The doublecortin-family kinase ZYG-8 DCLK1 regulates motor activity to achieve proper force balance in C. elegans acentrosomal spindles

  1. Emily R. Czajkowski
  2. Nikita S. Divekar
  3. Sarah M. Wignall

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Abstract

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. How proper force balance is achieved in these spindles is not known. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. Interestingly, we found that ZYG-8 depletion from oocytes resulted in spindles that were over-elongated, suggesting that there was excess outward force following ZYG-8 removal. Experiments with monopolar spindles confirmed this hypothesis and revealed a role for ZYG-8 in regulating the force-generating motor BMK-1/kinesin-5. Importantly, further investigation revealed that kinase activity is required for the function of ZYG-8 in both meiosis and mitosis. Altogether, our results support a model in which ZYG-8 regulates motor-driven forces within the oocyte spindle, thus identifying a new function for a doublecortin-family protein in cell division.

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    1. Point-by-point description of the revisions

    Reviewer #1:

    Evidence, reproducibility and clarity (Required):

    *In this manuscript Czajkowski et al explore the role of the doublecortin-family kinase ZYG-8 during meiosis in C. elegans Oocytes. First by studying available temperature-sensitive mutants and then by generating their own strain expressing ZYG-8 amenable to auxin-inducible degradation, they establish that defects in ZYG-8 lead to defects in spindle assembly, such as the formation of multipolar spindles, and spindle maintenance, in which spindles elongate, fall apart, and deform in meiosis. Based on these observations the authors conclude that ZYG-8 depletion leads to excessive outward force. As the lab had previously found that the motor protein KLP-18 generates outside directed forces in meiosis, Czajkowski et al initially speculate that ZYG-8 might regulate KLP-18. KLP-18 depletion generally leads to the formation of monopolar spindles in meiosis. Intriguingly, when the authors co-deplete ZYG-8 they find that in some cases bipolarity was reestablished. This led to the hypothesis that yet another kinesin, BMK-1, the homolog of the mammalian EG-5, could provide redundant outward directed forces to KLP-18. The authors then study the effect of ZYG-8 and KLP-18 co-depletion in a BMK-1 mutant background strain and observe that bipolarity is no longer reestablished under these conditions, suggesting that BMK-1 generates additional outward directed forces. The authors also conclude that ZYG-8 inhibits BMK-1. To follow up on this Czajkowski et al generate a ZYG-8 line that carries a mutation in the kinase domain, which should inhibit its kinase activity. This line shows similar effects in terms of spindle elongation but reduced impact on spindle integrity, reflected in minor effects on the number of spindle poles and spindle angle. The authors conclude that ZYG-8's kinase activity is required for the function of ZYG-8 in meiosis and mitosis. Overall, the paper is well written, and the data is presented very clearly and reproducible. The experiments are adequately replicated, and statistical analysis are adequate. **The observations are very interesting. However, the authors could provide some additional insight into the function of ZYG-8. This paper is strongly focused on motor generated forces within the spindle and tries to place ZYG-8 within this context, but there is compelling evidence from other studies that ZYG-8 also affects microtubule dynamics, which would have implications for spindle assembly and structure. The paper would strongly benefit from the authors exploring this role of ZYG-8 in the context of meiosis further. If the authors feel that this would extend beyond the scope of this paper, I would suggest that the authors rephrase some of their introduction and discussion to reflect the possibility that changes in microtubule growth and nucleation rates could explain some of the phenotypes (think of katanin) and effects and that therefore it can not necessarily be concluded that BMK-1 is inhibited by ZYG-8. *

    We thank the reviewer for these positive comments on our manuscript and on the rigor of our data. We also thank them for the excellent suggestion to explore a potential role for microtubule dynamics. As detailed below in response to the specific points, we performed new experiments to explore this possibility, and found via FRAP analysis that there were substantial changes in microtubule dynamics upon ZYG-8 depletion. We have therefore added these new data and have re-written major parts of the manuscript to incorporate a discussion of microtubule dynamics throughout the paper (introduction, results, model, discussion). Our data now support two roles for ZYG-8 in regulating acentrosomal spindle assembly and stability - one in modulating microtubule dynamics and the other in tuning forces (either directly or indirectly). We are grateful to the reviewer for motivating us to do these experiments, as they have added a whole new angle to the manuscript and have greatly increased its impact, as we now have a fuller understanding of how ZYG-8 contributes to oocyte meiosis.

    Major points:

    *1.) Zyg-8, as well as the mammalian homolog DCLK-1, has been reported to play an important role for microtubule dynamics. While the introduction mentions its previously shown role in meiosis and mitosis, it is totally lacking any background on the effect on microtubule dynamics. The authors mention these findings in the discussion, but it would be helpful to incorporate this in the introduction as well. As an example, Goenczy et al 2001 demonstrated that ZYG-8 is involved in spindle positioning but also showed its ability to bind microtubules and promote microtubule assembly. Interestingly, like the authors here, Goenczy et al concluded that while the kinase domain contributes to, it is not essential ZYG-8's function. Also, Srayko et al 2005 (PMID 16054029) demonstrated that ZYG-8 depletion led to reduced microtubule growth rates and increased nucleation rates in C. elegans mitotic embryos. And in mammalian cells DCLK-1 was shown to increase microtubule nucleation rate and decrease catastrophe rate, leading to a net stabilization of microtubules (Moores et al 2006, PMID: 16957770). It would be great if the authors could add to the introduction that ZYG-8 has been suggested to affect microtubule dynamics. *

    We agree that this is a great idea. As the reviewer suggested, we decided to explore the possibility that ZYG-8 impacts microtubule dynamics within the oocyte spindle. We depleted ZYG-8 and performed FRAP experiments to determine if there were effects on microtubule turnover. We found that loss of ZYG-8 caused a dramatic decrease in the spindle's ability to recover tubulin, both at the spindle center and at the spindle poles (shown in a new Figure 7). We made substantial changes to the manuscript when adding these new data - the manuscript now discusses ZYG-8's role in modulating microtubule dynamics in the introduction, results, discussion, and model (Figure 9), and we added all of the references suggested by the reviewer. We think that the manuscript is greatly improved due to these additions and changes.

    *2a.) The authors initially study two different ts alleles, or484ts and b235ts. The experiments clearly show a significant increase in spindle length in both strains. However, the or484 strain had been previously studied (McNally et al 2016, PMID: 27335123), and only minor effects on spindle length were reported (8.5µm in wt metaphase and 10µm in zyg-8 (or484)). How do the authors explain these differences in ZYG-8 phenotype. Even though the ZYG-8 phenotype is consistent throughout this paper it would be good to explain why the authors observe spindle elongation, fragmentation and spindle bending in contrast to previous observations. *

    The reviewer is correct that McNally et.al. (2016) noted only minor effects on spindle length and did not report observing spindle bending or pole defects. However, the images presented in their paper of spindles in the zyg-8(or484) mutant (in Figure 8B) only showed spindles after they had already shrunk in preparation for anaphase; it is possible that these spindles had pole or midspindle defects prior to this shrinking, and that the authors did not note those phenotypes because their analysis focused on anaphase. In contrast, since the goal of our study focused on how ZYG-8 impacts spindle assembly and maintenance, we looked carefully at spindle morphology and quantified a larger number of metaphase spindles (in their study, only 12 metaphase spindles were measured, since metaphase was not the focus of their manuscript). Recently (after we submitted our manuscript), another study from the McNally lab was published, where they did note metaphase defects following ZYG-8 inhibition (though they did not describe the defects in detail or explore why they happened). We now mention and cite this new paper (Li et.al., 2023) in our manuscript, to show that our findings are consistent with the work of others in the field.

    *2b.) As a general note, it would be helpful if the authors could indicate if the spindles are in meiosis I or II. The only time where this is specifically mentioned is in Video 7, showing a Meiosis II spindle, which makes me assume all other data is in Meiosis I. Adding this to the figures would also help to distinguish if some of the images, i.e. Figure 1B, show multipolar spindles due to failed polar body extrusion. If this is the case then the quantification of number of poles should maybe reflect different possibilities, such as fragmented poles vs. multiple poles because two spindles form around dispersed chromatin masses. *

    We agree that it is a good idea to clarify this issue. For all of our experiments, we analyzed both MI and MII spindles. However, there were no noticeable differences in phenotype between MI and MII spindles for any of our mutant/depletion conditions - we observed bent spindles, elongated spindles, and extra poles in both MI and MII following ZYG-8 inhibition. Therefore, for the quantifications presented in the manuscript (spindle length, spindle angle, number of poles), we pooled our MI and MII data. We have now added this information to the manuscript for clarity (lines 97-99 and 139-141). In addition, we have added new images to Figure 1B that show examples of MII spindles (both at the permissive and restrictive temperature), to show that the phenotypes are indistinguishable between MI and MII.

