Myosin VI lever arm rotation: fixed or variable?

Two recent articles addressed the power-stroke of myosin VI molecules during stepping. Although both groups measured the angles of fluorescent probes attached on the myosin VI molecule lever arm using polarized fluorescence techniques, they differ about whether the myosin VI lever arm rotation is fixed or variable. Here we discuss the causes of the discrepancy between the two studies and the implications for myosin VI processive motility. Introduction: Myosin VI, an unconventional myosin motor that walks toward the minus ends of actin filaments, has received great attention recently. Reifenberger et al. have presented evidence from Defocused Orientation and Positional Imaging (DOPI) assays that myosin VI lever arms tilt by an angle very close to 180° on each step. This contrasts with results from Sun et al. wherein we applied single molecule Polarized Total Internal Reflection Fluorescence (polTIRF) microscopy and found variable degrees of tilting both in the axial and azimuthal directions relative to actin. This variability is consistent with the known variable step size of myosin VI. There are several technical differences in the two papers that interested researchers should understand, and more importantly, substantive differences in the conclusions. Results and Discussion: Reifenberger et al. explained the difference in results between the two studies by a choice of analysis hemisphere, but their explanation is wrong. Fig. 1, here, shows lateral and axial views of a myosin molecule (M) that landed on actin (A) in a typical position away from the coverslip (blue). Although the fluorescence emission from the chromophore is symmetric about the dipole axis, the two ends of the bifunctional probe are different because they are linked to different cysteine residues (here 66 and 73 on calmodulin, in the same construct used by both studies). To indicate this asymmetry we draw the probe as a directional arrow. Neither DOPI nor polTIRF can intrinsically distinguish between the two ends of the dipole, so the choice of which one to report requires additional considerations. It is especially crucial to be sure that it's the same one before and after an angle change (e.g., after a motor step). For any measurement of polarized fluorescence, a hemisphere needs to be chosen that defines the range of valid angles reported. Fig. 1A shows the position of the probe before a step. Follow the pointed end of the arrow in Fig. 1 which is at the Cys end of the probe. If the lever arm rotates 180° axially (in a plane containing the actin), then the probe will be positioned as in Fig. 1B after the step. With an analysis hemisphere whose equator is parallel to the glass, as used by Reifenberger et al., the pointed end of the probe is positioned inside the hemisphere before (Fig. 1A middle panel) and outside the hemisphere after the step (Fig. 1B middle panel). In the latter case, the method will report the wrong angle. If the hemisphere is tilted around the axis of actin, as we did, then the relevant end of the dipole is reported correctly before (Fig. 1A lower panel) and after (Fig. 1B lower panel) the step. This idea was explained in our original paper on DOPI (Suppl. Fig. 5) and formally shows the error in Reifenberger et al.'s categorical claim that a horizontal hemisphere should always be used. N at ur e P re ce di ng s : d oi :1 0. 10 38 /n pr e. 20 10 .4 18 2. 1 : P os te d 26 J an 2 01 0