A Mixed-Kinetic Model Describes Unloaded Velocities of Smooth, Skeletal, and Cardiac Muscle Myosin Filaments in Vitro

Abstract

In vitro motility assays, where purified myosin and actin move relative to one another, are used to better understand the mechanochemistry of the actomyosin adenosine triphosphatase (ATPase) cycle. We examined the relationship between the relative velocity (V) of actin and myosin and the number of available myosin heads (N) or [ATP] for smooth (SMM), skeletal (SKM), and cardiac (CMM) muscle myosin filaments moving over actin as well as V from actin filaments moving over a bed of monomeric SKM. These data do not fit well to a widely accepted model that predicts that V is limited by myosin detachment from actin (d/t on ), where d equals step size and t on equals time a myosin head remains attached to actin. To account for these data, we have developed a mixed-kinetic model where V is influenced by both attachment and detachment kinetics. The relative contributions at a given V vary with the probability that a head will remain attached to actin long enough to reach the end of its flexible S2 tether. Detachment kinetics are affected by L/t on , where L is related to the tether length. We show that L is relatively long for SMM, SKM, and CMM filaments (59 ± 3 nm, 22 ± 9 nm, and 22 ± 2 nm, respectively). In contrast, L is shorter (8 ± 3 nm) when myosin monomers are attached to a surface. This suggests that the behavior of the S2 domain may be an important mechanical feature of myosin filaments that influences unloaded shortening velocities of muscle.

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain

“…Figure a shows unexchanged rhodamine‐labeled SMM filament suspension prespin, which was indistinguishable from images of filaments postspin and from SMM filaments exchanged with a 10‐fold excess native RLC (data not shown). Filaments appear as bright oblong shapes that we have previously characterized (Brizendine et al, ; Brizendine et al, ; Haldeman et al, ). The majority were single filaments (Figure (a), carets), but some filaments in small aggregates were also evident (arrows).…”

“…Activities of other controls with and without crosslinking were similar, as previously shown in (Haldeman et al, ). Assays for actin‐activated ATPase were at 30 °C as previously described (Brizendine et al, ) except the buffer contained 50 mM NaCl and [actin] = 50 μM. Phosphate was determined as described (Brizendine et al, ).…”

“…Assays for actin‐activated ATPase were at 30 °C as previously described (Brizendine et al, ) except the buffer contained 50 mM NaCl and [actin] = 50 μM. Phosphate was determined as described (Brizendine et al, ). (b) Velocity ( V ) vs N plot showing control SMM data (faded red) from (Brizendine et al, ).…”

“…After the exchange reaction, filaments were formed by dialysis and cross‐linked with EDC as previously described (Brizendine et al, ; Brizendine et al, ; Haldeman et al, ).…”

“…A/M f motility assays were performed as described (Brizendine et al, ; Brizendine et al, ). Actin was attached to a PEG surface and rhodamine‐labeled SMM filaments were observed moving along the actin.…”