    We agree with the reviewer that one of the spindles in the original Figure 1B looked like it could have resulted from failed polar body extrusion (the chromosomes appeared to be in two masses, something we did not originally notice, so theoretically each mass could have organized its own spindle). To determine if this was the case, we looked closely at the chromosomes in this image; we confirmed that there were only 6 chromosomes, and that all were bivalents (these can be distinguished from MII chromosomes based on size). Therefore, this spindle was not multipolar due to an issue with polar body extrusion. However, to prevent future confusion, we picked a different representative spindle (where the bivalents we not grouped into two masses), and we added a new column to the figure that shows the DNA channel in grayscale (so it is easier to see and count the chromosomes). We also now note in the materials and methods how we were able to distinguish between MI and MII in our experiments (chromosome count, size, presence of polar bodies), so that it is clear that none of our phenotypes result from failed polar body extrusion (lines 600-603).

    *3) The authors generate a line that carries a mutation leading to a kinase dead version of ZYG-8. It would be great if the authors could further test if this version is truly kinase dead. What is interesting is that the kinase dead version the authors create has less effect on the numbers of pole than the zyg-8 (b235)ts strain, which carries a mutation in a less conserved kinase region. Overall, it seems that the phenotypes are very similar, independent on mutations in the microtubule binding area, kinase area or after AID. This could of course be due to all regions being important, i.e. microtubule binding is required for localizing kinase-activity. Generating mutant versions of the target proteins, for example here BMK-1, that can not be phosphorylated or are constitutively active as well as assessment of changes in protein phosphorylation levels in the kinase dead strain would be helpful to provide deeper insight into potential regulation of proteins by ZYG-8. *

    We agree that it would be ideal to test whether the D604N mutant is truly kinase dead. However, in the interest of time, we ask to be allowed to skip that experiment. The analogous residue has been mutated in mammalian ZYG-8 (DCLK1), and has been shown to cause DCLK1 to be kinase dead in vitro; this is a highly conserved aspartic acid in the central part of the catalytic domain, so we infer that the mutation we made in ZYG-8 should be kinase dead as well. However, since we did not test this directly, we softened our language in the manuscript, explaining that we "infer" that it is kinase dead rather than stating definitively that it is. With regard to the zyg-8(b235)ts mutant having a stronger phenotype, we think that it is possible that this mutation destabilizes a larger portion of the protein (rather than just affecting the catalytic activity), since the phenotypes in this mutant are similar to depletion of the protein in the ZYG-8 AID strain. Therefore, we think that our D604N mutant reveals new information about the role of kinase activity, since it is a more specific mutation that should likely only affect catalytic activity and not the rest of the protein (based on the previous work on DCLK1).

    While we appreciate the suggestion from the reviewer to generate mutant versions of potential target proteins, we ask that this be considered beyond the scope of the study. Now that we know that ZYG-8 not only affects forces within the spindle (maybe BMK-1) but also microtubule dynamics, there are many potential targets - it would require a lot of work to figure out what the relevant targets are. Instead of exploring this experimentally in this manuscript, we added a new section to the discussion where we speculate on what some of these targets could be, to motivate future studies.

    *4a) The authors state that "BMK-1 provides redundant outward force to KLP18". Redundancy usually suggests that one protein can take over the function of another one when the other is not there. In these scenarios a phenotype is often only visible when both proteins are depleted as each can take over the function of the other one. Here however the situation seems slightly different, as depletion of BMK-1 has no phenotype while depletion of KLP-18 leads to monopolar spindles. If BMK-1 would normally provide outward directed forces, would this not be visible in KLP-18 depleted oocytes if they were truly redundant? I assume the authors hypothesize that ZYG-8 inhibits BMK-1 and thus it can not generate outward directed forces. In this case, do the authors envision that ZYG-8 inhibits BMK-1 prior to or in metaphase or only in anaphase or throughout meiosis? Do they speculate, that BMK-1 is inhibited in anaphase and only active in metaphase? *

    The reviewer makes an excellent point - we agree that we should not use the word "redundant" in this context, so we have removed this phrasing from the manuscript. We hypothesize that BMK-1 can provide outward forces during spindle assembly but is not capable of providing as much force as KLP-18 (the primary force-generating motor). We infer this based on our experiments where we co-deplete KLP-18 and ZYG-8 (using long-term depletion). Although BMK-1 is presumably activated under these conditions, it is not able to restore spindle bipolarity (there are outward forces generated, which results in minus ends being found at the periphery of the monopolar spindle, but spindles are not bipolar).

    Therefore, BMK-1 is not able to fully replace the function of KLP-18 during spindle assembly. Interestingly, our experiments imply that BMK-1 can better substitute for KLP-18 later on (when ZYG-8 is inhibited); when we remove ZYG-8 from formed monopolar spindles, bipolarity can be restored (an activity dependent on BMK-1). These findings suggest that ZYG-8 plays a more important role in suppressing BMK-1 activity after the spindle forms, to prevent spindle overelongation in metaphase. We have edited the manuscript to better explain these points.

    *4b) In addition, Figure S4 somewhat argues against a role for ZYG-8 in regulating BMK-1. ZYG-8 depletion supposedly leads to increased outward forces due to loss of BMK-1 inhibition, thus co-depletion of ZYG-8 with BMK-1 should rescue the increased spindle size at least to some extent, however neither increase in spindle length nor increase in additional spindle pole formation are prevented by co-depletion of BMK-1 suggesting that BMK-1 is not generating the forces leading to spindle length increase. Thus, arguing that after all ZYG-8 does not regulate BMK-1. This should be discussed further in the paper and the authors should consider changing the title. At this point the provided evidence that ZYG-8 is regulating motor activity is not strong enough to make this claim. *

    The reviewer is correct that Figure S4 shows that the effects of depleting ZYG-8 on bipolar spindles (spindle elongation and pole/midspindle defects) cannot solely be explained by a role for ZYG-8 in regulating BMK-1 - this was the point that we were trying to make when we included this data in the original manuscript. However, we previously did not know what this other role could be, and therefore we only speculated on other potential roles in the discussion. Now that we have done FRAP experiments and have found that ZYG-8 also affects microtubule dynamics in the oocyte spindle, we now have a better explanation for the data presented in Figure S4 - it makes sense that deleting BMK-1 would not rescue the effects of ZYG-8 depletion, since we have evidence that ZYG-8 also regulates microtubule dynamics. We now clearly explain this in the revised manuscript and we have changed the title to make it clear that ZYG-8 plays multiple roles in oocytes.

    *5) The authors are proposing that ZYG-8 regulates/ inhibits BMK-1, however convincing evidence for an inhibition is not provided in my opinion and the effect of ZYG-8 on BMK-1 could be indirect. To make a compelling argument for a regulation of BMK-1 the authors would have to investigate if ZYG-8 interacts and/ or phosphorylates BMK-1 (see 7) and if this affects its dynamics. In addition, given the reported role of ZYG-8 on microtubule dynamics it would be very important that the authors consider studying the effect of ZYG-8 degradation on microtubule dynamics. Tracking of EBP-2 would be good, however this is very difficult to do inside meiotic spindles due to their small size. In addition, the authors could maybe consider some FRAP experiments, which could provide insights into microtubule dynamics and motions, which could be indicative of outward directed forces/ sliding. *

    We thank the reviewer for these comments as they motivated us to explore a role for ZYG-8 in modulating microtubule dynamics. The reviewer is correct that tracking EBP-2 in the very small meiotic spindle is not possible due to technical limitations, so we took the suggestion to perform FRAP. These experiments revealed that microtubule turnover in the spindle is greatly slowed following ZYG-8 depletion, suggesting a global stabilization of microtubules (data presented in a new Figure 7). This change in dynamics could contribute to the observed spindle phenotypes, which we now explain in detail in the manuscript. Given these new findings, we also now note that the effects we see on BMK-1 activity could be indirect (i.e. maybe increasing the stability of microtubules allows motors to exert excess forces). We now clearly discuss these various possibilities in the discussion.

