Axial rotation of sliding actin filaments revealed by single-fluorophore imaging

Abstract

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...

A rotary molecular motor that can work at near 100% efficiency

“…A molecular machine must occasionally make mistakes. The 1208 steps have also been con¢rmed in F 1 without actin (K. Adachi, unpublished data), by attaching a £uorophore on g and observing the polarization of the £uorescence from the single £uorophore under a microscope (Sase et al 1997;Ha et al 1998). Thus, stepping is not an artefact caused by friction between the actin ¢lament and the stator cylinder with pseudo-threefold symmetry.…”

Stepping rotation of F 1 -ATPase visualized through angle-resolved single-fluorophore imaging

“…The emission was decomposed into vertically (V) and horizontally (H) polarized components in a dual-view apparatus, and the two were simultaneously projected onto the video camera (5,27). A vertically oriented fluorophore would show up in the V image, and a horizontal one would appear in H. Alternate appearance, as shown in Fig.…”

“…Individual behaviors can be assessed by single-fluorophore imaging (1)(2)(3)(4)(5), which is much less perturbing than imaging through a huge tag such as a plastic bead or actin filament (6). Real-time determination of fluorophore orientation (5,7,8) should be particularly useful, because a conformational change necessarily accompanies reorientation of one part against others. Thus far, however, successful applications of this potentially powerful method have been made infrequently.…”

Cytoskeletal Symmetry Breaking and Chirality: From Reconstituted Systems to Animal Development

“…Torque is clearly seen in in vitro assays: Nishizaka et al [145] showed that during myosin-driven sliding of actin filaments, a torque component can be observed that induce rotation of an actin filament around its long axis. Later, Sase et al [143] have confirmed that actin filaments undergo one revolution per sliding distance of approximately 1 µm. Similar rotation or twirling of actin filaments have been confirmed in more recent reports [144,146].…”

“…It is formed by actin protomers that assemble into a right-handed double helix with a full turn after 13 protomers or every 72 nm [142]. Sequential interactions of myosin motors with one of the helical strands can therefore generate a right-handed rotation of the filament around its axis [143]. Moreover, the myosin working stroke is not perfectly parallel to the axis of the actin filament [144] (and references therein), thus resulting in a small angular component that can generate torque.…”