Enhanced Processivity and Collective Force Production of Kinesin-1 at Low Radial Forces

Abstract

Kinesin-1 is a robust motor that carries intracellular cargos towards the plus ends of microtubules. However, optical trapping studies reported that kinesin-1 is a slippery motor that quickly detaches from the microtubule, and multiple kinesins are incapable of teaming up to generate large collective forces. This may be due to the vertical (z) forces that the motor experiences in a single bead trapping assay, accelerating the detachment of the motor from a microtubule. Here, we substantially lowered the z-force by using a long DNA handle between the motor and the trapped bead and characterized the motility and force generation of single and multiple kinesin-1s. Contrary to previous views, we show that kinesin-1 is a robust motor that resists microtubule detachment before it reaches high hindering forces, but it quickly detaches under assisting forces even at low z-forces. We also demonstrate highly efficient collective force generation by multiple kinesin-1 motors. These results provide an explanation for how multiple kinesins team up to perform cellular functions that require higher forces than a single motor can bear.

Figure 1.. The DNA tethered bead assay to reduce the z-force in the optical trap.

A) (Left) Conventional single-bead trapping assay where a truncated, biotinylated kinesin is attached to an 860-nm-diameter streptavidin-coated bead. (Right) The DNA-tethered bead assay, where full-length KIF5B is attached to a 510-nm-diameter bead via a 2800 bp DNA handle. B) Characteristic force-generating behavior of kinesin against a stationary trap in both a high and low z-force regime. Stalls are marked with black arrows, while premature detachments are marked with red arrows. C) Stall force of kinesin (mean ± s.e.m.) in the high and low z-force regimes. D) The inverse cumulative distribution function (1-CDF) of the peak forces (median ± s.e.m.) of every processive run that exceeded 1.5 pN hindering force in the high (N = 264) and low (N = 51) z-force regimes. E) 1-CDF of motor stall times. Solid curves represent fits to a double exponential decay to calculate the amplitude (A) and mean lifetime (τ; ± s.e.). The weighted averages of the two decays are 0.27 ± 0.02 s and 1.49 ± 0.08 s for high (N = 85) and low (N = 45) z-forces, respectively.

Figure 2.. Kinesin exhibits asymmetric slip bond behavior under force.

A) Example traces of kinesin motility under a 2 pN hindering force in high and low z-forces. The trap position (magenta) is updated to remain 100 nm behind the bead position along the microtubule long axis (blue). The trap stiffness is fixed at 0.02 pN nm⁻¹ so that this separation corresponds to 2 pN. B) Run lengths (fit parameter from single-exponential CDFs, ± s.e.), velocities (mean ± s.e.), and detachment rates (defined as the ratio of run length to velocity of the motor) for forces ranging from −4 pN to +4 pN under high (from left to right, N = 60, 69, 65, 50, 167, 100, 115 runs) and low (from left to right, N = 89, 76, 44, 42, 65, 32, 39 runs) z-force conditions. For −10 pN and −6 pN hindering forces, detachment rates were calculated from the run time distributions under high (N = 133, 167) and low (N = 43, 73) z-force regimes (Figure 2_figure supplement 2), as these conditions yielded minimal forward displacement. Negative and positive forces correspond to hindering (minus-ended) and assisting (plus-ended) directions, respectively. Under hindering forces, kinesin runs several-fold longer distances and has a lower detachment rate in the low z-force regime compared to the high z-force regime.

Figure 3.. Kinesin motors team up efficiently under low z-force conditions.

A) Schematic of the high and low z-force assays with three K560 motors bound to a DNA chassis. Left: The 3-motor DNA chassis binds directly to a streptavidin-coated bead. Right: The DNA chassis with a single-stranded DNA overhang hybridizes to the overhang on the long DNA handle attached to the bead. B) Mass photometry measurements with a DNA chassis are performed with a 3-fold excess of kinesin. The Gaussian mixture model identifies the mass and percentage of unbound kinesin (blue curves), chassis with one (blue curves), two (green curves), and three (red curves) motors. Mass populations of unbound motor and chassis with one motor bound cannot be distinguished from each other. C) Example trajectories of beads driven by two and three K560 motors in the high and low z-force regimes. These assays were performed with a 10 or 20-fold excess of kinesin for 2- or 3-motor DNA chassis, respectively. D) Forces (mean ± s.d.) produced by single kinesins or kinesins bound to 2- or 3-motor chassis under high (from left to right, N = 85, 66, 158) and low z-force conditions (from left to right, N = 37, 65, 66). The solid line represents a fit to a power function to calculate the scaling factor (c, ± s.e.). E) Dwell times before slips are plotted as 1-CDF. Weighted average time constants (τ, ± s.e.) are extracted from fitting to a double exponential decay.

Figure 4.. Kinesin resists microtubule detachment and backward processive movement under low z forces.

A) High z-forces inherent to optical trapping assays result in rapid detachment of kinesin from microtubules, whereas the motor experiences lower z-forces when carrying intracellular cargos and more persistently binds to microtubules under those conditions. B) (Top) The tug-of-war between kinesin and dynein results in pausing or stalling the cargo movement, as well as slow motility interspersed with frequent reversals, because either motor resists detachment or backward stepping under forces generated by its opponent. (Bottom) Coordination between the opposing motors, such as activation of one motor at a time (reciprocal activation) results in fast unidirectional transport in the direction of an active motor while the inactive motor is carried as a passive cargo.