Dynein achieves processive motion using both stochastic and coordinated stepping

Abstract

Processivity, the ability of single molecules to move continuously along a track, is a fundamental requirement of cargo-transporting molecular motors. Here, we investigate how cytoplasmic dynein, a homodimeric, microtubule-based motor, achieves processive motion. To do this, we developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we find that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use 'hand-over-hand' mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. This plasticity may allow tuning of dynein for its diverse cellular functions.

Figure 1

Dynein structure and constructs used in this study. (a) Linear diagrams of (1) native dynein’s domain structure, and constructs used in this study: (2) GST-dimerized dynein with an N-terminal HaloTag (H) for tail-labeled experiments, (3) GST-dimerized dynein with a C-terminal HaloTag for motor domain-labeled experiments, and (4) dynein monomer with an N-terminal SNAP-tag for DNA dimerization, and C-terminal HaloTag for motor domain labeling. MTBD is the microtubule-binding domain. (b) 2-D schematic of dimeric dynein. Dimerization (white box) can be achieved using the native protein dimerization domain, GST or complementary DNA oligomers attached via a SNAP-tag. (c) 3-D structure of yeast dynein (3QMZ) from Carter, Cho et al., filtered to 8 Å resolution. Views from left to right: the linker face, the opposite face of the ring containing the C-terminal (CT) alpha helix, and the side of the ring. Dimerization is achieved via GST (magenta) at the N terminus (NT).

Figure 2

Two-dimensional stepping analysis of GST–dynein homodimers. (a) Schematic of a GST–dynein homodimer labeled with a Qdot via an N-terminal (tail domain) HaloTag and a diagram of a microtubule showing on- and off-axis directions of movement. (b) Histograms of dynein’s step sizes in 1-D and 2-D. N = 1391 steps for all panels. (c) An angle histogram (or rose plot) of the step angles. The stepping angle is defined as the angle between the stepping vector and the direction of on-axis movement. Steps to the left or right of the direction of motion are between 0° and 180° or 180° and 360°, respectively. Steps between 90° and 270° are backwards steps. 77% of steps are forward steps. (d) Histogram of off-axis step sizes. Percentages of step sizes larger than 4, 6, and 8 nm are 53%, 38%, and 28%, respectively. (e) Histograms of leftward or rightward steps after a previous left or right step. Leftward and rightward steps are shown as steps with negative and positive off-axis components, respectively.

Figure 3

DNA-based dynein heterodimers are functional and step similarly to protein-based dynein homodimers. (a) Schematic of a DNA-based dynein heterodimer labeled with Atto647N (red star) and TMR (green star) via a C-terminal HaloTag (pink circles). The SNAP-tag and DNA oligomers (attached via the N-terminal SNAP-tag) are shown in blue. (b) LDS-PAGE gel showing dimerization of dynein monomers via DNA hybridization. (c) Kymograph of the motility of DNA-based dynein dimers labeled with TMR (green) and Atto647N (red), with overlapping, dual-labeled heterodimers in yellow. Scale bars: y (1 min), × (10 μm). (d) Histograms of the velocity of GST- and DNA-based dynein dimers. GST–dynein velocity is 134 ± 60.4 nm s⁻¹ (mean ± s.d., N = 943), and DNA–dynein velocity is 125 ± 56.1 nm s⁻¹ (mean ± s.d., N = 866). (e) Histograms of the run length of GST- and DNA-based dynein dimers. GST–dynein run length is 1.06 ± .044 μm (mean ± s.e.m., N = 943), and DNA–dynein run length is 1.45 ± .063 μm (mean ± s.e.m., N = 866). (f) Histograms of the 2-D step size of GST- and DNA-based dynein dimers labeled with a single Qdot 655 on the N-terminal tail domain. (g,h) Histograms of the dwell time distribution of GST–dynein homodimers (g) and DNA–dynein heterodimers (h) labeled with a single Qdot 655 on the N-terminal tail domain. The distributions are fit to single exponential functions with stepping rates of k = 1.78 ± 0.13 s⁻¹ and 1.43 ± 0.10 s⁻¹, respectively.