    *Summary: Additional requested experiments: *

    • Interaction/ phosphorylation of BMK-1 by ZYG-8, i.e. changes of BMK-1 phosphorylation in absence of ZYG-8, BMK-1 mutations that may prevent phosphorylation by ZYG-8.
    • Assessment of microtubule dynamics (EBP-2, FRAP, length in monopolar spindles...)
    • Kinase activity of the kinase dead ZYG-8 strain (OPTIONAL) We assessed the role of ZYG-8 in microtubule dynamics (bullet point #2). Because this new analysis revealed that ZYG-8 plays multiple roles in the spindle, we decided not to further investigate whether ZYG-8 phosphorylates BMK-1, since the manuscript now no longer argues that this is ZYG-8's major function. We also did not assess the kinase activity of the D604N mutant since this has been done previously for DCLK1, and instead we softened the language in manuscript when describing this mutant.

    Minor points:

    *1) In Figure 4C it seems that the ZYG-8 AID line as well as the zyg-8 (or848)ts already have a phenotype (increased ASPM-1 foci) in absence of auxin/ at the permissive temperature. Does this suggest that the ZYG-8 AID as well as the zyg-8 (or848) strains are after all slightly defective (even if Figure 1, S1 and S2 argue otherwise) and thus more responsive to the loss of KLP-18? *

    The reviewer is correct that the ZYG-8 AID strain (without auxin) and zyg-8(or848)ts strain (at the permissive temperature) are slightly defective in the klp-18(RNAi) monopolar spindle assay. To more rigorously determine whether these strains were also defective in other assays, we generated new graphs comparing the spindle lengths and angles of the two temperature sensitive strains at the permissive temperature to wild-type (N2) worms. These data are now shown in Figure S1 (new panels F and G). A comparison of our ZYG-8 AID strain to a control strain (both in the absence of auxin) are shown in Figure S2 (panels C and D). In this analysis, there wasn't a significant difference for either of these comparisons (i.e. the spindle lengths and angles were all equivalent). We do not know why these strains appear to be slightly defective in the monopolar spindle assay, though perhaps this assay is more sensitive and can detect very mild defects in protein function.

    *2) The authors observe that in preformed monopolar spindles degradation of ZYG-8 can sometimes restore bipolarity. This observation is very interesting but why do the authors not observe a similar phenotype in long-term ZYG-8 AID; klp-18 (RNAi) or zyg-8(or484)ts; klp-18(RNAi). In the latter conditions bipolarity does not seem to occur at all. Do the authors think this is due to differences in timing of events? *

    We thank the reviewer for highlighting this point. We do think that our data suggest that ZYG-8 plays a more important role maintaining the spindle that it does in spindle formation; we have now more clearly explained this in the manuscript (detailing the differences in phenotypes we observe when we deplete ZYG-8 prior to spindle assembly or after the spindle has already formed, lines 180-189 and 227-231). To emphasize this point further, we have also included a graph in Figure S3G that directly compares the number of poles per spindle in long-term auxin treated spindles to short-term auxin treated spindles (with and without metaphase arrest).

    *3) Based on the Cavin-Meza 2022 paper it looks like depletion of KLP-18 in a BMK-1 mutant background does not look different from klp-18 (RNAi) alone. However, looking at Video 8, it looks like spindles "shrink" in absence of KLP-18 and BMK-1. Or is this due to any effects from the ZYG-8 AID strain? This can also be seen in Video 9. *

    The reviewer highlights a fair point that was not clearly explained in our manuscript. In normal monopolar anaphase, chromosomes move in towards the center pole as the spindle gets smaller (C. elegans oocyte spindles shrink in both bipolar and monopolar anaphase); this was previously described in Muscat et.al. 2015, and, as the reviewer noted, in Cavin-Meza et.al. 2022 (in a strain with the bmk-1 mutation). We see this same monopolar anaphase behavior in the ZYG-8 AID strain (Figure 6). We have now better explained normal monopolar anaphase progression and we have cited the Muscat et.al. paper in the relevant sections of the manuscript (lines 221-223 and 714-717).

    *4) Line 311: " ZYG-8 loads onto the spindle along with BMK-1, and functions to inhibit BMK-1 from over elongating microtubules during metaphase." Maybe this sentence could be re-phrased as it currently sounds like BMK-1 elongates (polymerizes) microtubules. *

    In re-writing the manuscript and emphasizing that there are multiple for ZYG-8 (in addition to regulating forces within the spindle), we removed this sentence.

    *5) Line 313: "Intriguingly, in C. elegans oocytes and mitotically-dividing embryos, BMK-1 inhibition causes faster spindle elongation during anaphase, suggesting that BMK-1 normally functions as a brake to slow spindle elongation (Saunders et al., 2007; Laband et al., 2017). Further, ZYG-8 has been shown to be required for spindle elongation during anaphase B (McNally et al., 2016). Our findings may provide an explanation for this phenotype, since if ZYG-8 inhibits BMK-1 as we propose, then following ZYG-8 depletion, BMK-1 could be hyperactive, slowing anaphase B spindle elongation." This paragraph could be modified for better clarity. It is not clear how the findings of the authors, BMK-1 provides outward force but is normally inhibited by ZYG-8, align with the last sentence saying "following zyg-8 depletion, BMK-1 could be hyperactive slowing anaphase B spindle elongation", should it not increase elongation according to the authors observations? *

    In re-writing the manuscript to incorporate our new data showing that ZYG-8 plays a role in modulating microtubule dynamics, we also re-wrote this discussion so that there would be less emphasis on the potential connection between ZYG-8 and BMK-1. In making these edits to expand the focus of the manuscript, we removed this section of the discussion.

    *Reviewer #1 (Significance (Required)): * *In this manuscript Czajkowski et al explore the role of the doublecortin-family kinase ZYG-8 during meiosis in C. elegans Oocytes. The authors conclude that BMK-1 generates outward directed force, redundant to forces generated by KLP-18, and that ZYG-8 inhibits BMK-1. The authors conclude that ZYG-8's kinase activity is required for the function of ZYG-8 in meiosis and mitosis. This research is interesting and provides some novel insight into the role of ZYG-8. In particular the observed spindle elongation and subsequent spindle fragmentation are novel and had not yet been reported. Also, the observation that degradation of ZYG-8 in monopolar klp-18(RNAi) spindles can restore bipolarity is novel and interesting, as well as the observation that this is somewhat dependent on the presence of BMK-1. This will be of interest to a broad audience and provides some new insight into the role of importance of ZYG-8 and BMK-1. The limitation of the study is the interpretation of the results and the lack of solid evidence that the observed phenotypes are due to ZYG-8 regulation motor activity, as the title claims. To support this some more experiments would be required. In addition, ZYG-8 has been reported to affect microtubule dynamics, which can certainly affect the action of motors on microtubules. This line of research is not explored in the paper but would certainly add to its value.

    Field of expertise: Research in cell division *

    We thank the reviewer for their positive comments on the impact and novelty of our findings. We hope that the additional experiments we performed and the revisions we made to the text thoroughly address the reviewer's concerns and that they deem the revised manuscript ready for publication.

    *Reviewer #2 (Evidence, reproducibility and clarity (Required)): * *In this manuscript, the authors explore the requirement for doublecortin kinase Zyg8 in C elegans oocytes. Oocytes build meiotic spindles in the absence of centrosomes, and therefore unique regulation occurs during this process. Therefore, how spindles are built and its later stability are an area of active investigation in the field. Using mutant alleles of Zyg8 and auxin-induced degron alleles, the authors demonstrate that this kinase is required to negatively regulate outward pole forces through BMK1 kinesin and that it has other functions to still explore. Overall, I find that this study takes an elegant genetic approach to tackling this important question in oocyte biology. I have some comments to consider for making the MS clear to a reader. *

    We thank the reviewer for these positive comments on our approach and the importance of our research question. We have attempted to address all of the reviewer's suggestions and we think that they increase the clarity of the manuscript.

