Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors

Abstract

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

Fig. 1.

Self-assembly and collective motility of kinesins. (A) Schematic representation of DNA–motor construction (not drawn to scale). The typical spacing between motors is 22.7 nm, and the lengths of kinesin, SNAP-tag, and HaloTag are ∼17, 4.3, and 4.8 nm, respectively. (B) SDS/PAGE analysis of the DNA–motor assemblies. SDS/PAGE was performed on a 3% to 10% polyacrylamide gel. The numbers at the top of each lane represent the number of molecules engaged to the DNA scaffolds. Bands with asterisks denote an excessive amount of unreacted dimers. Note that the dimers consist of two different polypeptides, one of which carries a single enzyme tag. Unreacted dimers are not visible by fluorescence microscopy. M, markers; C, control lanes without DNA scaffold. (C) Velocities and run lengths of the assemblies including one, two, three, or four kinesin-1 dimer(s) linked by flexible DNA scaffolds (22.7-nm spacing). Open circles and blue bars show experimental data. Run lengths are corrected for photobleaching. The velocity distributions of the two groups (single and four kinesin-1 motors) differed significantly (Mann–Whitney test, P < 0.0001, two-tailed). Triangles connected by the dashed line and bars in gray show the simulated data. Error bar represents SEM. The simulated data were obtained by averaging the three data sets, each consisting of 1,000 traces. (D) Velocities and run lengths of the Ncd assemblies. The DNA scaffolds and the legend are the same as in C. Note that the scored run length for single Ncd (asterisk) is an overestimation (see text). (E) Kymographs showing the motion of the assemblies including (from top to bottom) one, two, three, or four Ncd dimer(s) linked by flexible DNA scaffolds with 22.7-nm spacing (red) on Cy3-labeled MTs (green). Plus and minus symbols at right refer to the polarity of the MT. (Scale bars, 3 μm.)

Fig. 2.

Monte Carlo simulations of collective motility. (A) Schematic of the in vitro experiment (Upper) and the simulation model (Lower). (B) Kymographs showing simulated motion of assemblies including (from top to bottom) one, two, three, or four Ncd dimer(s) linked by flexible DNA scaffolds (22.7-nm spacing). Plus and minus symbols at right refer to the polarity of the MT. (Scale bar, 3 μm.) (C) Velocities (Left) and run lengths (Right) of two coupled dimers of Ncd. The experimental data for flexible (orange circles) and rigid (gray squares) DNA scaffolds are plotted vs. intermotor spacing. Red solid line represents the model in which on-rate depends on intermotor spacing (on-rate = 68.0 × L^−0.7, where L is intermotor spacing; model 1). Gray dashed line shows the model with constant on-rate (model 2). Each simulated plot was calculated from 1,000 traces at intervals of 1 nm.

Fig. 3.

Optical trapping assays. (A) Schematic of the typical experimental setup for optical trapping assays (not drawn to scale). (B) Time traces of optical trapping assays for (from top to bottom) one, two, three, or four kinesin-1 dimer(s) linked by rigid DNA scaffolds (∼6-nm spacing). Traces were acquired at 10 kHz (gray) and median-filtered to 25 Hz (black). The positive value corresponds to the force toward the MT plus-end (bead diameter, 0.45 μm; trap stiffness, 0.05–0.182 pN⋅nm⁻¹). (C) Time traces of optical trapping assays for Ncd linked by rigid DNA scaffolds (∼6-nm spacing). Arrowheads show examples of binding events (bead diameter, 0.21 μm; trap stiffness, 0.014–0.027 pN⋅nm⁻¹). (D) Histogram of the average maximum force generated by kinesin-1. The motors are linked by rigid DNA scaffolds (∼6-nm spacing). The numbers in the legend represent the number of motors in the assemblies. Inset: Average maximum forces (mean ± SEM) are plotted vs. motor number. Black and red colors represent intermotor spacings of 6.1 to 7.0 nm and 22.1 to 22.7 nm, respectively (SI Appendix, Fig. S4). The spacing between motors did not appear to affect the on-rate unlike in the low load condition, probably because of the difference in the arrangement of the motors on the trapped bead. (E) Histogram of the average maximum force generated by Ncd. The motors are linked by rigid DNA scaffolds (∼6-nm spacing). The color scheme and symbol are the same as in D. N.D., not determined.

Fig. 4.

Ratchet-like mechanism of Ncd. (A) Schematic of the simulation model for optical trapping assays. (B) Fitted parameters for optical trapping assays determined by an automated scanning algorithm (SI Appendix, p. 15). Each plot represents the parameter set obtained from each trial of the simulated annealing optimization. The free parameters were on-rate, off-rate, and critical detachment rate (SI Appendix, Table S7). The size of the circles represents the goodness of fit, which is inversely proportional to the sum of residuals between experimental and simulated data. Triangles and crosses denote the parameters in low load conditions (SI Appendix, Figs. S16 and S18). (C) Typical simulated traces of the force generated by four Ncds. Parameters are as follows: bead diameter, 0.21 μm; trap stiffness, 0.015 pN⋅nm⁻¹. The other parameters are listed in SI Appendix, Table S7. (D) Typical time trace of the unbinding force of single Ncd (black) and displacement of the stage (blue). The positive value of force corresponds to the applied force toward the MT plus-end. The trapped beads were dragged back and forth over MTs in the presence of 1 mM ATP (trap stiffness, 0.018 pN⋅nm⁻¹). (E) Unbinding force of single Ncd as a function of loading rate (1 mM ATP). Values represent mean ± SEM. The total number of unbinding events and the number of MTs for each plot was 242 to 518 and 7 to 11, respectively. Because we measured the impulse rather than the force, the unbinding force here is dependent on the loading rate. Thus, the actual force that unbinds Ncd from the MT would be larger than the plotted values. (F) Velocities for the tug-of-war between single kinesin-1 and several Ncds. The motors are linked by a flexible DNA scaffold (22.7-nm spacing). Green and gray bars correspond to experimental and simulated data, respectively. The parameter sets are shown in SI Appendix, Table S6. Each bar represents the mean velocity ± SEM toward the MT plus-ends.