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In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of Ϸ72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through f luorescence polarization imaging of individual tetramethylrhodamine f luorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of Ϸ1 m, which is much greater than the 72 nm pitch. Thus, myosin does not ''walk'' on the helical array of actin protomers; rather it ''runs,'' skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each ''kick'' of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single f luorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.The actin filament is an array of actin protomers arranged in the form of two-start, right-handed helices with a pitch of Ϸ72 nm containing Ϸ13 protomers (1). If myosin tends to interact sequentially with one of the helical strands (the binding site on the other strand being on the opposite side), right-handed rotation of a sliding actin filament around its axis is expected. Indeed, in an in vitro motility assay in which the front end of a sliding filament was fixed on a surface, the middle part formed a left-handed superhelix, indicating right-handed rotation of the sliding rear part (2). However, in another assay where a marker (bead aggregate) was attached at the tail of a freely sliding filament, the filament slid over a long distance without rotating the tail beads (3). Quantitative resolution of this issue is important for the mechanism of motor function, because axial rotation is an indication of (i) sequential interaction of a myosin molecule with successive (or closely apposed) actin protomers, as stated above, and͞or (ii) the presence of a genuine torque component in the individual myosin-actin interaction not necessarily related to the helical structure. In the first assay above (2) where the superhelix formation was observed, the number of axial rotations could not be determined in the video images of limited resolution. A complication with the bead-tailed actin (3) was that the beads produced a large rotational, but not translational, friction that may have impeded the axial rotation. Here we show that the amount of rotation is small even in the absence of the external asymmetric load. Neither i nor ii appear to be essential components of the actomyosin motor. Quantitative measurement of the axial rotation without an impeding marker was achieved by continuo...

Relatively simple modifications of an ordinary epifluorescence microscope have greatly reduced its background luminescence, allowing continuous and real time imaging of single fluorophores in an aqueous medium. Main modifications were changing the excitation light path and setting an aperture stop so that stray light does not scatter inside the microscope. A simple and accurate method using actin filaments is presented to establish the singularity of the observed fluorophores. It was possible, at the video rate of 30 frames/s, to image individual tetramethylrhodamine fluorophores bound to actin filaments sliding over heavy meromyosin. The successful imaging of moving fluorophores demonstrates that conventional microscopes may become a routine tool for studying dynamic interactions among individual biomolecules in physiological environments.

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