    Major Comments:

    *1.) Although I like the graphs describing the altered angles of the spindles, it falls short in fully assessing the phenotype in a meaningful statistical way. Could the authors also graph the data to show statistical significance in the angles between conditions? Perhaps by grouping them into angle ranges and performing an Anova test? This is important in Figure 2E where it is not obvious that there is a difference. *

    The reviewer makes a good point - we have now addressed this concern by performing ANOVA tests to compare conditions on each of the angle graphs. Results of these tests have been reported in the corresponding figure legends. This analysis has confirmed all of the statements we made in the original manuscript. In Figure 1D and S1D, spindle angles were significantly different in the zyg-8 temperature sensitive mutants at the restrictive temperature, and in Figure 2, the angles were significantly different between the "minus auxin" and "plus auxin" conditions. This differs from Figure 7, where there was no significant difference in spindle angle between control spindles and kinase dead mutant spindles (p-value >0.1).

    *2.) The authors do not discuss the significance of the altered spindle angles which I think is an interesting phenotype. Would this be a problem upon Anaphase onset? What is known about spindle angle and aneuploidy or cell viability? Has this phenotype been described before in oocytes or somatic cells? Does depletion of other kinesin motors cause this? *

    The reviewer brings up a good point that warrants more discussion in the manuscript. We agree that the angled spindles are an interesting phenotype; we believe that they could be a result of the spindle elongating to a point where the spindle center becomes weakened, suggesting that the severity of the angle is representative of the severity of spindle elongation. Alternatively, the angled spindles could be a result of the loss of spindle stability factors, such as the doublecortin domain of ZYG-8. This domain is known to have microtubule binding activity; this could be required to maintain stable crosslinked microtubules in the spindle center, such that when ZYG-8 is depleted, the spindle more easily comes apart as the spindle elongates. We now discuss these possibilities in the revised manuscript.

    To the reviewer's second point, we did not examine anaphase outcomes in our manuscript. However, this was recently explored by another lab (in a study that was published after we submitted our manuscript). This study showed that spindles lacking ZYG-8 were able to initiate anaphase and segregate chromosomes (McNally et.al., 2023, https://doi.org/10.1371/journal.pgen.1011090). Perhaps when the spindle shrinks at anaphase onset, the spindle is able to reorganize and largely correct the angle defect, enabling bi-directional chromosome segregation. Interestingly, however, McNally et.al. did report conditions under which spindle bending in anaphase resulted in polar body extrusion errors. The authors reported that BMK-1, which is known to act as a brake to prevent spindle oveelongation in anaphase, is required to prevent bent spindles during anaphase by resisting the forces of cortical myosin on the spindle. Thus, there is precedence for the idea that spindle needs to remain straight throughout anaphase, to ensure proper chromosome segregation.

    *3.) How is embryo spindle positioning determined? It is not clear from the images that there is a defect so I'm not sure what to look for. Is there a way to quantify this? *

    In the original manuscript, spindle positioning within the embryo was determined qualitatively by eye, which we agree was not a precise measure. To address the reviewer's comment, we re-analyzed our images and assessed the position of the spindle within each embryo quantitatively - these data are now shown in Figure 8H and Figure S2B. Spindle position was quantified by analyzing images using Imaris software. The center of the spindle was set by creating a Surface of the DNA signal, and finding the center of that signal. The cell center was determined by measuring the length of the embryo along the long axis and the width of the embryo along the short axis, and setting the center as the halfway point of the total length and width of the embryo. Distance from spindle center to cell center was then measured and graphed. This quantification confirmed the claims we made in our original manuscript - both auxin-treated ZYG-8 AID spindles and ZYG-8 kinase-dead mitotic spindles were significantly mispositioned. The details of how we performed this quantification have been added to the materials and methods.

    *4.) In Figure 1, it appears that there are 2 spindles. Are these MI and MII spindles or ectopic spindles? How do the authors know which one to measure? *

    We thank the reviewer for pointing this out. Reviewer 1 had a similar comment, and we now understand that using that image was misleading, as it looked like as if were two separate MII spindles formed following a failed polar body extrusion event. We have gone through all of our images to stage the oocytes by looking at their chromosome morphology (i.e., to distinguish MI and MII) - the image in question had 6 bivalents and was therefore in Meiosis I; we think that this was a single spindle where the chromosomes happened to cluster into two masses. However, to prevent further confusion, we have replaced this image with a different representative image. In spindles like this with multiple poles, we measure the dominant axis of the spindle (if there are multiple poles, we pick the most prominent ones for the angle measurement). For additional details please see our response to Reviewer 1 major point #2b.

    *5.) The authors show depletion of Zyg8 by western (long) and loss of Gfp (long and short), but don't do so for the acute treatment. I'm guessing this is because the Gfp tag is taken by the spindle marker. The authors should either demonstrate or explain how they know that the acute depletion is effective in removing Zyg8 protein. *

    The reviewer makes a valid point. However, we are unable to see ZYG-8 depletion via acute auxin treatment using live imaging, as ZYG-8 localization is too dim and diffuse to see on the spindle using our typical live imaging parameters (we attempted to do this in a version of the ZYG-8 AID strain that has mCherry::tubulin and GFP::ZYG-8, so that there was no other spindle protein tagged with GFP). To see any GFP::ZYG-8 signal, we had to increase the laser power and exposure time well above what we typically use for live imaging - in doing this, we noticed that there was a limit to how high we could go before the cell began dying during the imaging time course, evident by a lack of chromosome movement, lack of tubulin turnover, and a general increase in tubulin signal throughout the cytoplasm. We do believe that ZYG-8 is being depleted using acute auxin treatment, however, as we see spindle defects very quickly upon dissection of the oocytes into auxin - we just unfortunately don't have a good way of quantifying this given these technical limitations. We have now added information to the materials and methods noting that we cannot see GFP::ZYG-8 under our live imaging conditions (lines 552-561), so that the reader better understands this caveat.

    *6.) In video 2, the chromosome signal is dimmer in the auxin treatment compared to video 1. Why is this? Is it just an experimental artifact or is there something significant about this? If it is because of video choice, consider replacing this one. *

    We thank the reviewer for their keen observations. The chromosome signal being dimmer in the auxin treatment is an experimental artifact - the brightness of the signal can vary depending on how far the spindle is from the slide (this can vary from video to video, and can also change over the time course of one video if the spindle moves during filming). Because of this, movies taken at the same intensity and exposure conditions may appear to have varying levels of brightness. So that readers of the manuscript can better see the chromosomes in this video, we have brightened the chromosome channel in this movie and noted this in the materials and methods (lines 549-551).

    7.) Please consider color palette changes for color-blind readership.

    We agree that it is important to present data in a way that can be appreciated by color-blind readers. Although we would prefer not to have to alter every image in our paper at this point, we have provided all important individual channels in grey scale. We are also planning to adopt a change in color palette for future papers.

    Reviewer #2 (Significance (Required)):

    *The strengths of this manuscript include use of multiple genetic approaches to establish temporal requirements of ZYG8 and which pathway it is acting through. Additionally, the videos and images make the phenotypes clear to evaluate. A minor limitation is that we don't know if the ZYG8 and BMK1 genetic interaction is a direct phosphorylation or not. This MS is an advancement to the field of spindle building and stability, and is particularly relevant to human oocyte quality and fertility. Previous work has shown that human oocyte spindles are highly unstable, but it is challenging to dissect genetic interactions and to conduct mechanistic studies in human oocytes. Therefore, the work here, although conducted in a nematode, can shed light on mechanism as to why human oocyte spindles are unstable and associated with high aneuploidy rates. Based on my expertise in mammalian oocyte biology, I am confident that work presented here will be of high interest to people in the field of meiotic spindle building, aneuploidy and fertility. It also will have broader interest to folks in the areas of kinesin biology, general microtubule and spindle biology. *

    We thank the reviewer for these positive comments on the strength of our data and the significance of our findings reported in our original manuscript. We think that the improvements that we have made in response to suggestions from all three reviewers has further increased this impact.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    *Summary: The focus of this paper is the function of a relatively understudied (at least in meiosis) kinase in acentrosomal spindle assembly (Zyg-8, or DCLK1 in mammals) in C. elegans oocytes. The authors use existing ts alleles and a newly generated GFP-Auxin fusion protein, and find that the ts alleles and auxin degron have similar phenotypes. They also examine the interaction with two related kinesins, KLP-18 and BMK-1 in order to investigate the mechanism behind the zyg-8 mutant phenotype. One can probably debate the significance and focus of their conclusions (force balance on the spindle). However, this is an important study because its the first on the meiotic function of a ZYG-8 kinase, and it may open the way to further studies of this kinase and how it regulates multiple kinesins and meiotic spindle assembly. *

    We thank the reviewer for these positive comments and for pointing out the potential future impacts of our work. In revising the manuscript, we have broadened the focus of the manuscript - we no longer solely focus on force balance within the spindle. Thus, our revisions have substantially increased the significance and impact of our work, since the manuscript is no longer narrowly focused.

