Observing the Behavior of a Single Myosin Head Within a Myosin Filament Moving on Actin

Abstract

We developed a novel unloaded in vitro motility assay in which myosin filaments move over surface-attached actin (Brizendine et al, PNAS 2015). For three types of muscle myosin II (smooth, skeletal, and cardiac), the relationship between myosin filament velocity (V) and the number of myosin heads available to interact with actin (N) can be accounted for by a novel mixed-kinetic model where V is influenced by both attachment and detachment kinetics (see Brizendine et al, Abstract this meeting). A key model variable is the flexibility and effective length (L) of the subfragment 2 (S2) region of the myosin tail, which can presumably bend or buckle to allow actin-attached myosin heads to place very little drag load on the moving filament. This model predicts that a given myosin head within a moving filament will appear stationary for the duration of time it remains attached to actin (ton), followed by a jump forward a distance L relative to the filament backbone. To test these predictions, we prepared three different smooth muscle myosin (SMM)-derived QD-labeled constructs (regulatory light chain (RLC), light meromyosin (LMM), and S2) and assembled them individually and simultaneously into co-filaments with unlabeled native SMM. We will track the QD motion with nm accuracy during filament motility to compare the behavior of myosin heads (RLC-QD) and the headless S2-QD relative to the global filament motion (LMM-QD). As [ATP] is varied, ton is predicted to change according to ton = (k-AD)−1 + (kt[ATP])−1, where k-AD is the ADP release rate and kT is the rate of attachment of ATP to acto-myosin, whereas L will be invariant at ∼60 nm, a value based upon our previous work (Brizendine et al, PNAS 2015).