Figure 4

High-precision, two-color tracking of dynein stepping. (a) Representative two-color stepping trace of a DNA–dynein heterodimer. The raw 2-D positions (black dots in left and center panels) from a DNA–dynein heterodimer labeled with Cy3B (left panel, blue line) and Atto647N (center panel, red line). Co-alignment of the motor domain traces from each channel is shown in the right panel, with darker solid blue (Cy3B) and red (Atto647N) dots representing steps determined by a 2-D step finding algorithm, and larger, lighter-colored blue and red circles representing the s.d. of individual steps. (b) 1-D on-axis projection of the 2-D data from (a), with lighter blue and red bars representing the s.d. of individual steps along the projection axis. Ovals highlight examples of hand-over-hand (orange) or inchworm (green) steps. Y-axis grid lines are spaced 16 nm apart in all panels. See Supplementary Figures 3c–f for additional stepping traces. (c) Examples of 1-D on-axis projections of two-color stepping trace pairs from dual-labeled DNA-dynein heterodimers. The grey arrows indicate the start of each trace. Pairs of solid blue and red lines represent the 1-D on-axis projection of steps, determined by a 2-D step finding algorithm for the Cy3B and Atto647N traces, respectively. Lighter blue and red bars represent the s.d. of individual steps along the projection axis. Examples of the four different types of steps are as follows: light green and red asterisks represent alternating and passing steps (“hand-over-hand”); dark green and red asterisks represent alternating and not passing steps (“inchworm”); light green and light orange asterisks represent not alternating and passing steps; dark green and light orange asterisks represent not alternating and not passing steps. (d) Temporal analysis of the relative frequency of stepping events. Alternating events are defined as current and previous stepping events originating from different heads, while non-alternating events are current and previous stepping events originating from the same head. N = 268. (e) Spatial analysis of the relative frequency of passing or not passing stepping events. N = 233. (f) Combined temporal and spatial analysis of stepping events. N =135.

Figure 5

Spatial arrangement of dynein motor domains during the two-head-bound state. (a) Schematic of dual-labeled DNA-dimerized dynein bound to a microtubule. Other arrangements of the motor domains are possible. Fluorophores are represented by red and green stars. (b) Histogram of dynein’s head-to-head distances during the two-head-bound state. N = 523. (c) Contour plot showing the left and right asymmetry between the leading and lagging heads. Orientation of the microtubule axis is vertical, as indicated by − and +, with the centroid position of each dynein molecule placed at the origin of the axes (white X). The number of occurrences of each position is indicated by the color bar on the right, with bin edges at 8 nm increments from −32 nm to +32 nm on both axes. N = 256 dimers or 512 heads. (d) On the left, an angle histogram of the position of the leading and lagging heads of individual dynein dimers relative to their respective centroid position (placed at the origin of the axes). Locations to the left or right of the direction of motion are between 0° and 180°, or 180° and 360°, respectively. 64% of the leading heads are to the right of the microtubule axis. N = 256 dimers or 512 heads. On the right, the angular distributions of the next step taken by the leading (top) or lagging (bottom) head. Steps between 90° and 270° are backwards steps.

Figure 6

Dynein steps are stochastic at short head-to-head spacing and coordinated as head-to-head spacing increases. (a) Histograms of the duration of the two-head-bound states that are terminated by a leading head stepping event or a lagging head stepping event. The mean durations are 4.3 s or 5.4 s for two-head-bound states terminated by a lagging head stepping (N = 228) or leading head stepping (N = 119), respectively; the means are significantly different (P = 9.7e−5, alpha 0.05, one-tailed KS test). (b) Relative stepping frequency of the leading and lagging heads as a function of the on-axis distance between motor domains. Error bars represent the s.e.m. and were generated by bootstrapping each bin. N = 352. (c) The duration of the two-head-bound state plotted as a function of the on-axis head-to-head distance. Mean durations ± s.e.m. are shown (*P = 0.0139, **P = 0.0094; two-tailed KS test, alpha value 0.05, N = 485). (d) Model for the dynein stepping mechanism. The 3-D structure of dynein (3QMZ) filtered to 8 Å resolution was used to generate two microtubule-bound models of dimeric GST–dynein. The dynein rings are shown parallel to the long-axis of the microtubule and parallel to each other based on electron microscopy reconstructions^-. Stepping is stochastic when dynein’s motor domains are close together (left panel). Large distances between the two motor domains result in a tension-based mechanism that coordinates stepping (right panel).