Evidence of myosin swaying heads in relaxed thick filaments from tarantula striated muscle
Riggs, Claire L.
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Myosin swaying heads are integral to the proposed model of thick filament activation. This study investigates the presence of swaying heads in tarantula striated muscle, an ideal model system, due to high filament order and image resolution. Primarily regulated by phosphorylation of the myosin regulatory light chain (RLC), thick filament activation extends myosin heads, allowing for interaction with actin to result in contraction. Each myosin molecule has two heads, organized as a free and a blocked head on the filament. In the relaxed state, calcium absent, a basal mono-phosphorylation is present on Ser35 of the free head RLC which allows these heads to sway away; these are the swaying heads. Upon activation, calcium activates the myosin light chain kinase (MLCK), yielding bi-phosphorylation and thereby full extension of swaying heads, exposing their partner blocked head for phosphorylation. Though these additionally activated heads extend and increase contractile force, swaying heads alone support actin movement under relaxing conditions. In the prepared thick filament homogenate, SDS-PAGE confirmed the presence of expected proteins, and urea-glycerol (UG) gels revealed non- and mono-phosphorylated RLC under relaxing and activating conditions, and the additional bi-phosphorylated RLC under activating conditions. Thick filaments in the relaxed state supported actin movement, detected by In Vitro Motility Assay (IVMA). This movement was also supported under MLCK inhibition. Since this treatment effectively maintains myosin filaments under relaxing conditions, only swaying heads can account for the movement since they are basally present. Three-dimensional reconstruction of electron micrographs of thick filaments under relaxing conditions were obtained, however refinement of the technique is necessary to reveal the swaying heads.