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

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.

Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling

“…Because chromosomes are not bioriented on the spindle during initial SPB separation, they do not exert significant inward force. This result is consistent with our previous work, which demonstrated that initial bipolar spindle assembly can occur in a model lacking chromosomes [63,65,66].…”

“…Most spindle models have started with a bipolar structure or separated spindle poles, and most previous chromosome models have begun with chromosomes attached to the spindle or near a pre-formed spindle. Because we seek to model simultaneous spindle assembly and chromosome alignment with few imposed constraints, we developed a new model, building on previous work on spindle assembly in the absence of chromosomes and kinetochore-microtubule attachments [63,65,66].…”

“…However, constraining SPBs to a fixed radius alters force balance on the spindle and may alter spindle length. Therefore, we tested a model of a soft nuclear envelope by allowing the SPBs to move radially in a potential that mimics the energy required to deform the nuclear envelope [65,66] (Methods, Appendix A). The results show that a soft nuclear envelope leads to slightly longer spindles ( Figure 3D, Video S3), but for a physically realistic nuclear envelope force of around 17 pN, spindle length remains near 3 µm, as measured experimentally.…”

“…Our group has developed a simulation framework for microtubule-motor active matter and mitotic spindle self-assembly [52,62,63,65,66]. The computational scheme alternates between Brownian dynamics (BD) and kinetic Monte Carlo (kMC) steps to evolve the system forward in time.…”

“…Because we study de novo spindle assembly and chromosome alignment, we could not use previous models that started with an already-bipolar structure and/or chromosomes attached to the spindle. Therefore we extended a previous model of spindle assembly in the absence of chromosomes and kinetochore-microtubule attachments [63,65,66] to include chromosomes and kinetochores.…”