Smooth muscle myosin species used in this study. A, schematic drawing of smooth muscle myosin species with different phosphorylation states. The heavy meromyosin versions of the smooth muscle myosin species were also used for solution kinetics experiments. B, preparation of the single-head phosphorylated smooth muscle myosin. The sample of each preparation step was examined by SDS-PAGE and stained by Coomassie Brilliant Blue. Lane 1, smooth muscle myosin; lane 2, RLC depleted smooth muscle myosin; lane 3, SHPMII containing a phosphorylated RLC-FLAG and a dephosphorylated RLC-hexahistidine. C, in vitro actin translocating activities of smooth myosin species. Actin filament movement was observed in 25 mm KCl, 25 mm HEPES-KOH, pH 7.5, 5 mm MgCl₂, 1 mm EGTA, 0.5% methylcellulose, 4.5 mg/ml glucose, 216 μg/ml glucose oxidase, 36 μg/ml catalase, and 2 mm ATP at 25 °C. All of the values of velocity are the means ± S.D. (n = 100–150). The movement of actin was observed for more than 30 s, and the average velocities were calculated.

Histogram of bead displacement induced by DHPMII, SHPMII, and UPMII. A, the average displacements of DHPMII (pp), SHPMII (pFdH), and UPMII (dd) were 24.8 ± 36.4, 26.4 ± 26.2, and 3.6 ± 20.5 nm, respectively. For SHPMII (B) and UPMII (C), the displacement distributions were analyzed for faster and slower actomyosin interactions. The average displacements of the shorter dwell time component (<0.1 s) and the longer dwell time component (≥0.1 s) of SHPMII were 27.6 ± 25.1 and 22.6 ± 29.2 nm, respectively. The average displacement of the shorter dwell time component (<0.2 s) and the longer dwell time component (≥0.2 s) of UPMII were 3.6 ± 18.8 and 2.8 ± 20.2 nm, respectively. The trapping force of the bead was 0.02 pN/nm. Thus the force required for displacing the bead, e.g. ∼10 nm is just ∼0.2 pN (0.02 pN/nm × 10 nm) that is much smaller than the force exerted by actomyosin strong binding (∼10 pN). Therefore, almost all actomyosin interactions observed in our nanometry experiment should be the results of forward stepping, although we cannot distinguish forward steps from backward steps. The apparently negative displacements are due to the thermal fluctuation of actin filament loosely trapped by the optical beads (0.02 pN/nm). Depending on the position of the first weak association of myosin on the actin filament, the apparent position of the bead after the forward stepping becomes sometimes negative. However, this does not necessarily mean it is due to backward displacement.

Single molecule mechanical measurement of smooth muscle myosin with optical trap nanometry. A, the nanometry setup for measuring actin displacements by a single smooth muscle myosin molecule. B, typical recordings of actin displacements by DHPMII, SHPMII, and UPMII. The white dots represent raw data. The data passed through a low pass filter of 30-Hz bandwidth is shown by black lines. Note the differences in the time scale among the recordings. C, dwell time distribution of the actomyosin interaction. The integrated frequencies of DHPMII (pp) (n = 2051), SHPMII (pFdH) (n = 704), and UPMII (dd) (n = 237) were plotted against the observed dwell times. Both single exponential and double exponential fitting were tested, and the residuals are shown in the inset graphs. Note that the differences in the amplitude of the residuals among the smooth muscle myosin species came from their different sampling sizes (n = 2051, 704, and 237 for DHPMII, SHPMII, and UPMII, respectively). The single exponential fitting gave apparent rates of 29.0 ± 0.2, 21.1 ± 0.4, and 2.5 ± 0.1 s^–1 for DHPMII, SHPMII, and UPMII, respectively, whereas the double exponential fittings gave 28.8 ± 1.3 s^–1 (97%) and 1.3 ± 1.6 s^–1 (3%) for DHPMII, 24.2 ± 0.6 s^–1 (92%) and 1.0 ± 0.3 s^–1 (8%) for SHPMII, and 12.3 ± 0.7 s^–1 (41%) and 1.1 ± 0.1 s^–1 (59%) for UPMII. The percentage values are the amplitude contributions of the different rate components. The amplitudes of the fitting curves were greater than the total integrated frequencies because displacements with very short dwell times were not detected because of the 30-Hz low pass filtering of the raw data. (The shortest possible events we can detect with the 30-Hz low pass filter can be calculated as 1/2 P_i/30 =∼5 ms. Thus the dead time of our measurement is ∼5 ms.) Note that the dwell time distribution of DHPMII is fairly explained by a single exponential kinetics, whereas those of SHPMII and UPMII are well explained by double exponential.

Simulation of the production of smooth muscle myosins with different phosphorylation states (UPMII, SHPMII, and DHPMII) in the smooth muscle fiber after agonist stimulation. A, time-dependent relative activities of MLCK and MLCP during the contraction of tonic smooth muscle. B, fractions of UPMII, SHPMII, and DHPMII during a tonic contraction of smooth muscle.