Myosin Heads Walk Along Actin Filaments in Discrete Steps

Unraveling the mechanism of myosin-powered movement was greatly aided by development of in vitro motility assays. In one such assay, the sliding-filament assay, the movement of fluorescence-labeled actin filaments along a bed of myosin molecules is observed in a fluorescence microscope. Because the myosin molecules are tethered to a coverslip, they cannot move; thus any force generated by interaction of myosin heads with actin filaments forces the filaments to move relative to the myosin (Figure 19-17a). If ATP is present, added actin filaments can be seen to glide along the surface of the coverslip; if ATP is absent, no filament movement is observed. This movement is caused by a myosin head (bound to the coverslip) "walking" toward the (+) end of a filament; thus filaments move with the (—) end in the lead. [The one exception is myosin VI, which moves in the opposite direction, toward the (—) end; so the (+) end of a moving filament is in the lead.] The rate at which myosin moves an actin filament can be determined from video camera recordings of sliding-filament assays (Figure 19-17b). The velocity of filament movement can vary widely, depending on the myosin tested and the assay conditions (e.g., ionic strength, ATP and Ca2+ concentrations, temperature).

▲ EXPERIMENTAL FIGURE 19-17 Sliding-filament assay is used to detect myosin-powered movement.

(a) After myosin molecules are adsorbed onto the surface of a glass coverslip, excess myosin is removed; the coverslip then is placed myosin-side down on a glass slide to form a chamber through which solutions can flow. A solution of actin filaments, made visible by staining with rhodamine-labeled phalloidin, is allowed to flow into the chamber. (The coverslip in the diagram is shown inverted from its orientation on the flow chamber to make it easier to see the positions of the molecules.) In the presence of ATP; the myosin heads walk toward the (+) end of filaments by the mechanism illustrated in Figure 3-25. Because myosin tails are immobilized, walking of the heads causes sliding of the filaments. Movement of individual filaments can be observed in a fluorescence light microscope. (b) These photographs show the positions of three actin filaments (numbered 1, 2, 3) at 30-second intervals recorded by video microscopy. The rate of filament movement can be determined from such recordings. [Part (b) courtesy of M. Footer and S. Kron.]

The most critical feature of myosin is its ability to generate a force that powers movements. Researchers have used a device called an optical trap to measure the forces generated by single myosin molecules (Figure 19-18). The results of optical-trap studies show that myosin II moves in discrete steps, approximately 5-10 nm long, and generates 3-5 pico-newtons (pN) of force, approximately the same force as that exerted by gravity on a single bacterium. This force is sufficient to cause myosin thick filaments to slide past actin thin filaments during muscle contraction or to transport a membrane-bounded vesicle through the cytoplasm. With a step size of 5 nm, myosin would bind to every actin subunit on one strand of the filament. Some evidence suggests that ATP hydrolysis and myosin walking are closely coupled, with myosin taking a discrete step for every ATP molecule hydrolyzed.

▲ EXPERIMENTAL FIGURE 19-18 Optical trap determines force generated by a single myosin molecule. In an optical trap, the beam of an infrared laser focused by a light microscope on a latex bead (or any other object that does not absorb infrared light) captures and holds the bead in the center of the beam. The strength of the force holding the bead is adjusted by increasing or decreasing the intensity of the laser beam. In this experiment, a bead is attached to the end of an actin filament. With the optical trap turned off, the actin filament and its attached bead move in response to the force generated by myosin adsorbed on

The neck domains in different myosins vary in length and number of associated light chains. Generally, the longer the neck domain of a myosin, the greater its step size (i.e., the distance traveled along an actin filament in one step). Because the neck region is the lever arm of myosin, a longer neck would lead to a longer distance traveled by the arm. For instance, myosin II, with a short neck, has from 5- to 10-nm steps, whereas myosin V, with a long neck, has much longer 36-nm steps. The correlation between step size and neck length has been further supported by experiments in which the neck domain is lengthened by recombinant methods. However, the correlation between neck length and step size is not absolute, as evidenced by myosin VI, which moves in 30-nm steps, although it has a neck domain shorter than that of myosin II.

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