Changes in microtubule overlap length regulate kinesin-14-driven microtubule sliding

Abstract

Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for the remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at a constant velocity until no overlap remains between them, leading to the breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14 HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback using the local interaction kinetics of HSET with overlapping microtubules that cause retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force that antagonizes the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through the local alteration of their individual interaction kinetics.

Figure 1. HSET-driven microtubule sliding slows down when microtubules start to slide apart.

(a) Schematic representation of GFP-HSET-driven sliding of a transport microtubule (sliding direction indicated by the grey arrow) along a surface-immobilized template microtubule. (b) Time-lapse fluorescence micrographs of a transport microtubule (bright magenta) sliding along a template microtubule (dim magenta) at 1.5 nM GFP-HSET (green) in solution. Fully overlapping microtubules initially slide at constant velocity. Sliding slows down when the microtubules start to slide apart. The dashed line indicates the position of the minus end of the template microtubule. The schematic diagrams indicate the positions of the template (orange) and the transport (magenta) microtubules at the beginning and at the end of the experiment, respectively. Microtubule plus-ends are indicated. (c) Multichannel kymographs representing the time-lapse fluorescence data presented in panel (b). The slope of movement is initially constant, indicating a constant sliding velocity, until the transport microtubule reaches the end of the template microtubule, causing a drastic decrease in the sliding velocity. Scale-bars are 2 µm horizontal and 5 min vertical.

Figure 2. HSET-driven microtubule sliding slows down with increasing motor density.

(a) The density of GFP-HSET increases as the microtubule overlap shortens when two microtubules slide apart (events as presented in Fig. 1). GFP-HSET density and overlap length were normalized to unity at the moment when the microtubules started to slide apart. Blue data points indicate combined measurements from 33 sliding events. Red crosses indicate binned and averaged values (± SD). (b) The increase in GFP-HSET density (same raw data as in panel a) correlates with a decrease in the velocity of microtubule sliding. Blue data points indicate the combined measurements. Red data crosses indicate binned and averaged values (± SD). Time was set to zero, when the microtubules started to slide apart. (c) The increase in GFP-HSET density also correlates with a decrease in the velocity for fully overlapping microtubules. Blue data points indicate combined measurements from 67 sliding events. Red data crosses indicate binned and averaged values (± SD). (d) The gliding velocity of microtubules driven by surface-immobilized HSET molecules decreases upon increasing the HSET concentration in solution used for the surface coating, i.e. with an increasing HSET density on the coverslip surface. For microtubules longer than 1.5 µm, which were analyzed here, the gliding velocity was independent of the microtubule length (Supplementary Fig. 2a). Gliding experiments were performed at three different HSET concentrations, namely 25 nM (n = 88 microtubules), 85 nM (n = 75) and 850 nM (n = 82) and the observed velocities are presented in box and whisker plots.

Figure 3. Full-length GFP-HSET diffuses with different diffusion constants on single microtubules and in microtubule overlaps.

(a) Kymograph showing single full-length GFP-HSET molecules at a concentration of 0.1 nM diffusing along a single microtubule and in a microtubule overlap in the presence of 1 mM ATP. The position of the microtubule overlap is indicated by the grey transparent box overlaid with the kymograph. Occasionally, single GFP-HSET molecules were observed to enter (red arrows) or leave (black arrow) the overlap. (b) Kymograph showing single GFP-HSET molecules at concentration of 0.15 nM diffusing along a single microtubule and in a microtubule overlap in the presence of 1 mM ADP (0 mM ATP). Diffusion did not depend on the nucleotide state of HSET - compare to panel a. (c) Schematic representation of the HSET amino-acid sequence. (d) and (e) Kymographs showing the interaction of 0.1 nM GFP-HSET-tail (d) and 0.1 nM GFP-HSET-motor (e) with single microtubules and microtubule overlaps. Microtubule overlaps were formed using 0.15 nM unlabeled HSET. In contrast to full-length GFP-HSET, GFP-HSET-tail diffused freely across the overlap ends (d; events marked by blue arrows) and GFP-HSET-motor interacted only shortly with both, single microtubules and microtubule overlaps (e). Scale bars are 2 µm horizontal and 2 s vertical.