    Major points:

    *1.) The main concern is the focus that the main defect in zyg-8 depleted oocytes is on outward forces (eg line 134, 277, but many other places in Results and Discussion). The arguments in favor (eg line 269-271) are reasonable. However, these data are not conclusive, and do not rule out regulation of other motor activities, such as bundling, depolymerization or chromosome movement. These are complex phenotypes, and a kinase could have multiple targets and there are often multiple interpretations. This is briefly alluded to in line 372-373 but the authors could do more. Spindle length changes could be caused by different rates of depolymerization or polymerization at the poles or chromosomes. Its not clear how poleward force regulation explains the multiple pole phenotype, although a lack of central spindle integrity could do that. In most of the Results and Discussion, it is not clearly stated on what structures these outward forces are acting. Are these forces effecting kinetochore associated microtubules, or antiparallel overlap microtubules? What do the authors mean by proper force balance? Figure 8 suggests the defect is associated with the amount of overlap and force among antiparallel microtubules - that the forces effected are from the sliding of these microtubules. *

    We agree that our original manuscript was too narrowly focused on the idea of force balance and that we did not discuss other potential roles for ZYG-8 in enough detail (except for briefly in our discussion). In response to both this comment and to a suggestion by Reviewer #1, we decided to investigate a potential role for ZYG-8 in modulating microtubule dynamics (which could be another explanation for some of the phenotypes we observed). We performed FRAP to measure the rate of tubulin turnover within the spindle near the center and at the poles. Interestingly, these experiments revealed that loss of ZYG-8 slows the rate of tubulin turnover, suggesting a general stabilization of microtubules. Thus, we have re-written our manuscript to clearly explain that ZYG-8 plays multiple roles in oocyte spindles - with these changes throughout the manuscript (in the introduction, results, and discussion), the paper is now no longer focused primarily on forces. We hypothesize in the discussion that the phenotypes we observe could be a combination of the effects on microtubule dynamics and spindle forces; if microtubules become more stable and motors produce excess outward forces, this may cause stress on the spindle structure that could cause the midspindle to bend and the poles to split (lines 379-382). We also now more clearly explain that the effect ZYG-8 has on spindle forces could be either direct or indirect (e.g., ZYG-8 could directly regulate motors or, by affecting the microtubule tracks themselves, it could affect their ability to exert forces). As for which population of microtubules are affected, we hypothesize that the excess forces act primarily on overlapping antiparallel microtubules (these microtubules run laterally alongside chromosomes in this system), as is represented in the model figure (Figure 9); we attempted to more clearly explain this in the re-written manuscript.

    *2.) Based on differences between the long term and short term knockdown phenotypes, the authors suggest ZYG-8 is more important for spindle maintenance. For example, in line 299 the authors note that there is a more severe phenotypes with zyg-8 removed from pre-formed spindles. The authors could improve the presentation of this to allow the reader to appreciate this observation. The data is spread between Figures 2 and 3 without a direct comparison of the data. One solution would be to graph the data (eg # of poles) together in one graph and indicate if there is statistical significance. In the Discussion, the authors could refer to specific figure panels. *

    The reviewer is correct that our data suggests that ZYG-8 is more important for spindle maintenance than it is for assembly. As suggested, we made a graph that includes all the pole data from Figures 2 and 3 (long-term auxin, short-term auxin, and metaphase-arrested short-term auxin) - this is now shown in Figure S3D. This makes it easier for the reader to compare these data and appreciate this point. In addition, we added text to the results section, to more clearly explain our rationale for thinking that ZYG-8 plays a more prominent role in spindle maintenance than in assembly (lines 180-189 and 227-231).

    *3.) What is the practical difference between acute and short term depletion. Does acute show weaker phenotypes because there is more residual protein? Unfortunately, the effectiveness of Auxin treatment does not appear to be measured for acute or short term. If the acute depletion adds little to Figure 3, or is not much different than long term, then its not clear what it adds to the paper. Later, in Figure 6, why is only short term and acute analyzed. In general, the authors need to provide better rationale for the different auxin conditions, particularly acute and short term (eg. line 135). If they don't add anything, they should consider not presenting them because readers may get confused by the different conditions, why they were done, and what is learned from each one. *

    The reviewer brings up a fair point that we agree requires clarification. Descriptions of the different types of auxin experiments is provided in Figure 1A. Long-term AID depletes proteins overnight, so the protein of interest is already missing from the oocyte when the spindle begins to form - this allows us to assess whether the protein is required for spindle assembly. However, to determine if a protein is required to stabilize pre-formed spindles, we need to remove the protein quickly after the spindle forms (using either acute or short-term AID). Acute AID is performed by dissecting oocytes directly into auxin-containing media; this allows us to watch what happens to the spindle live, as the protein is being depleted. However, one limitation is that we can only film for a short time before the oocytes begin to die (oocytes become unhappy with extended light exposure, so we cut off the videos after 15 minutes or so, to ensure that we are not filming past the point where they begin to arrest or die). Therefore, to assess what happens to spindles beyond this point, we perform short-term auxin treatment, where whole worms are soaked in auxin containing solution for 30-45 minutes and then the oocytes are dissected for immunofluorescence; this technique allows us to look at what happens to the spindle after more extended protein depletion (since we are not limited to the 10-15 minute window of filming). We have now clarified this in the manuscript by adding these details to the materials and methods. Unfortunately, it is not technically possible to quantify the extent of protein depletion in acute AID via western blotting since we would not be able to easily collect enough dissected oocytes to make a protein sample. (It is also technically challenging to quantify this via imaging; see our response to Reviewer #2, point #5). However, we assume that we are depleting ZYG-8 since we see dramatic spindle defects immediately upon dissection into auxin.

    Minor points:

    *3.) I am a little confused about imaging for GFP::tubulin in auxin experiments. Doesn't the ZYG-8 protein also have GFP? Should this be visible in controls? Is it measurable in the experiments? *

    The reviewer is correct that the ZYG-8 protein is also tagged with GFP in the GFP::tubulin; mCherry::histone live imaging experiments. However, we found that the GFP::ZYG-8 signal is undetectable using the live imaging conditions we are using. We determined this by analyzing a version of the ZYG-8 AID strain in which tubulin was tagged with mCherry (and thus the only GFP-tagged protein was ZYG-8). Using the same live imaging parameters we use for our movies of GFP::tubulin (same exposure time, laser power, etc), we did not detect any GFP::ZYG-8. We have now added this information to the materials and methods (lines 552-561) to clarify these points for the reader, to prevent further confusion.

    *4.) It is nice that the authors validated the results in an emb-30 background with unarrested oocytes. The authors note that the wild-type oocytes undergo anaphase (line 150). The images seem to suggest the auxin treated oocytes do not. Can the authors comment on anaphase in the depletion experiments. Even better, would be to comment on the accuracy of chromosome alignment and segregation. If zyg-8 mutant oocytes complete meiosis, is there any aneuploidy? These are important questions because otherwise the defects in zyg-8 mutants have less significance. *

    We thank the reviewer for their comment. Previous work on ZYG-8 in C. elegans examined a role for ZYG-8 in anaphase and showed that this protein is required for anaphase B spindle elongation (McNally et.al. 2016); because this was known when we launched our study, we purposely did not extensively study ZYG-8 in anaphase and instead focused on understanding how ZYG-8 contributes to spindle formation and stability. Our fixed imaging long-term AID experiments revealed that spindles were able to go through anaphase and segregate chromosomes bidirectionally despite the metaphase spindle phenotypes, consistent with this previous work (McNally et.al. 2016) and with another recent paper from the same lab (McNally et.al. 2023). However, we did not examine whether there were chromosome segregation errors. Given that anaphase is not the focus of our paper, we ask that this be deemed beyond the scope of our study.

    5.) Later, in line 184, the authors indicate that zyg-8 bipolar spindles "segregate chromosomes". Which images show anaphase I? As noted above, a limitation of these studies is not knowing the outcome of meiosis in these Zyg-8 depletions.

