An in vitro motility assay system retaining the steady-state force-velocity characteristics of muscle fibers under positive and negative loads

Abstract

To investigate in vitro myofilament sliding in conditions close to those in muscle fibers, we developed a new assay system in which a polystyrene bead (diameter, 4 μm; specific gravity, 1.3), with a single to a few thick filaments of a molluscan smooth muscle attached, was made to slide along actin filament arrays (actin cables) in the internodal cell of an alga, mounted on the rotor of a centrifuge microscope. The bead moving along actin cables in the presence of ATP was subjected to centrifugal forces either opposite to the bead movement (positive loads) or in the same direction as the bead movement (negative loads). With positive loads increasing from zero to the maximum isometric force P 0, the velocity of bead movement decreased gradually to zero, exhibiting the hyperbolic force-velocity relation except for loads above 0.8 P 0. With further increase of positive loads above P 0, the bead was forced to move in the direction of centrifugal force, being eventually detached from actin cables at a load of ∼1.4 P 0. These features are very similar to the force-velocity relation in intact single muscle fibers. With negative loads increasing from zero to a certain value, on the other hand, the bead moved faster than the maximum unloaded velocity (1.5–3.5 μm/s, 20–25°C), until it was eventually detached from actin cables. These results indicate that our assay system is extremely promising for future combined biochemical and physiological studies on the mechanism of muscle contraction.