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 microm/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 approximately 0.3 microm excursions at approximately 0.02 microm/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.
Tao
Li
L
Laboratory of Cell and Computational Biology, Center for Genetics and Development, University of California, Davis, Davis, California 95616, USA.
Mogilner
Alex
A
Civelekoglu-Scholey
Gul
G
Wollman
Roy
R
Evans
James
J
Stahlberg
Henning
H
Scholey
Jonathan M
JM
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D020285:Cryoelectron Microscopy; D019281:Dimerization; D004330:Drosophila / Q000196:embryology*; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology*; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000502:physiology; D008870:Microtubules / Q000737:chemistry*; D009068:Movement; D011994:Recombinant Proteins / Q000302:isolation & purification; D008941:Spindle Apparatus / Q000502:physiology
D029721:Drosophila Proteins; C084034:Klp61F protein, Drosophila; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; C066696:ncd protein, Drosophila; D000255:Adenosine Triphosphate; D016547:Kinesins