    We agree that in the original manuscript it was difficult to see that chromosomes were segregating bidirectionally in our movies and in the still timepoint images presented in Figure 5. Therefore, we brightened the chromosome channel in the relevant videos to make it easier to see the segregating chromosomes. Video 6 shows an oocyte in Meiosis II, as the first polar body can be seen near the spindle in this movie. At 2 minutes, the monopolar spindle becomes bipolar and begins to shrink as it goes into anaphase. Chromosomes begin to move apart and then the spindle elongates. At 11 minutes, you can see that the chromosomes have segregated bidirectionally. Thus, when monopolar spindles reorganize into bipolar spindles under these conditions, they can drive bidirectional chromosome segregation. We did not assess the fidelity of chromosome segregation under these conditions (i.e., whether chromosomes segregated accurately), as the question we were trying to answer in this experiment was whether outward forces sufficient to re-establish bipolarity could be activated upon ZYG-8 depletion (as explained above in response to point #4, we focused our study on trying to understand the effects of ZYG-8 depletion on the spindle, rather than on anaphase). We agree that analyzing anaphase outcomes would be interesting, but we ask that it be considered beyond the scope of this study.

    *6.) Line 206 suggests that ZYG-8 inhibits BMK-1. Is a simple explanation that BMK-1 is required for the bipolar spindles observed in the klp-18 zyg-8 AID oocytes? *

    Yes, the reviewer is correct that BMK-1 is required for the generation of bipolar spindles in the klp-18(RNAi) ZYG-8 AID conditions. In the original manuscript we extrapolated this result to propose that ZYG-8 regulates BMK-1. However, this comment, as well as feedback from the other reviewers and our new experiments (showing that ZYG-8 also modulates microtubule dynamics) has made us re-think the way we discuss this result, as we now agree that it does not prove this regulation (it is only suggestive). Therefore, in the revised manuscript, we no longer definitely claim that ZYG-8 regulates BMK-1 - we have switched to softer language (stating that ZYG-8 "may regulate" BMK-1, etc.). In the results section we now describe our conclusions as follows: "These data demonstrate that BMK-1 produces the outward forces that are activated upon ZYG-8 and KLP-18 co-depletion and raise the possibility that ZYG-8 regulates BMK-1 either directly or indirectly" (lines 250-252).

    *7.) Given that many mitotic and meiotic kinases are localized to specific regions or domains of the spindle, there is only limited discussion of the ZYG-8 localization pattern. Does the ZYG-8 localization pattern provide any insights into its mechanism of promoting spindle assembly? *

    The reviewer makes a good point - while we did report ZYG-8 localization, the discussion on the importance of its localization pattern was limited. To address this, we now remind readers in the discussion that ZYG-8 and BMK-1 co-localize throughout meiosis, consistent with the possibility that ZYG-8 could regulate BMK-1. Notably, this localization pattern is also consistent with the observation that ZYG-8 modulates microtubule dynamics across the spindle; this is now also noted in the discussion (lines 358-361).

    *8.) Line 96-97 - how much is the ZYG-8 depletion? *

    To address this question, we have quantified the amount of ZYG-8 protein in our ZYG-8 AID strain in control, long-term, and short-term auxin treated conditions. The western blot was quantified by comparing the raw intensity of the bands and subtracting the background signal. Short-term auxin depletion resulted in an ~63% reduction in ZYG-8 GFP signal, and long-term depletion resulted in an ~93% reduction in ZYG-8 GFP signal. This has now been reported in the manuscript on lines 785-786.

    *9.) Line 140: the authors say spindle length could not be measured, but perhaps it makes more sense to measure half spindle (chromosome to spindle pole). The images do give the impression that the chromosome to pole distance is shorter. *

    While we liked this idea and tried to perform these measurements, it turned out to be difficult in practice, since the spindle length measurements are obtained by finding the distance from pole (center of the ASPM-1 staining) to center of the chromosome signal. If you look carefully at our images you will notice that the chromosomes lose alignment following short-term AID; therefore, the chromosomes do not form one mass, which made it very difficult to determine an accurate "center" of the DNA signal. Additionally, in most cases the poles are disrupted such that ASPM-1 is found in many separate masses and/or is diffusely localized around the periphery of the spindle. Because of this, we unfortunately felt that these measurements would not be very accurate and would be hard to interpret.

    *10.) Don't see the point of lines 323-330. Could be deleted? *

    In revising our manuscript, we have rephrased these lines in an attempt to provide more context. Because DCLK1 has been shown to be upregulated in a wide variety of cancers, there are ongoing efforts to find chemical inhibitors that specifically block the kinase activity of this protein to be used as cancer therapeutics. However, no one has previously shown that the kinase activity of DCLK1 is important for its in vivo function (in any organism). Therefore, we were trying to make the point that, since we demonstrated that kinase activity is important for the functions of a DCLK1 family member in vivo, this suggests that these kinase inhibitors may in fact be beneficial in knocking down DCLK1 activity.

    11.) Figure 1: Because ts alleles could have a defective phenotype at "permissive" temperature, a wild-type control should be included. This data does appear in a later figure.

    The reviewer is correct that this data does appear in a later figure, but we agree this direct comparison would provide clarity to the reader. To address this comment, we compared the spindle lengths and angles of the two temperature-sensitive (TS) strains (at both the permissive and restrictive temperatures) to wild-type (N2) worms - these data have been added to Figure S1 (new panels F and G). The spindle lengths of both TS strains at the permissive temperature did not significantly differ from wild-type spindle lengths (p>0.1), while both TS strains at the restrictive temperature were significantly different than wild-type (p0.1), but there was a significant difference between wild-type spindles and the TS mutants at the restrictive temperature (doublecortin domain mutant (p Reviewer #3 (Significance (Required)): The strengths of this paper are the novelty of studying Zyg-8. It also addresses important questions regarding acentrosmal spindle assembly in oocytes. The weakness is mostly in the limited interpretation of results and not enough consideration of alternative interpretations. Related to this, the authors only test the force balance hypothesis with the knockout of two related kinesins. They don't experimentally investigate other mechanisms for the zyg-8 phenotype. This research should be of broad interest to anyone interested in oocyte spindle assembly, and also in a more specialized way to those who study kinases or Zyg-8 homologs in other cell types or organisms.

    We thank the reviewer for these positive comments on the strengths and novelty of our manuscript. We also appreciate the constructive suggestions of all three reviewers, which motivated us to perform new experiments that revealed additional functions for ZYG-8 - these revisions have greatly improved the manuscript and have broadened its impact.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The focus of this paper is the function of a relatively understudied (at least in meiosis) kinase in acentrosomal spindle assembly (Zyg-8, or DCLK1 in mammals) in C. elegans oocytes. The authors use existing ts alleles and a newly generated GFP-Auxin fusion protein, and find that the ts alleles and auxin degron have similar phenotypes. They also examine the interaction with two related kinesins, KLP-18 and BMK-1 in order to investigate the mechanism behind the zyg-8 mutant phenotype. One can probably debate the significance and focus of their conclusions (force balance on the spindle). However, this is an important study because its the first on the meiotic function of a ZYG-8 kinase, and it may open the way to further studies of this kinase and how it regulates multiple kinesins and meiotic spindle assembly.

    Major:

    1. The main concern is the focus that the main defect in zyg-8 depleted oocytes is on outward forces (eg line 134, 277, but many other places in Results and Discussion). The arguments in favor (eg line 269-271) are reasonable. However, these data are not conclusive, and do not rule out regulation of other motor activities, such as bundling, depolymerization or chromosome movement. These are complex phenotypes, and a kinase could have multiple targets and there are often multiple interpretations. This is briefly alluded to in line 372-373 but the authors could do more. Spindle length changes could be caused by different rates of depolymerization or polymerization at the poles or chromosomes. Its not clear how poleward force regulation explains the multiple pole phenotype, although a lack of central spindle integrity could do that. In most of the Results and Discussion, it is not clearly stated on what structures these outward forces are acting. Are these forces effecting kinetochore associated microtubules, or antiparallel overlap microtubules? What do the authors mean by proper force balance? Figure 8 suggests the defect is associated with the amount of overlap and force among antiparallel microtubules - that the forces effected are from the sliding of these microtubules.

    2. Based on differences between the long term and short term knockdown phenotypes, the authors suggest ZYG-8 is more important for spindle maintenance. For example, in line 299 the authors note that there is a more severe phenotypes with zyg-8 removed from pre-formed spindles. The authors could improve the presentation of this to allow the reader to appreciate this observation. The data is spread between Figures 2 and 3 without a direct comparison of the data. One solution would be to graph the data (eg # of poles) together in one graph and indicate if there is statistical significance. In the Discussion, the authors could refer to specific figure panels.

