinery of this superfast muscle assembles during development, how it dissembles during remodeling of muscle tissues, how tension are transmitted during muscle activities and how muscles malfunction in nemaline myopathy and other muscle diseases.
