Mechanics of actomyosin bonds in different nucleotide states are tuned to muscle contraction

Abstract

Muscle contraction and many other cell movements are driven by cyclic interactions between actin filaments and the motor enzyme myosin. Conformational changes in the actin-myosin binding interface occur in concert with the binding of ATP, binding to actin, and loss of hydrolytic by-products, but the effects of these conformational changes on the strength of the actomyosin bond are unknown. The force-dependent kinetics of the actomyosin bond may be particularly important at high loads, where myosin may detach from actin before achieving its full power stroke. Here we show that over a physiological range of rapidly applied loads, actomyosin behaves as a "catch" bond, characterized by increasing lifetimes with increasing loads up to a maximum at approximately 6 pN. Surprisingly, we found that the myosin-ADP bond is possessed of longer lifetimes under load than rigor bonds, although the load at which bond lifetime is maximal remains unchanged. We also found that actomyosin bond lifetime is ultimately dependent not only on load, but loading history as well. These data suggest a complex relationship between the rate of actomyosin dissociation and muscle force and shortening velocity. The 6-pN load for maximum bond lifetime is near the force generated by a single myosin molecule during isometric contraction. This raises the possibility that all catch bonds between load-bearing molecules are "mechanokinetically" tuned to their physiological environment.

Fig. 1.

Ribbon representation of the myosin motor domain, featuring the actin binding cleft. The upper and lower 50,000 (50k) domains are shown in black, and the A- and R-sites are highlighted in gray. The figure is based on coordinates from ref. .

Fig. 2.

Force spectroscopy of actomyosin bonds. (A) Schematic showing the arrangement for actomyosin force spectroscopy using the laser trap. (B) Illustration of the expected responses of catch, slip, and catch–slip bonds to imposed loads when measured in terms of bond lifetime.

Fig. 3.

The characteristic rupture force of actomyosin bonds over a range of loading rates. At higher loading rates, actomyosin–ADP bonds (●) appear to be stronger than rigor bonds (○). Corresponding data from single-headed HMM in each state (♦, ◇) are superimposed. The dashed line indicates the rigor behavior predicted from step load data (see text). Error bars represent SEM.

Fig. 4.

Catch–slip behavior in actomyosin bonds. (A) Actomyosin–ADP bonds (●) appear to be stronger than rigor bonds (○), and both states demonstrate the catch–slip behavior illustrated in Fig. 2B. Corresponding data taken in the presence of PP_i (▴) are superimposed. Control data (□) were collected in the presence of HMM but without actin on the beads. Error bars represent SEM. (B) Bond survival curves at two discrete forces demonstrate single exponential decays (n = 50 in each case). (C) Example raw data trace showing the onset of a loading step at time 0. A bond results in the bead remaining displaced from trap center for a period t (the bond lifetime). Bond rupture allows the bead to return to a new baseline. The baselines before and after the step are slightly offset because of an optical artifact when the trapped bead is positioned against the target bead (see text).