Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Acceptance summary:

This study investigates the mechanism of spindle elongation in the model system fission yeast using quantitative live cell imaging and genetic manipulations. The authors present compelling evidence for the kinesin Klp9 being important for the control of both microtubule sliding and microtubule growth during anaphase B, providing new insight into the mechanism coordinating both processes during cell division.

Decision letter after peer review:

Thank you for submitting your article "Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Stefan Westermann (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. Results, line 130: A major finding of the study seems to be that microtubule growth speed in anaphase does not depend on spindle bipolarization. Does this imply that growth speed depends on microtubule length – in which case one might expect that Klp9 accumulation at the ends of monopolar spindle bundles is similar to accumulation of Klp9 at the ends of bipolar microtubule overlaps (but this does not seem to be the case) – or growth speed could instead simply depend on time of mitosis (possibly depending on the kinetics of dephosphorylations)? If data are already available, they could be shown to answer these questions. Otherwise, adding a discussion would be considered appropriate.

2. Results, line 253 and following: The reviewers consider it important that the experiments performed with the sad1-1 allele need clarification. It is a point mutation located in the SUN domain that is very sensitive to temperatures above 32 °C. The experiments are carried out at 37 °C, so presumably the protein disappears completely and this blocks the insertion of SPB into the nuclear envelope. Monopolar spindles formation is likely to be observed due to the only insertion of the mother SPB in those cells where some Sad1.1 molecules still persist. It is important to know the percentage of monopolar spindles in a sad1.1 setting observed in the experimental conditions where the experiments have been carried out to see if sad1.1 is a robust tool to explore monopolar spindles formation at 37C. On the other hand, the Sad1 and the SPB insertion itself could affect the spindle elongation mechanisms during anaphase. Can the authors rule out this possibility?

3. The section on phospho-regulation of Klp9 and Dis1 is confusing (starting around line 345). The concern here is that the interpretation is entirely based on localization data and previously reported effects of phospho-alleles, while more direct means to follow the phosphorylation state of the respective proteins (e.g. shift on an SDS- or Phos-tag gel etc,) and how they are influenced by other alleles are not used. The microscopy and the genetic combination of alleles may simply reach limits here in terms of what can be learned mechanistically. A more satisfying explanation for the relationship between Dis1 and Klp9 would require additional biochemical methods. Some questions that don't appear fully answered are: Is Clp1 really responsible for dephosphorylation of Dis1? Is it really the phosphorylation state of Klp9 that is influenced by the Dis1-6A mutation?

Minimally, this section should be re-written to make it more accessible for the reader and more clearly explain the proposed mechanism. Ideally, a few key points should be tested experimentally: e.g. Phosphorylation state of Klp9 in different mutants by western blotting.

4. Related to the previous point, it was considered a pity that the authors went away from the "half-spindle" assay in the later part of the paper (after Figure 3). Maybe some of the regulatory aspects would become more clear in this setting: Is Clp1 required for polymerization of the half spindle? What about the recruitment of Klp9 to the tip of the bundle? Is that impaired in Dis1-6A, is it increased in the Klp9-3A mutant, etc..? The authors could use this simplified setting more. It seems that the endpoint of the mechanism, presumably involving a dephosphorylation cascade that originates at Dis1, is really Klp9. The observation that Klp9-3A can override the Dis1-6A phenotype would support this notion. Therefore a closer investigation of Klp9-3A or -3E in this setting could be informative.

5. Results, Figure 6. Compared to the experiments performed in cells, the in vitro work appears somewhat preliminary.

(a) Please show a Coomassie gel of purified Klp9.

Additional experiments may be worth considering to answer the following 2 points:

(b) Is Klp9 processive, does it accumulate at microtubule ends? Is its motor activity required for its effect on microtubule dynamics? Please use fluorescently labelled Klp9 to visualize it's localization/motility?

(c) Is Klp9 soluble at the higher tubulin concentration? Could it be that the effect of slowing down microtubule growth could be due protein insolubility (which could easily be seen when fluorescently labelled Klp9 is used)

The following issue should be addressed in the Discussion:

(d) How relevant is the observed behavior at 20 μm tubulin where the microtubule growth speed is significantly faster as in cells? In cells Klp9 clearly only accelerates microtubule growth speed according to the data presented in this study.

Reviewer 1:

The authors investigate the roles of kinesin-6 Klp9 and the microtubule polymerase Dis1 for anaphase spindle elongation in fission yeast. Using live cell fluorescence microscopy imaging of bipolar and artificially monopolarized spindles, they find that in cells Klp9 promotes microtubule growth in anaphase. recruitment of Klp9 to microtubules depends on Klp9 dephosphorylation that in turn depends on cdc14 phosphatase Clp1 and in a parallel pathway on Dis1 that itself needs to be phosphorylated in pro/metaphase and is then dephosphorylated in anaphase. The authors also show first experiments investigating how purified Klp9 affects microtubule dynamics in vitro (in the presence of purified tubulin), showing an activity that agrees at low tubulin concentrations with observations in cells, but that differs at higher tubulin concentrations.

