Crossbridge and tropomyosin positions observed in native, interacting thick and thin filaments

Tropomyosin movements on thin filaments are thought to sterically regulate muscle contraction, but have not been visualized during active filament sli ding. In addition, although 3-D visualization of myosin crossbridges has be en possible in rigor, it has been difficult for thick filaments actively in teracting with thin filaments. In the current study, using three-dimensiona l reconstruction of electron micrographs of interacting filaments, we have been able to resolve not only tropomyosin, but also the docking sites for w eak and strongly bound crossbridges on thin filaments. In relaxing conditio ns, tropomyosin was observed on the outer domain of actin, and thin filamen t interactions with thick filaments were rare. In contracting conditions, t ropomyosin had moved to the inner domain of actin, and extra density, refle cting weakly bound, cycling myosin heads, was also detected, on the extreme periphery of actin. In rigor conditions, tropomyosin had moved further on to the inner domain of actin, and strongly bound myosin heads were now obse rved over the junction of the inner and outer domains. We conclude (1) that tropomyosin movements consistent with the steric model of muscle contracti on occur in interacting thick and thin filaments, (2) that myosin-induced m ovement of tropomyosin in activated filaments requires strongly bound cross bridges, and (3) that crossbridges are bound to the periphery of actin, at a site distinct from the strong myosin binding site, at an early stage of t he crossbridge cycle. (C) 2001 Academic Press.