Cytoplasmic dynein moves through uncoordinated stepping of the AAA+ ring domains

Abstract

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos toward the microtubule minus end. How the two catalytic head domains interact and move relative to each other during processive movement is unclear. Here, we tracked the relative positions of both heads with nanometer precision and directly observed the heads moving independently along the microtubule. The heads remained widely separated, and their stepping behavior varied as a function of interhead separation. One active head was sufficient for processive movement, and an active head could drag an inactive partner head forward. Thus, dynein moves processively without interhead coordination, a mechanism fundamentally distinct from the hand-over-hand stepping of kinesin and myosin.

Fig. 1

(A) Step-size measurements of tail- and head-labeled GST-Dyn_331kD with a single QD-655 at 2-msec temporal resolution. The QD position (blue traces) was fit by a step-finding algorithm (solid red lines). (B) Multiple Gaussian fits to step-size histograms reveal two major peaks at 9.3 and 17.5 nm for the head and 4.8 and 8.7 nm for the tail. (C) Dwell-time histogram of head-stepping fitted to a convolution of two unequal rate constants and that of tail-stepping fitted to a model assuming uncoordinated stepping between two heads, each with two unequal rate constants.

Fig. 2

(A) Stepping trace of GST-Dyn_331kD labeled with QD-585 (blue) and QD-655 (red) shows that the heads move independently of each other during processive runs. The heads are separated by 28.4 ± 10.7 nm (bottom inset). (B) Examples of nonalternating and lead-head stepping (arrows) show that dynein stepping deviates from the HoH mechanism. (C) Histogram of the angles between the interhead separation vector (red arrow) and the microtubule long axis (blue arrow).

Fig. 3

(A) The on-axis step size (blue dots) of GST-Dyn_331kD decreases linearly (red line) as a function of interhead separation and is biased forward by 9.1 ± 0.6 nm. The leading head takes shorter (d = 1.5 nm) steps with more frequent (p_BW = 0.45) backward steps, compared with the trailing head (d = 17.5 nm; p_BW = 0.14) (bar graphs). (B) Off-axis step sizes show a linear dependence on interhead separation without a bias to move toward the right (positive) or left (negative). (C) Fraction of the steps taken by the leading and trailing heads at different interhead separations (mean ± SEM). (D) Tethered excursion model for the dynein stepping mechanism. Either the leading or the trailing head can hydrolyze ATP and release from the microtubule. A diffusional search of the trailing head (green) is biased forward by interhead tension and the linker swing, resulting in a large forward step. In contrast, linker swing and tension bias the diffusion of the leading head (blue) in opposing directions, resulting in either a backward step or a short forward step.

Fig. 4

(A) Stepping trace of the WT_h\Mut_h heterodimer. Mut_h and WT_h were labeled with QD-585 (blue trace) and QD-655 (red trace), respectively. WT_h mostly remains in the lead during a short backward run (insert 1) and a long forward run (insert 2). (B) Plot of on-axis step size (blue dots) of WT_h and Mut_h versus interhead separation. The step size decreases linearly (red line) with increased interhead separation and is biased forward by 10.9 ± 0.8 nm for WT_h and 7.1 ± 0.7 nm for Mut_h. Step-size histograms (bar graphs) show that dWT = 6.5 nm whereas d_Mut = −2.6 nm in the lead. (C) Histogram of the angles between interhead separation and stepping vectors of Mut_h and WT_h. (D) Model for WT_h\Mut_h processivity. ATP binding to WT_h triggers its release from the microtubule and the subsequent diffusional search is biased forward by the power stroke of the WT_h linker. The weakly attached Mut_h moves toward WT_h under linker tension.