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Visualization of single actin filaments by fluorescence microscopy led to the development of new in vitro assays for analysing actomyosin-based motility at the molecular level. The ability to manipulate actin filaments with a microneedle or an optical trap combined with position-sensitive detectors has enabled direct measurements of nanometre displacements and piconewton forces exerted by individual myosin molecules. To elucidate how myosin generates movement, it is necessary to understand how ATP hydrolysis is coupled to mechanical work at the level of the single molecule. But the most sensitive microscopic ATPase assay available still requires over 1,000 myosins. To enhance the sensitivity of such assays, we have refined epifluorescence and total internal reflection microscopies to visualize single fluorescent dye molecules. We report here that this approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions. In contrast to previously reported single fluorescent molecule imaging methods, which used specimens immobilized on an air-dried surface, our method allows video-rate imaging of single molecules in aqueous solution, and hence can be applied to the study of many types of enzymes and biomolecules.

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

Filamentous structures are abundant in cells. Relatively rigid filaments, such as microtubules and actin, serve as intracellular scaffolds that support movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depend critically on its flexibility—for example, DNA-binding proteins can induce sharp bends in the helix1. The mechanical characterization of such filaments has generally been conducted without controlling the filament shape, by the observation of thermal motions2,3,4,5 or of the response to external forces6,7,8,9 or flows10,11,12. Controlled buckling of a microtubule has been reported13, but the analysis of the buckled shape was complicated. Here we report the continuous control of the radius of curvature of a molecular strand by tying a knot in it, using optical tweezers to manipulate the strand's ends. We find that actin filaments break at the knot when the knot diameter falls below 0.4 µm. The pulling force at breakage is around 1 pN, two orders of magnitude smaller than the tensile stress of a straight filament. The flexural rigidity of the filament remained unchanged down to this diameter. We have also knotted a single DNA molecule, opening up the possibility of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.

Kinesin is a two-headed motor protein that powers organelle transport along microtubules. Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.

The myosin molecule consists of two heads, each of which contains an enzymatic active site and an actin-binding site. The fundamental problem of whether the two heads function independently or cooperatively during muscle contraction has been studied by methods using an actomyosin thread, superprecipitation and chemical modification of muscle fibres. No clear conclusion has yet been reached. We have approached this question using an assay system in which sliding movements of fluorescently labelled single actin filaments along myosin filaments can be observed directly. Here, we report direct measurement of the sliding of single actin filaments along one-headed myosin filaments in which the density of heads was varied over a wide range. Our results show that cooperative interaction between the two heads of myosin is not essential for inducing the sliding movement of actin filaments.