Abstract
Much of Howard's Review Article1 concerns the results of experiments with single myosin molecules and actin filaments. It was a huge surprise when reports of such experiments first appeared (see, for example, ref. 2), and much is being learnt from them that cannot be deduced from experiments on whole muscle fibres, whether intact or after removal of the membrane. But single-molecule experiments do not yet approach the time resolution or the freedom from brownian noise that are easily attainable on larger assemblies of myosin and actin filaments, and their interpretation is subject to many uncertainties — due, for instance, to compliance in the actin filaments and in their attachments to beads or other force-measuring components, and the attachment of myosin molecules or fragments to the base. No doubt the time course of the working stroke of a single myosin head will one day be recorded, but until that is achieved the results of experiments on whole fibres and myofibrils deserve more careful attention than has been given to them by Howard.
Main
Many of Howard's statements can be challenged. Here I will mention only three, of particular relevance to my own work. First, Howard uses a value of 4 nm for the “working distance” of a myosin molecule, defined as the distance over which a myosin head remains attached during a single interaction with an actin filament. But he uses the same value of 4 nm for the “working stroke”, the distance over which an attached crossbridge exerts positive force. In rapid shortening, the former is presumably greater than the latter as crossbridges remain attached for a short time after the force they exert has fallen to zero. Howard justifies the use of this value on the grounds that it is the amount of sudden shortening per half-sarcomere needed to bring tension to zero from its value in an isometric tetanus of an isolated frog muscle fibre (Fig. 26 of ref. 3).
However, this quantity is usually interpreted as the average amount by which compliant structures in the half-sarcomere had been strained by the active tension before application of the shortening step. The working stroke is represented in such experiments by the rapid recovery of much of the tension that was present before the shortening step. The slack in a fibre caused by sudden shortening of 13 nm per half-sarcomere is the maximum taken up by this quick recovery process (Fig. 13 of ref. 3). In simulations of the response to a shortening step, this maximum is about equal to the assumed working stroke plus the contribution from linear compliance, whether it is assumed that the working stroke is performed by crossbridges that were already attached before the step (as in my own4 and many other simulations) or by fresh crossbridges that attach very rapidly after the step. In the latter case, if a stroke of 4 nm is assumed the model cannot account for the 13 nm of shortening observed (Fig. 7 of ref. 5).
Second, Howard points out correctly that “the discovery that the actin filaments contribute about half the compliance of muscle [during isometric contraction] means that the stiffness is not proportional to the number of attached heads” and concludes that “a low duty ratio is therefore not inconsistent with the stiffness measurements” (p. 564 of ref. 1).
The stiffness of an isolated frog fibre contracting under zero load is about one- third of its stiffness during isometric contraction6, implying 20% of heads attached if it is assumed that all heads are attached in rigor. This is very different from the 1% claimed by Howard.
Last, Howard writes of “a paradox” — that the amount of filament sliding that occurs in the time required by each myosin molecule to hydrolyse one ATP is much larger than the “working distance”, referring to two papers (refs 7 and 8 here). He claims to resolve this “paradox” by supposing that much of the sliding takes place while the myosin is detached from actin.
But for many years (for example, ref. 9) it has been supposed that each myosin head acts intermittently and that continuous sliding is brought about by asynchronous action of many myosin molecules, although estimates of the sliding distance per ATP hydrolysed have varied. The controversy raised by refs 7 and 8 was different: those papers report that when a myosin head interacts with an actin filament during rapid shortening, it remains attached for a distance of 60 nm (ref. 7) or 40 nm (ref. 8). Staying attached for such large distances remains difficult to explain, and Howard's “resolution” of the paradox is irrelevant because his central postulate is that a myosin head remains attached for only 4 nm, much smaller than 40 or 60 nm.
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Huxley, A. How molecular motors work in muscle. Nature 391, 239 (1998). https://doi.org/10.1038/34567
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DOI: https://doi.org/10.1038/34567
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