    3. What is the practical difference between acute and short term depletion. Does acute show weaker phenotypes because there is more residual protein? Unfortunately, the effectiveness of Auxin treatment does not appear to be measured for acute or short term. If the acute depletion adds little to Figure 3, or is not much different than long term, then its not clear what it adds to the paper. Later, in Figure 6, why is only short term and acute analyzed. In general, the authors need to provide better rationale for the different auxin conditions, particularly acute and short term (eg. line 135). If they don't add anything, they should consider not presenting them because readers may get confused by the different conditions, why they were done, and what is learned from each one.

    Minor:

    1. I am a little confused about imaging for GFP::tubulin in auxin experiments. Doesn't the ZYG-8 protein also have GFP? Should this be visible in controls? Is it measurable in the experiments?

    2. It is nice that the authors validated the results in an emb-30 background with unarrested oocytes. The authors note that the wild-type oocytes undergo anaphase (line 150). The images seem to suggest the auxin treated oocytes do not. Can the authors comment on anaphase in the depletion experiments. Even better, would be to comment on the accuracy of chromosome alignment and segregation. If zyg-8 mutant oocytes complete meiosis, is there any aneuploidy? These are important questions because otherwise the defects in zyg-8 mutants have less significance.

    3. Later, in line 184, the authors indicate that zyg-8 bipolar spindles "segregate chromosomes". Which images show anaphase I? As noted above, a limitation of these studies is not knowing the outcome of meiosis in these Zyg-8 depletions.

    4. Line 206 suggests that ZYG-8 inhibits BMK-1. Is a simple explanation that BMK-1 is required for the bipolar spindles observed in the klp-18 zyg-8 AID oocytes?

    5. Given that many mitotic and meiotic kinases are localized to specific regions or domains of the spindle, there is only limited discussion of the ZYG-8 localization pattern. Does the ZYG-8 localization pattern provide any insights into its mechanism of promoting spindle assembly?

    6. Line 96-97 - how much is the ZYG-8 depletion?

    7. Line 140: the authors say spindle length could not be measured, but perhaps it makes more sense to measure half spindle (chromosome to spindle pole). The images do give the impression that the chromosome to pole distance is shorter.

    8. Don't see the point of lines 323-330. Could be deleted?

    9. Figure 1: Because ts alleles could have a defective phenotype at "permissive" temperature, a wild-type control should be included. This data does appear in a later figure.

    Significance

    The strengths of this paper are the novelty of studying Zyg-8. It also addresses important questions regarding acentrosmal spindle assembly in oocytes. The weakness is mostly in the limited interpretation of results and not enough consideration of alternative interpretations. Related to this, the authors only test the force balance hypothesis with the knockout of two related kinesins. They don't experimentally investigate other mechanisms for the zyg-8 phenotype. This research should be of broad interest to anyone interested in oocyte spindle assembly, and also in a more specialized way to those who study kinases or Zyg-8 homologs in other cell types or organisms.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors explore the requirement for doublecortin kinase Zyg8 in C elegans oocytes. Oocytes build meiotic spindles in the absence of centrosomes, and therefore unique regulation occurs during this process. Therefore, how spindles are built and its later stability are an area of active investigation in the field. Using mutant alleles of Zyg8 and auxin-induced degron alleles, the authors demonstrate that this kinase is required to negatively regulate outward pole forces through BMK1 kinesin and that it has other functions to still explore. Overall, I find that this study takes an elegant genetic approach to tackling this important question in oocyte biology. I have some comments to consider for making the MS clear to a reader.

    Major Comments:

    1. Although I like the graphs describing the altered angles of the spindles, it falls short in fully assessing the phenotype in a meaningful statistical way. Could the authors also graph the data to show statistical significance in the angles between conditions? Perhaps by grouping them into angle ranges and performing an Anova test? This is important in Figure 2E where it is not obvious that there is a difference.

    2. The authors do not discuss the significance of the altered spindle angles which I think is an interesting phenotype. Would this be a problem upon Anaphase onset? What is known about spindle angle and aneuploidy or cell viability? Has this phenotype been described before in oocytes or somatic cells? Does depletion of other kinesin motors cause this?

    3. How is embryo spindle positioning determined? It is not clear from the images that there is a defect so I'm not sure what to look for. Is there a way to quantify this?

    4. In Figure 1, it appears that there are 2 spindles. Are these MI and MII spindles or ectopic spindles? How do the authors know which one to measure?

    5. The authors show depletion of Zyg8 by western (long) and loss of Gfp (long and short), but don't do so for the acute treatment. I'm guessing this is because the Gfp tag is taken by the spindle marker. The authors should either demonstrate or explain how they know that the acute depletion is effective in removing Zyg8 protein.

    6. In video 2, the chromosome signal is dimmer in the auxin treatment compared to video 1. Why is this? Is it just an experimental artifact or is there something significant about this? If it is because of video choice, consider replacing this one.

    7. Please consider color palette changes for color-blind readership.

    Significance

    The strengths of this manuscript include use of multiple genetic approaches to establish temporal requirements of ZYG8 and which pathway it is acting through. Additionally, the videos and images make the phenotypes clear to evaluate. A minor limitation is that we don't know if the ZYG8 and BMK1 genetic interaction is a direct phosphorylation or not.

    This MS is an advancement to the field of spindle building and stability, and is particularly relevant to human oocyte quality and fertility. Previous work has shown that human oocyte spindles are highly unstable, but it is challenging to dissect genetic interactions and to conduct mechanistic studies in human oocytes. Therefore, the work here, although conducted in a nematode, can shed light on mechanism as to why human oocyte spindles are unstable and associated with high aneuploidy rates.

    Based on my expertise in mammalian oocyte biology, I am confident that work presented here will be of high interest to people in the field of meiotic spindle building, aneuploidy and fertility. It also will have broader interest to folks in the areas of kinesin biology, general microtubule and spindle biology.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript Czajkowski et al explore the role of the doublecortin-family kinase ZYG-8 during meiosis in C. elegans Oocytes. First by studying available temperature-sensitive mutants and then by generating their own strain expressing ZYG-8 amenable to auxin-inducible degradation, they establish that defects in ZYG-8 lead to defects in spindle assembly, such as the formation of multipolar spindles, and spindle maintenance, in which spindles elongate, fall apart, and deform in meiosis. Based on these observations the authors conclude that ZYG-8 depletion leads to excessive outward force. As the lab had previously found that the motor protein KLP-18 generates outside directed forces in meiosis, Czajkowski et al initially speculate that ZYG-8 might regulate KLP-18. KLP-18 depletion generally leads to the formation of monopolar spindles in meiosis. Intriguingly, when the authors co-deplete ZYG-8 they find that in some cases bipolarity was reestablished. This led to the hypothesis that yet another kinesin, BMK-1, the homolog of the mammalian EG-5, could provide redundant outward directed forces to KLP-18. The authors then study the effect of ZYG-8 and KLP-18 co-depletion in a BMK-1 mutant background strain and observe that bipolarity is no longer reestablished under these conditions, suggesting that BMK-1 generates additional outward directed forces. The authors also conclude that ZYG-8 inhibits BMK-1. To follow up on this Czajkowski et al generate a ZYG-8 line that carries a mutation in the kinase domain, which should inhibit its kinase activity. This line shows similar effects in terms of spindle elongation but reduced impact on spindle integrity, reflected in minor effects on the number of spindle poles and spindle angle. The authors conclude that ZYG-8's kinase activity is required for the function of ZYG-8 in meiosis and mitosis.

    • Overall, the paper is well written, and the data is presented very clearly and reproducible. The experiments are adequately replicated, and statistical analysis are adequate. The observations are very interesting. However, the authors could provide some additional insight into the function of ZYG-8. This paper is strongly focused on motor generated forces within the spindle and tries to place ZYG-8 within this context, but there is compelling evidence from other studies that ZYG-8 also affects microtubule dynamics, which would have implications for spindle assembly and structure. The paper would strongly benefit from the authors exploring this role of ZYG-8 in the context of meiosis further. If the authors feel that this would extend beyond the scope of this paper, I would suggest that the authors rephrase some of their introduction and discussion to reflect the possibility that changes in microtubule growth and nucleation rates could explain some of the phenotypes (think of katanin) and effects and that therefore it can not necessarily be concluded that BMK-1 is inhibited by ZYG-8.

