Muscle contraction is driven by the cyclical interaction of myosin with actin, coupled to the breakdown of ATP. Studies of the interaction of filamentous myosin and of a double-headed proteolytic fragment, heavy meromyosin (HMM), with actin have demonstrated discrete mechanical events, arising from stochastic interaction of single myosin molecules with actin. Here we show, using an optical-tweezers transducer, that a single myosin subfragment-1 (S1), which is a single myosin head, can act as an independent generator of force and movement. Our analysis accounts for the broad distribution of displacement amplitudes observed, and indicates that the underlying movement (working stroke) produced by a single acto-S1 interaction is approximately 4 nm, considerably shorter than previous estimates but consistent with structural data. We measure the average force generated by S1 or HMM to be at least 1.7 pN under isometric conditions.

Muscle contraction is brought about by the cyclical interaction of myosin with actin coupled to the breakdown of ATP. The current view of the mechanism is that the bound actomyosin complex (or "cross-bridge") produces force and movement by a change in conformation. This process is known as the "working stroke." We have measured the stiffness and working stroke of a single cross-bridge (kappa xb, dxb, respectively) with an optical tweezers transducer. Measurements were made with the "three bead" geometry devised by Finer et al. (1994), in which two beads, supported in optical traps, are used to hold an actin filament in the vicinity of a myosin molecule, which is immobilized on the surface of a third bead. The movements and forces produced by actomyosin interactions were measured by detecting the position of both trapped beads. We measured, and corrected for, series compliance in the system, which otherwise introduces large errors. First, we used video image analysis to measure the long-range, force-extension property of the actin-to-bead connection (kappa con), which is the main source of "end compliance." We found that force-extension diagrams were nonlinear and rather variable between preparations, i.e., end compliance depended not only upon the starting tension, but also upon the F-actin-bead pair used. Second, we measured kappa xb and kappa con during a single cross-bridge attachment by driving one optical tweezer with a sinusoidal oscillation while measuring the position of both beads. In this way, the bead held in the driven optical tweezer applied force to the cross-bridge, and the motion of the other bead measured cross-bridge movement. Under our experimental conditions (at approximately 2 pN of pretension), connection stiffness (kappa con) was 0.26 +/- 0.16 pN nm-1. We found that rabbit heavy meromyosin produced a working stroke of 5.5 nm, and cross-bridge stiffness (kappa xb) was 0.69 +/- 0.47 pN nm-1.

A prerequisite for using muscle mutants to study contraction in Drosophila melanogaster is a description of the mechanics of wild-type muscles. Here we describe the mechanics of two different wild-type muscles; the dorsal longitudinal flight muscle which is asynchronous (nerve impulses are not synchronised with each contraction), and a leg muscle, the tergal depressor of the trochanter, which is synchronous. We have compared their mechanics to those of the asynchronous flight and the synchronous leg muscle from the giant waterbug Lethocerus indicus. We found that the mechanics of the asynchronous flight muscles from the two species were similar. At rest length both muscles had a high relaxed stiffness, were partially activated by Ca2+ (low steady-state active tension) and, once activated, had a large delayed increase in tension, which was well maintained, in response to a rapid stretch. The rate constant for the delayed increase in tension was about 10 times greater for D. melanogaster than for L. indicus under the same conditions. The mechanics of the synchronous leg muscles from both species were different from those of the flight muscles and resembled those of other synchronous muscles such as vertebrate striated muscle. At rest length, both muscles had a lower relaxed stiffness than the flight muscles, were fully activated by Ca2+ (high steady-state active tension) and, once activated, had a small delayed increase in tension, which was less well maintained, in response to a rapid stretch. The rate constant for the delayed increase in tension was similar for the leg muscles of both species. The different mechanical properties of the flight and leg muscles must arise from differences in their contractile proteins. The demonstration that satisfactory mechanical responses can be obtained from the small (less than 1 mm long) muscles of D. melanogaster will enable future responses from mutant muscles to be tested.

In this paper we suggest and test a specific hypothesis relating the attachment-detachment cycle of cross bridges between actin (I) and myosin (A) filaments to the measured length-tension dynamics of active insect fibrillar flight muscle. It is first shown that if local A-filament strain perturbs the rate constants in the cross-bridge cycle appropriately, then exponentially delayed tension changes can follow imposed changes of length; the latter phenomenon is sufficient for the work-producing property of fibrillar muscle, as measured with small-signal forcing of length and at low Ca(2+) concentration, and possibly for related effects described recently in frog striated muscle. It is not clear a priori that the above explanation of work production by fibrillar muscle will remain tenable when the viscoelastic complexity of the heterogeneous sarcomere is taken into account. However, White's (1967) recent mechanical and electron microscope study of the passive dynamics of glycerinated fibrillar muscle has produced a model of the distributed viscoeleastic structure sufficiently explicit that alternative schemes for cross-bridge force generation in this muscle can now be tested more critically than previously. Therefore, we derive and solve third-order partial-differential equations which relate local interfilament shear forces associated with the perturbed cross-bridge cycles to the over-all length-tension dynamics of an idealized sarcomere. We then show (a) that the starting hypothesis can account approximately for the small-signal dynamics of glycerinated muscle in the work-producing state over two decades of frequency and (b) that the rate constants for cross-bridge formation and breakage, restricted solely by fitting of the model to the mechanical data, determine a cycling rate of cross bridges in the model compatible with recent measurements of ATP hydrolysis rate vs. stretch in this muscle. Finally, the formulation is extended tentatively to the large-signal nonlinear case, and shown to compare favorably with previous suggestions for the origin of the work-producing dynamics of fibrillar flight muscle.

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