Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro

Abstract

Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.

Figure 1

Schematic representation of the experimental geometry (not to scale). A His-tagged Eg5 homodimer (dark blue) is attached to a bead (light blue) maintained in an optical trap (pink) as it walks along a surface-attached microtubule (green). Details of the Eg5-bead linkage are shown on the top right. His-tagged motors were linked stereospecifically to streptavidin-coated polystyrene beads through a biotinylated anti-pentaHis antibody. The remaining binding sites were quenched by the addition of excess biotin. Details of the microtubule-surface linkage are shown on the bottom right. The coverglass surface was treated with biotinylated poly(l-lysine)–graft–polyethylene glycol block copolymers (PLL–PEG–biotin) to prevent non-specific adsorption of Eg5. Positively charged PLL blocks self-assemble through non-specific electrostatic interactions, while the neutral PEG chains form a dense layer that extends into solution, providing a barrier against protein adsorption. Biotinylated microtubules were specifically bound to this copolymer surface through streptavidin linkages.

Figure 2

Single Eg5–513–5His dimers are processive. (a–f) Representative records of bead motion in the force clamp driven by single Eg5–513–5His dimers showing 8-nm steps. Occasional back-steps are also observed at high loads (grey arrows). Median-filtered bead position (25-point window, black trace) is superimposed on the unfiltered position (grey trace). Trap position (black trace) is offset from bead position by computer feedback, supplying constant load. Experimental conditions: load = −4 pN and ATP concentration = 8 μM in a, b and d; load = −4 pN and ATP concentration = 2 mM in c; load = −4 pN and ATP concentration = 31 μM in e; load = +4 pN and ATP concentration = 2 mM in f. (g) Fraction of Eg5–513–5His-coated beads moving as a function of the relative motor concentration. If a single dimer suffices to transport a bead, then the fraction of moving beads, ρ, corresponds to the Poisson probability that the bead carries one or more dimers, given by ρ(C) = 1−exp(−λC), where λ is a single parameter. The data fit this function (χ_ν² = 0.5; ν = 5; P(χ_ν²) = 0.78). Values are expressed as ρ ± [ρ(1−ρ)/n]^1/2 (N = 323 total measurements; n = 43–66 at each concentration). If two or more dimers were required, then ρ (C) = 1−exp(−λC)−λCexp(−λC). This fit was significantly poorer (χ²_ν = 2.0; ν = 5; P(χ_ν²) = 0.075). (h) Histogram of run lengths for Eg5–513–5His dimers at zero load (N = 1,592). Fit to a single exponential (excluding first 3 bins) returned a mean run length of 67 ± 7 nm (χ_ν² = 0.57; ν = 12; P(χ_ν²) = 0.84, 2 parameters).

Figure 3

Eg5 velocity as a function of ATP concentration and force with global model fits. Negative forces denote hindering loads. Both force-clamped data (circles) and unloaded video-tracked data (triangles) are plotted. (a) Velocity (weighted mean ± s.e.m.) versus ATP concentration at F = 0 pN (solid triangles; n = 22–67) and F = −4 pN (open circles; n = 29–92). (b) Velocity (weighted mean ± s.e.m.) versus applied force at ATP concentration = 2 mM (solid points; n = 70–212) and ATP concentration = 16 μM (open points; n = 57–80). (c) Reaction pathway for a minimal, three-state model of the Eg5 mechanochemical cycle. Reversible nucleotide binding is followed by an irreversible load-dependent translocation step and an irreversible biochemical transition with no associated displacement. An alternative model explicitly incorporating a reversible, load-dependent transition returned a best-fit rate of zero for the back reaction (k₋₂). Therefore, this step was taken to be irreversible. Rate constants and transition state distances derived from a global fit of this model to the data are shown in Table 1.