Figure 4. HSET confined in partial microtubule overlaps generates entropic forces.

(a) Schematic representation of the experiment with HSET-mediated microtubule sliding in the presence of 1 mM ADP (0 mM ATP). (b) Time-lapse fluorescence micrographs of the transport microtubule sliding along a template microtubule (magenta) driven by the presence of ADP-bound GFP-HSET (green) confined in the microtubule overlap. All unbound GFP-HSET was removed from solution before the beginning of imaging. Transport microtubules always moved in the direction of increasing overlap length. The dashed line indicates the position of the minus end of the template microtubule. (c) Multichannel kymograph representing the time-lapse fluorescence data presented in panel b. Scale-bars are 3 µm horizontal and 5 min vertical. (d) The absolute velocities of overlap expansion (binned and averaged values, mean ± SD) decreased hyperbolically with the lengths of the microtubule overlaps (n = 8 microtubule pairs; overlap expansion is expressed using negative values because the microtubules moved in opposite direction compared to motor-driven sliding). The hyperbolic dependence is a signature of entropic forces generated by diffusible crosslinkers confined in the overlap.

Figure 5. Simulation of diffusible motors confined in a microtubule overlap explains the regulatory feedback by HSET.

(a) Microtubules are modeled as one-dimensional array of binding sites. Motors are simulated as harmonic springs, whose ends hop randomly (tail-domain) and directionally (motor-domain) between neighboring binding sites on the microtubules (Methods). (b) Kymograph showing the simulated diffusion of individual motors along a single microtubule and in a microtubule overlap. The fast diffusion on the single microtubule is mediated exclusively by the tail-domain. The slow diffusion in the overlap (dark grey area) is explained by the high binding and unbinding rates of the motor-domain (compare to experimental data in Fig. 3a). The cartoon on top of the kymograph represents the positions of the template (orange) and transport (magenta) microtubules. Scale bars are 2 µm horizontal and 2 s vertical. (c) The simulated sliding velocity of fully overlapping microtubules decreases with increasing motor density in the overlap (n = 22 simulated events; blue points indicate the simulated data, black crosses indicate the binned and averaged values (± SD); compare to experimental data in Fig. 2c). (d) Simulated kymograph of microtubule sliding driven by HSET in presence of ADP (compare to experimental data in Fig. 4c). ATP-independent microtubule sliding was simulated using the molecular motors described in b, which had their maximum force set to zero (Methods). Scale bars are 1 µm horizontal and 5 s vertical. (e) The absolute values of the simulated velocities of overlap expansion (binned and averaged values (± SD)) decrease hyperbolically with the lengths of the microtubule overlaps in events as presented in d (n = 9 simulated events; velocity is expressed using negative values; compare to experimental data in Fig. 4d).

Figure 6. Kinesin-14-driven microtubule sliding is regulated by changes in microtubule overlap length.

(a) Kymograph representing a simulated event of HSET-driven sliding of a transport microtubule over the end of a template microtubule. In agreement with our experimental data, we observed that the transport microtubule slowed down as the two microtubules started to slide apart (compare to experimental data in Fig. 1c). Scale bars are 0.5 µm horizontal and 5 s vertical. (b) The motor density in the overlap increases as the microtubules start to slide apart in events as presented in a (n = 15 simulated events; blue points indicate the simulated data, black crosses indicate the binned and averaged values (± SD)). Motor density and overlap length were normalized to unity at the moment when the microtubules started to slide apart (compare to experimental data in Fig. 2a). (c) The increasing motor density in shortening microtubule overlaps in events as shown in a correlates with a decreasing sliding velocity (n = 15 simulated events; blue points indicate the simulated data, black crosses indicate the binned and averaged values (± SD); compare to experimental data in Fig. 2b). (d) Tethering the HSET tail-domain to a microtubule increases the effective net binding rate of the HSET motor-domain to an adjacent microtubule. (e) Schematic representation of the regulatory feedback mechanism by HSET: as microtubules start to slide apart, the diffusible HSET molecules are retained within the microtubule overlap, leading to a density-dependent decrease in the sliding velocity (grey arrows) and an increase in an entropic force counteracting the sliding (blue arrows).