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Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5-2IQ). Electron microscopy of this chimera (Myo5-2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5-6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for MyoS-6IQ but much greater than for Myo5-2IQ. Myo5-2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5-6IQ in in-vitro single molecule assays. In comparison, Myo5-2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5-6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins. ATPase | electron microscopy | optical trap | single alpha helix doi/10.1073/pnas.0909851106

Rat liver myosin-I of relative molecular mass 130,000 and chick intestinal brush-border myosin-I, two members of the myosin-I family, produce a working stroke that takes place in two phases. Research in this area has used an optical-tweezers transducer to measure the mechanical transitions made by a single myosin head while it is attached to actin. It is likely that the two phases are linked to different biochemical states of the actomyosin cycle. It has been established that the slower myosins have longer working strokes.

The single-headed myosin-I's are thought to be involved in diverse functions such as membrane protrusion and tensioning, endocytosis and vesicle transport (Ruppert et al. 1995. J. Cell Sci. 108:3775). Here we have studied the cellular dynamics of the 130kD myosin-I, (rat myr1b). Myr1b has an [NH.sub.2]-terminal motor domain, a regulatory domain with 5 IQ motifs and a COOH-terminal lipid binding domain. Its slow biochemical kinetics (Coluccio and Geeves. 1999. J. Biol. Chem. 274:21575) and ADP-induced shape change (Veigel et al. 1999. Nature. 398:530) and localization in cells suggest that it may be important in maintaining tension between membrane and cytoskeleton. We used total internal reflection fluorescence microscopy (TIRF) to investigate the behavior of myr1b fused to GFP in cultured mouse myoblasts. Expression levels were low enough to visualize individual GFP-tagged molecules using TIRF. We used time-lapse recordings to identify molecules bound to the basal membrane of leading lamellae. We developed a computer algorithm to automatically detect single fluorophores using `DISH' criteria: Diffraction-limited image size, Intensity similar to single fluorophore control values, Single step photobleaching, Halftime proportional to illumination intensity. The last criterion distinguishes between diffusive motion of diffraction-limited objects or aggregates and single-step photobleaching. Our two-pass algorithm detects stepwise intensity changes of spots of appropriate size and intensity by performing a running t test on a diffraction limited pixel area over adjacent 1-s time windows of image data. Then the average lifetime of detected spots were measured at different laser powers. The density of myr1b-GFP on the basal membrane of the leading lamellae was sufficiently low to allow the observation of single fluorophores, 0.5-1 molecule/[micro][m.sup.2]. We are now developing methods to track the position of individual myr1b-GFP molecules in the leading lamellae of migrating myoblasts. (Myr1b clone was a gift of Martin Bahler; work funded by BBSRC.)

Class I myosins are actin-based molecular motors believed to be involved in motile events in the cell. Several class I myosins exist in higher cells. To assist in determining whether these isoforms have unique or similar roles, we have been investigating the properties of class I myosins from rat liver. Using steady-state and transient kinetic analyses, we have recently observed that rat liver 130-kD myosin I (also known as MI130, MYR 1, or MM1a) interacts with nucleotide and actin in much the same way qualitatively as other myosins; but, it is much slower. Also, acto.MI130 has a high affinity for ADP. These results led us to propose that MI130 is designed for efficient tension maintenance (Coluccio and Geeves. 1999. J. Biol. Chem. 274:21575-21580). Furthermore, we showed in single-molecule experiments that, unlike skeletal muscle myosin II, MI130 interacts with actin in two-steps (Veigel et al., 1999. Nature. 398: 530-533). The second step may be a consequence of the high affinity of acto.MI130 for ADP. Here, we examine MI110, a myr 2 (or MM1b) gene product, which is also widely expressed. The steady-state [Mg.sup.2+]-ATPase activity of MI110 is activated in [Ca.sup.2+]. Purified rat liver MI110 translocates actin filaments in vitro and, unlike MI130, the rate of translocation is greater at pCa 4 than at pCa 7. In transient kinetic studies, we have observed that the major phase of the ATP-induced dissociation of actin-MI110 ([K.sub.1] [k.sub.+2] = 0.0035 [micro][M.sup.-1][s.sup.-1]) is due largely to the smaller value of the maximal observed rate, [k.sub.+2]=2 [s.sup.-1]. This is 10 times slower than MI130 and places MI110 among the slowest myosins so far examined; 1/[K.sub.1] is comparable to that of other myosins. The affinity of ADP for A.MI110 is ~5 [micro]M and [Ca.sup.2+] insensitive. The 3-D structure of actin filaments decorated with MI110 under conditions of rigor or in the presence of ADP shows an ADP-induced conformational change, a characteristic of actomyosin complexes having a high affinity for ADP. Results from optical trapping of this myosin I as it interacts with actin indicate that MI110 exhibits a two-step power stroke similar to that exhibited by MI130. The study of these myosins has resulted in a new view of how motors work. Furthermore, they indicate that although these two myosins share some similar characteristics, they possess some unique mechanochemical properties which might permit them to carry out distinct functions. (Supported by NIH, Royal Society, and the Wellcome Trust.)

We have used single molecule techniques to study the mechanism of force production by myosin I. Myosin I's are ideal for investigating the myosin motor mechanism, because of their monomeric structure, slow biochemical kinetics, and long, two-phase power stroke. Using optical tweezers, we have made mechanical recordings from individual myosin-I molecules. We found that myr2 (with 3 IQ motifs) has an overall working stroke of ~4 nm compared with 11nm for myr1 (with 6 IQ's). To analyze these mechanical recordings, we developed methods based on Page's test, which we found gave better timing accuracy and robustness of event detection than other widely used methods (Knight et al. 2001. Progr. Biophys. Mol. Biol. In press). Using TIRFM (Total Internal Reflection Fluorescence Microscopy), we observed the binding and release of single molecules of Cy3-EDA-ATP/ADP by myosin I molecules on a surface. We found that the ADP release rate of brush border myosin I (BBMI), under similar conditions to the mechanical experiments above, is 0.16 [s.sup.-1]. We are combining this method with optical tweezers to perform correlated single molecule measurements to test if the second power stroke of myosin I correlates with ADP release. By observing fluorescent actin filaments as they swivel on individual myosins, we found the torsional stiffness of BBMI to be 31.0 [+ or -] 4.7pN [multiplied by] nm [multiplied by] [rad.sup.-1] (corresponding to an r.m.s. rotation of ~21 [degrees]). From these numbers, it is likely that a myosin molecule would be able to bind to an actin filament even if it were presented orthogonally to the axis of the myosin molecule. This finding is important in the context of the living cell, in single molecule experiments and motility assays. (Supported by the Wellcome Trust, BBSRC, and the Royal Society.)