Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast

Sergio A Rincon,Robert Blackwell,Phong T Tran,Meredith D Betterton,Adam Lamson,Anne Paoletti,Vincent Fraisier,Viktoriya Syrovatkina

doi:10.1038/ncomms15286

Abstract

Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Δpkl1Δ spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Δpkl1Δ spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors.

Highlights

Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein
Initial spindle pole separation is driven by kinesin-5, based on its unique properties of crosslinking and sliding antiparallel MTs apart[2,3,4,5,7]
Recent reports show that spindle bipolarity can be supported in the absence of kinesin-5, especially on depletion of inward forces produced by minus-end directed motors[8,9,11,13,14,15,16]

Summary

Introduction

Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. This suggests that the delayed transition to anaphase in cut7Dpkl1D cells is due to chromosome bi-orientation defects In agreement with these results, deletion of the spindle assembly checkpoint (SAC) component Mad[2] in cut7Dpkl1D cells resulted in colonies growing very poorly (Fig. 1f), indicating that cut7Dpkl1D cells require the additional time provided by the SAC to properly capture and segregate chromosomes. These data indicate that if the SAC is active, fission yeast cells manage to assemble a functional mitotic spindle able to properly segregate chromosomes when the counteracting kinesins Cut[7] and Pkl[1] are deleted

Methods

Findings

Conclusion