Isolation, Characterization and Reconstruction of Vertebrate Striated Muscle Myosin Filaments

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Project Summary The long term goal of this research project is to understand the molecular mechanism of force production through 3-D visualization of myosin molecular motors in their natural environment. This research project focuses on extending methods used and results obtained in structural studies of muscle filaments isolated from the large waterbug Lethocerus sp. to vertebrate striated muscle, specifically skeletal muscles obtained from rabbits, Oryctolagus cuniculus. We have obtained a near atomic resolution 3-D image of thick filaments from Lethocerus flight muscle, which have a helical structure with 4-fold rotational symmetry. No coiled-coil protein of the size of myosin had been imaged previously at the resolution we have achieved (4.2Å) in the backbone of the myosin filament. There is now more known about the 3-D structure of Lethocerus thick filaments than those from any other animal. Recent advancements in detector technology, robotic electron microscopes and high throughput data collection, have made this possible. We now propose to extend these methods to the much more difficult vertebrate skeletal muscle thick filament, which is not a helical assembly and has only 3-fold rotational symmetry. The high-resolution structure of Lethocerus thick filaments suggests that studies of invertebrate thick filaments can inform familial muscle diseases caused by myosin rod mutations. About 40% of disease-causing myosin mutations occur in the myosin coiled-coil domain. However, how well invertebrate thick filaments can inform human disease depends on how similar their filament backbones are structured like those of vertebrates. In the current funding period, we have obtained unprecedented resolution and detail of the relaxed state of thick filaments from Lethocerus flight muscle. This advance provides opportunity to investigate the mechanism whereby myosin rod mutations can affect muscle function. The head folding of myosin II produces a head conformation called the interacting heads motif that sequesters the myosin heads from interaction with the thin filament. In filaments of smooth and non-muscle myosin, the head folding leads to filament instability and formation of a soluble conformation, called 10S, incapable of polymerizing. This phenomenon has been hypothesized to be due to changes in the rod structure brought on by the head folding. Put simply, the structure of the myosin rod and the myosin heads are coupled in some way. Recent muscle research has pointed to the possibility that tension applied either internally by myosin heads or externally by a stretch, can affect the structure of the thick filament. Thus, the thick filament may function as a tension transducer, but the molecular mechanism by which this occurs is unknown. We hypothesize that tension applied to the thick filament affects the structure of the myosin heads and vice versa, that the myosin heads affect the structure of the myosin tails. This hypothesis can be tested using cryoEM by comparing a naturally formed super-relaxed filament to one with the myosin heads disordered.