The long term goal of this research project has been and continues to be an understanding of the molecular mechanism of muscle function. The existing project, funded continuously since 1983, has evolved in parallel with the capabilities of both microscopes and methods for 3-D image reconstruction from electron microscopes. Originally focused on understanding myosin-actin interactions in situ in muscle using chemically fixed, plastic embedded and sectioned muscle, it now proposes to take advantage of the resolution revolution in cryoEM to study the major structural elements of the muscle at the highest resolution possible using isolated components, followed by a return to imaging actin-myosin interactions in situ in frozen live muscle cells. Striated muscles have four major components: actin-containing thin filaments, myosin- containing thick filaments, a Z-disk to crosslink antiparallel thin filaments and a connecting filament to link the thick filaments to the Z-disk. The least understood of these four elements are the thick filament, the Z-disk and the connecting filament, whose interactions with the thick filament and Z-disk are its least understood elements. Thus, our study of Z-disk and thick filament can make major contributions to an understanding of all three elements. Following a major breakthrough of ours that showed that coiled-coil tail domain of myosin can be imaged at subnanometer resolution, even near atomic resolution, the project concentrates initially on subnanometer resolution imaging of thick filaments from several species, to examine the generality and structural conservation of the ?curved molecular crystalline layers? across species and muscle types. The project will utilize the fruit fly, Drosophila melanogaster, to investigate how genetic removal of certain component proteins affects how the myosin tails interact with non-myosin proteins to affect thick filament properties. We will utilize mutations in the myosin tail of Drosophila that correspond to established disease causing mutations in human striated muscle. The Z-disk will be studied using methodology developed in our lab to isolate Z-disks from invertebrates applied to determination of the Drosophila melanogaster Z-disk. The experimental system will facilitate decoration of the Z-disks with various signaling proteins. Ultimately, the utility of these studies on components needs development within the myofibril. We will investigate by cryoelectron tomography frozen-hydrated myofibrils of Lethocerus and Drosophila thinned using FIB/SEM in states produced using various nucleotides to see how myosin heads interact with the thin filament in situ. Ultimately, we will apply what we have learned methodologically from these myofibril studies to studies of live cultured smooth and cardiac muscle cells fast frozen, thinned via FIB/SEM to visualize active interactions between thick and thin filaments. This work will open to future structural investigation all the structures present in a muscle cell within their natural context.
The least understood parts of the striated muscle sarcomere are the thick filament, connecting filament and the Z-disk. Almost 40% of myopathy mutations occur in the thick filament backbone in which the coiled-coil myosin tails pack in order to bear the force produced by the myosin heads while another group of disease-causing mutations occur among proteins of the Z-disk, which anchors thin filaments into a bipolar array. This project seeks to understand through 3-D imaging of these three muscle components how mutations alter their structures resulting in defects of muscle function.