Experiments in living cells are performed very carefully and comprehensively, quantitative analysis is performed to a high standard and conclusions are supported by the data. A limitation is that the mechanism by which Dis1 promotes dephosphorylation of Klp9 in a phosphorylation-dependent manner in cells, thereby essentially regulating its function for spindle elongation, remains unclear. Interesting is also the first observation of an unusual effect of purified Klp9 on the dynamic properties of microtubule dynamics, even if several mechanistic questions remain open.

Reviewer 2:

In this article, Krüger et al. identified a new role for Kinesin-6 using interesting tools to explore microtubule dynamics during anaphase B in fission yeast, for example, the monopolar spindles generated by the cut7-24 and sad1-1 mutations. Interestingly, they found that kinesin-6 (klp9 in fission yeast) promotes microtubule polymerization in vivo in a dose-dependent manner. The authors suggest that Klp9 could act as a cruise control for microtubule growth during anaphase B. They also found that the XMAP215 family member, Dis1, regulates recruitment of Klp9 to spindle microtubules in a dephosphorylation-dependent manner. In my opinion, these data add important information about a new role for kinesin-6 and its regulation throughout the cell cycle in S. pombe that are of interest to cell biologists in general.

Experiments performed with the sad1-1 allele need clarification. It is a point mutation located in the SUN domain that is very sensitive to temperatures above 32 °C. The experiments are carried out at 37 °C, so presumably the protein disappears completely and this blocks the insertion of SPB into the nuclear envelope. Monopolar spindles formation is likely to be observed due to the only insertion of the mother SPB in those cells where some Sad1.1 molecules still persist. It is important to know the percentage of monopolar spindles in a sad1.1 setting observed in the experimental conditions where the experiments have been carried out to see if sad1.1 is a robust tool to explore monopolar spindles formation at 37C. On the other hand, I think the removal of the Sad1 and the SPB insertion itself could affect the spindle elongation mechanisms during anaphase and it would be good if the authors could rule out this possibility.

Reviewer 3:

In this manuscript Krüger, Tran and colleagues investigate molecular mechanisms of anaphase spindle elongation in the model system fission yeast, using a combination of genetics and live cell microscopy, complemented by experiments with the purified motor Klp9 in vitro. In particular the authors are interested in understanding the coordination between midzone sliding and microtubule polymerization during anaphase elongation, which is required to maintain a stable midzone length. They use a clever genetic trick to generate monopolar half-spindles in fission yeast, which allows one to follow polymerization events usually masked in the midzone bundle. With this, they identify the Kinesin-6 Klp9 and the XMAP-215 protein Dis1 as key factors promoting microtubule polymerization in the half-spindle. They go on to show that these proteins are regulated by the Cdc14 phosphatase Clp1 and that Klp9 association with the midzone requires prior phosphorylation/dephosphorylation of Dis1 on Cdc2 consensus-sites. Finally, the authors show that Klp9 can either promote or attenuate microtubule polymerization in vitro, depending on the concentration of free tubulin.

Overall, the authors tackle an interesting problem, the execution of the genetic and imaging experiments, the display of the data and the writing/discussion, all follow a very high standard.

The section on phospho-regulation of Klp9 and Dis1 is confusing. My overall concern here is that the interpretation is entirely based on localization data and previously reported effects of phospho-alleles, while more direct means to follow the phosphorylation state of the respective proteins (e.g. shift on an SDS- or Phos-tag gel etc,) and how they are influenced by other alleles are not used. The microscopy and the genetic combination of alleles may simply reach limits here in terms of what can be learned mechanistically. A more satisfying explanation for the relationship between Dis1 and Klp9 would require additional biochemical methods. Some questions that don't appear fully answered to me are: Is Clp1 really responsible for dephosphorylation of Dis1? Is it really the phosphorylation state of Klp9 that is influenced by the Dis1-6A mutation?

I found it a bit of a pity, that the authors went away from the "half-spindle" assay in the later part of the paper (after Figure 3). Maybe some of the regulatory aspects would become more clear in this setting: Is Clp1 required for polymerization of the half spindle? What about the recruitment of Klp9 to the tip of the bundle? Is that impaired in Dis1-6A, is it increased in the Klp9-3A mutant, etc..?. The authors could use this simplified setting more. It seems that the endpoint of the mechanism, presumably involving a dephosphorylation cascade that originates at Dis1, is really Klp9. The observation that Klp9-3A can override the Dis1-6A phenotype would support this notion. Therefore a closer investigation of Klp9-3A or -3E in this setting could be informative.