Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast
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 cut7Dpkl1D 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 cut7Dpkl1D 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.
Results
Figure 1
[x] cut7D pkl1D shorter spindles
[x] cut7D pkl1D delayed meta-ana transition
[x] cut7D pkl1D 30% reduction elongation spindle speed anaphase B
[x] cut7D pkl1D mad2D severe reduction of growth
Figure S1
pkl1OE makes monopolar spindles (this was known)
[x] pkl1OE cut7OE rescues
Figure 2
[x] cut7D pkl1D klp9D lethal (tetrad disection).
[x] klp9 is recruited to the spindle at anaphase B onset
[x] cut7D pkl1D klp9off
[x] cut phenotype (100% of cells)
[x] abolished spindle elongation
[x] cut7D pkl1D klp9OE
[x] still short spindle at anaphase onset
[x] normal spindle elongation rate during Anaphase B
[x] cut7-24 klp9D lethal
[x] cut7Dpkl1Dklp2Dklp6Dklp5D
[ ]
Figure 3
[x] Ase1 recruited to the spindle before anaphase. -> Supp. Fig. 3D (add in GO annotation)
[x] cut7D pkl1D ase1D lethal
[x] cut7D pkl1D ase1off:
[x] 40% abolished elongation -> cut phenotype
[x] 40% short elongation -> cut phenotype
[x] 20% elongation and no cut
[x] Did not include ase1 truncation because it does not go to the nucleus.
[x] cut7D pkl1D cls1off
[x] 15% spindle collapse
[x] 25% deffective spindle elongation during anaphase (vague)
Figure 4
Figure S4
[x] ase1OE slower spindle anaphase B elongation
[x] Cls1OE rescues cut7-24.(high temp)
[x] pka1D partially rescues cut7-24 (high temp)
Figure 5
FYPO
GO
Spindle elongation during anaphase B should probably be a term.
Annotate cut7 and klp9 here.
Existing annotations.
Ask the authors:
cut7D pkl1D cls1-36 tetrad dissection was done at normal temp?
Discuss with Val
[x] What phenotype to put in cut7OE pkl1OE rescue of bipolarity. If the only thing they say is that bipolarity is reached. This is a rescue so it could be added only as genetic interaction, but probably more would be better.
[X] Is the growth on plate a "cell growth assay"? Yes
[X] What kind of evidence is tetrad dissection for lethality? Also what to write for lethality? Inviable cell? Cell growth assay.
PMID:28513584
Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast
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 cut7Dpkl1D 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 cut7Dpkl1D 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.
Results
Figure 1
cut7D pkl1D shorter spindlescut7D pkl1D delayed meta-ana transitionFigure S1
Figure 2
Figure 3
Figure 4
Figure S4
Figure 5
FYPO
GO
Existing annotations.
Ask the authors:
Discuss with Val