A Homotetrameric Kinesin-5, KLP61F, Bundles Microtubules and Antagonizes Ncd in Motility Assays

Background: Mitosis depends upon the cooperative action of multiple microtubule (MT)-based motors. Among these, a kinesin-5, KLP61F, and the kinesin-14, Ncd, are proposed to generate antagonistic-sliding forces that control the spacing of the spindle poles. We tested whether purified KLP61F homotetramers and Ncd homodimers can generate a force balance capable of maintaining a constant spindle length in Drosophila embryos. Results: Using fluorescence microscopy and cryo-EM, we observed that purified full-length, motorless, and tailless KLP61F tetramers (containing a tetramerization domain) and Ncd dimers can all cross-link MTs into bundles in MgATP. In multiple-motor motility assays, KLP61F and Ncd drive plus-end and minus-end MT sliding at 0.04 and 0.1 μm/s, respectively, but the motility of either motor is decreased by increasing the mole fraction of the other. At the "balance point," the mean velocity was zero and MTs paused briefly and then oscillated, taking ∼0.3 μm excursions at ∼0.02 μm/s toward the MT plus end and then the minus end. Conclusions: The results, combined with quantitative analysis, suggest that these motors could act as mutual brakes to modulate the rate of pole-pole separation and could maintain a prometaphase spindle displaying small fluctuations in its steady-state length.