Figure 4

Eg5 randomness as a function of ATP and force. (a) Randomness (weighted mean ± s.e.m.) versus ATP concentration for F = −4 pN (open squares; n = 27–86). In saturating ATP conditions (2 mM), where nucleotide binding is rapid and therefore cannot be rate-limiting, the value of r asymptotes to appromimately 0.5, indicative of at least two rate-limiting transitions in the reaction cycle, in addition to nucleotide binding. For limiting ATP concentrations, randomness increases towards unity, implying that one ATP molecule is hydrolysed per step. (b) Randomness (weighted mean ± s.e.m.) versus force for ATP concentration = 2 mM (solid squares; n = 62–212) and ATP concentration = 16 μM (open squares; n = 53–73). At limiting ATP concentrations, randomness is near unity for nearly all applied loads, reflecting that ATP binding remains the solitary, rate-limiting transition. At saturating ATP concentrations and high hindering loads, r increases as the completion time of the force-dependent transition begins to dominate the reaction cycle. For saturating ATP concentrations and high assisting loads, where neither nucleotide binding nor translocation are rate-limiting, r asymptotes to approximately 0.4, indicative of the presence of at least two additional steps in the reaction cycle.

Figure S1

Analytical gel filtration profiles for Eg5 motors purified from E. coli. (a) Elution profiles monitoring intrinsic fluorescence vs. time for Eg5-513 (green), Eg5-513-5His (red), and Eg5-367 (brown). The void volume eluted at 16 min and the included volume at 51.5 min. The predicted value for M_r, based on the amino acid sequence for monomers is 57,644 Da for HsEg5-513, 58,787 Da for HsEg5-513-5His, and 41,667 Da for HsEg5-367. The oligomeric state was determined from plots of the partition coefficient, K_av, vs. log M_r (not shown). HsEg5-513: M_r = 189,688 Da; HsEg5-513-5His: M_r = 181,036 Da. (b) Gel filtration of HsEg5-513-5His at varying protein concentrations indicates that the dimeric state remains stable at low concentrations.

Figure S2

Histogram of step sizes for single Eg5-513-5His dimers (bin width, 1 nm; N = 257). A Gaussian fit to this distribution yielded a mean step size of 8.1 ± 0.1 nm, a distance consistent with the spacing of tubulin heterodimers along the microtubule protofilament. Note the absence of fractional or multiple step sizes, which can be generated when beads carry multiple motors.

Figure S3

Run lengths are relatively insensitive to the applied load or to the ATP concentration. (a) Global histogram compiled from run lengths for all applied forces and ATP concentrations (N = 1,602; min. bin width 10 nm; all bins widths scaled to contain >6 counts). A small number of runs >200 nm exceeded the region reliably monitored by our instrument and were excluded. An exponential fit to this distribution (excluding the first three bins) yielded a mean run length of 63 ± 3 nm, in statistical agreement with the unloaded mean run length determined by video tracking (67 ± 7 nm)(data in Fig. 2h). (b) Run length as a function of force at [ATP] = 2 mM (solid diamonds; n = 70 – 212 for each point) and [ATP] = 16 μM (open diamonds; n =57 – 80 for each point), determined by exponential fits to histograms of run length at each load. (c) Run length as a function of [ATP] at F = 0 (solid diamonds, n = 22 – 67 for each point) and F = −4 pN (open diamonds, n = 29 – 92 for each point), as determined by exponential fits to histograms of run length at each [ATP].

Figure S4

Stall force measurements with a fixed-position trap. Single Eg5 dimers step processively from the trap centre under monotonically increasing, hindering loads. (a, b) Representative records of bead motion. Median-filtered bead position (15-point window, black trace) is superimposed on the unfiltered position (grey trace). (a) Optical trap stiffness, k = 0.09 pN/nm. Beads moved out to ~65 nm from the centre of the trap, sustaining loads up to 6 pN. (b) Optical trap stiffness, k = 0.035 pN/nm. The bead moved 120 nm from the centre, sustaining a load up to 4 pN, before dissociating. Note that noise decreases at higher loads due to increased stiffness associated with the kinesin-bead linkage. As a consequence, 8-nm-sized steps are more apparent at higher loads.