Processive movement of single 22S dynein molecules occurs only at low ATP concentrations

Abstract

We have analyzed the movement of single 22S dynein molecules from Tetrahymena cilia by using a nanometer measuring system equipped with optical tweezers. Statistical analysis proved that a single molecule of 22S dynein can move processively and develop force at low concentrations of ATP (<20 microM). The maximum force was approximately 4.7 pN, and the force-velocity curve was convex down. During force development, dynein molecules showed stepwise displacement of approximately 8 nm and frequently exhibited backward steps of approximately 8 nm. At higher concentrations of ATP (>/=20 microM) single molecules of 22S dynein were not observed to move processively. Twenty-two S dynein seems to switch over from a processive mode to a nonprocessive mode, sensing a subtle change of ATP concentrations. These observations indicate that the processivity, maximum force, and step size of dynein are similar to those of kinesin, but the ATP concentration-dependence, force-velocity relationship, and backward steps are clearly distinct from kinesin.

Figure 1

Fraction of beads binding (□) and moving (●) in the absence and the presence of 3 μM ATP, respectively, at various molar ratios of 22S dynein molecules to beads at the mixing. The plots are fitted to 1 − exp(−x/27.9) (broken line) and 1 − exp(−x/39.2) (solid line), where x is the molar ratio of 22S dynein to beads at the mixing. The dotted line indicates the probability, including geometric considerations, of a bead adsorbing two or more 22S dynein molecules that can simultaneously interact with a microtubule.

Figure 2

Time courses of bead displacement driven by single 22S dynein molecules at various concentrations of ATP. ATP concentrations were as indicated. The molar ratio of 22S dynein to beads was 30:1 at the mixing, and the trap stiffness was 0.010 pN/nm. The arrows indicate signals of active movements, judging from the decrease of Brownian noise.

Figure 3

Displacement and force of beads coated with single 22S dynein molecules at high trap stiffness. (a) Time course of displacement of a 22S dynein-coated bead at 3 μM ATP. The bead was prepared by mixing 22S dynein molecules and beads at 10:1 molar ratio. The trap stiffness was 0.050 pN/nm. Force (right-hand scale) was calculated from the displacement multiplied by the trap stiffness (20). (b) Histogram of the maximum force developed by single molecules of 22S dynein. The maximum force was measured where the bead stayed more than 0.2 s at the maximal level of displacement without taking further steps. Trap stiffness was 0.050–0.20 pN/nm. The mean and standard deviation are 4.7 and 0.64 pN, respectively (n = 92). (c) Force-velocity relationship of single 22S dynein molecules. Twelve runs of bead displacements were averaged, and the displacement of 22S dynein molecules was calculated according to Kojima et al. (20). The velocities were calculated from the slopes of the displacement. Error bars indicate confidence intervals of the velocities at a confidence level of 95%. The curve fit (v + 1.70)(y + 74.9) = 504 refers to Hill (33).

Figure 4

Stepwise movement of 22S dynein molecules. (a) Time course of displacement of 22S dynein at 3 μM ATP. The bead was prepared by mixing 22S dynein molecules and beads at a 30:1 molar ratio. The trap stiffness was 0.010 pN/nm, and the stiffness of the proteins was 0.20 pN/nm. The displacement corresponding to 22S dynein movement is obtained by correcting the bead displacement multiplied by the elastic correction factor, 1.05, due to the compliance of proteins (20) and passed through a median filter with 12.5-ms window. The displacement of 22S dynein was calculated at a force of more than 0.4 pN to avoid a large amount of noise in the attenuation factor at low force. (b) Histogram of step size. The window of 100-ms interval was moved every 5 ms on a and another similar example, and the net displacements in a given interval were scored. The histogram was fitted to multiple Gaussian curves as indicated by integer of i = −2 to 3 of A_i ⋅ exp{−(x − i⋅D)²/(2σ²)}, where D is the elementary step size and A_i and σ are the amplitude and the standard deviation. D and σ are 8.5 and 3.2 nm, respectively. (c) Ratio of backward steps to forward steps at different levels of force.