    Major comments:

    1. Zyg-8, as well as the mammalian homolog DCLK-1, has been reported to play an important role for microtubule dynamics. While the introduction mentions its previously shown role in meiosis and mitosis, it is totally lacking any background on the effect on microtubule dynamics. The authors mention these findings in the discussion, but it would be helpful to incorporate this in the introduction as well. As an example, Goenczy et al 2001 demonstrated that ZYG-8 is involved in spindle positioning but also showed its ability to bind microtubules and promote microtubule assembly. Interestingly, like the authors here, Goenczy et al concluded that while the kinase domain contributes to, it is not essential ZYG-8's function. Also, Srayko et al 2005 (PMID 16054029) demonstrated that ZYG-8 depletion led to reduced microtubule growth rates and increased nucleation rates in C. elegans mitotic embryos. And in mammalian cells DCLK-1 was shown to increase microtubule nucleation rate and decrease catastrophe rate, leading to a net stabilization of microtubules (Moores et al 2006, PMID: 16957770).

    It would be great if the authors could add to the introduction that ZYG-8 has been suggested to affect microtubule dynamics.

    1. The authors initially study two different ts alleles, or484ts and b235ts. The experiments clearly show a significant increase in spindle length in both strains. However, the or484 strain had been previously studied (McNally et al 2016, PMID: 27335123), and only minor effects on spindle length were reported (8.5µm in wt metaphase and 10µm in zyg-8 (or484)). How do the authors explain these differences in ZYG-8 phenotype. Even though the ZYG-8 phenotype is consistent throughout this paper it would be good to explain why the authors observe spindle elongation, fragmentation and spindle bending in contrast to previous observations.

    As a general note, it would be helpful if the authors could indicate if the spindles are in meiosis I or II. The only time where this is specifically mentioned is in Video 7, showing a Meiosis II spindle, which makes me assume all other data is in Meiosis I. Adding this to the figures would also help to distinguish if some of the images, i.e. Figure 1B, show multipolar spindles due to failed polar body extrusion. If this is the case then the quantification of number of poles should maybe reflect different possibilities, such as fragmented poles vs. multiple poles because two spindles form around dispersed chromatin masses.

    1. The authors generate a line that carries a mutation leading to a kinase dead version of ZYG-8. It would be great if the authors could further test if this version is truly kinase dead. What is interesting is that the kinase dead version the authors create has less effect on the numbers of pole than the zyg-8 (b235)ts strain, which carries a mutation in a less conserved kinase region. Overall, it seems that the phenotypes are very similar, independent on mutations in the microtubule binding area, kinase area or after AID. This could of course be due to all regions being important, i.e. microtubule binding is required for localizing kinase-activity. Generating mutant versions of the target proteins, for example here BMK-1, that can not be phosphorylated or are constitutively active as well as assessment of changes in protein phosphorylation levels in the kinase dead strain would be helpful to provide deeper insight into potential regulation of proteins by ZYG-8.

    1. The authors state that "BMK-1 provides redundant outward force to KLP18". Redundancy usually suggests that one protein can take over the function of another one when the other is not there. In these scenarios a phenotype is often only visible when both proteins are depleted as each can take over the function of the other one. Here however the situation seems slightly different, as depletion of BMK-1 has no phenotype while depletion of KLP-18 leads to monopolar spindles. If BMK-1 would normally provide outward directed forces, would this not be visible in KLP-18 depleted oocytes if they were truly redundant? I assume the authors hypothesize that ZYG-8 inhibits BMK-1 and thus it can not generate outward directed forces. In this case, do the authors envision that ZYG-8 inhibits BMK-1 prior to or in metaphase or only in anaphase or throughout meiosis? Do they speculate, that BMK-1 is inhibited in anaphase and only active in metaphase? In addition, Figure S4 somewhat argues against a role for ZYG-8 in regulating BMK-1. ZYG-8 depletion supposedly leads to increased outward forces due to loss of BMK-1 inhibition, thus co-depletion of ZYG-8 with BMK-1 should rescue the increased spindle size at least to some extent, however neither increase in spindle length nor increase in additional spindle pole formation are prevented by co-depletion of BMK-1 suggesting that BMK-1 is not generating the forces leading to spindle length increase. Thus, arguing that after all ZYG-8 does not regulate BMK-1. This should be discussed further in the paper and the authors should consider changing the title. At this point the provided evidence that ZYG-8 is regulating motor activity is not strong enough to make this claim.

    2. The authors are proposing that ZYG-8 regulates/ inhibits BMK-1, however convincing evidence for an inhibition is not provided in my opinion and the effect of ZYG-8 on BMK-1 could be indirect. To make a compelling argument for a regulation of BMK-1 the authors would have to investigate if ZYG-8 interacts and/ or phosphorylates BMK-1 (see 7) and if this affects its dynamics. In addition, given the reported role of ZYG-8 on microtubule dynamics it would be very important that the authors consider studying the effect of ZYG-8 degradation on microtubule dynamics. Tracking of EBP-2 would be good, however this is very difficult to do inside meiotic spindles due to their small size. In addition, the authors could maybe consider some FRAP experiments, which could provide insights into microtubule dynamics and motions, which could be indicative of outward directed forces/ sliding.

    Summary:

    Additional requested experiments:

    • Interaction/ phosphorylation of BMK-1 by ZYG-8, i.e. changes of BMK-1 phosphorylation in absence of ZYG-8, BMK-1 mutations that may prevent phosphorylation by ZYG-8. -Assessment of microtubule dynamics (EBP-2, FRAP, length in monopolar spindles...) -Kinase activity of the kinase dead ZYG-8 strain (OPTIONAL)

    Minor comments:

    1. In Figure 4C it seems that the ZYG-8 AID line as well as the zyg-8 (or848)ts already have a phenotype (increased ASPM-1 foci) in absence of auxin/ at the permissive temperature. Does this suggest that the ZYG-8 AID as well as the zyg-8 (or848) strains are after all slightly defective (even if Figure 1, S1 and S2 argue otherwise) and thus more responsive to the loss of KLP-18?

    2. The authors observe that in preformed monopolar spindles degradation of ZYG-8 can sometimes restore bipolarity. This observation is very interesting but why do the authors not observe a similar phenotype in long-term ZYG-8 AID; klp-18 (RNAi) or zyg-8(or484)ts; klp-18(RNAi). In the latter conditions bipolarity does not seem to occur at all. Do the authors think this is due to differences in timing of events?

    3. Based on the Cavin-Meza 2022 paper it looks like depletion of KLP-18 in a BMK-1 mutant background does not look different from klp-18 (RNAi) alone. However, looking at Video 8, it looks like spindles "shrink" in absence of KLP-18 and BMK-1. Or is this due to any effects from the ZYG-8 AID strain? This can also be seen in Video 9.

    4. Line 311: " ZYG-8 loads onto the spindle along with BMK-1, and functions to inhibit BMK-1 from over elongating microtubules during metaphase." Maybe this sentence could be re-phrased as it currently sounds like BMK-1 elongates (polymerizes) microtubules.

    5. Line 313: "Intriguingly, in C. elegans oocytes and mitotically-dividing embryos, BMK-1 inhibition causes faster spindle elongation during anaphase, suggesting that BMK-1 normally functions as a brake to slow spindle elongation (Saunders et al., 2007; Laband et al., 2017). Further, ZYG-8 has been shown to be required for spindle elongation during anaphase B (McNally et al., 2016). Our findings may provide an explanation for this phenotype, since if ZYG-8 inhibits BMK-1 as we propose, then following ZYG-8 depletion, BMK-1 could be hyperactive, slowing anaphase B spindle elongation." This paragraph could be modified for better clarity. It is not clear how the findings of the authors, BMK-1 provides outward force but is normally inhibited by ZYG-8, align with the last sentence saying "following zyg-8 depletion, BMK-1 could be hyperactive slowing anaphase B spindle elongation", should it not increase elongation according to the authors observations?

    Significance

    In this manuscript Czajkowski et al explore the role of the doublecortin-family kinase ZYG-8 during meiosis in C. elegans Oocytes. The authors conclude that BMK-1 generates outward directed force, redundant to forces generated by KLP-18, and that ZYG-8 inhibits BMK-1. The authors conclude that ZYG-8's kinase activity is required for the function of ZYG-8 in meiosis and mitosis. This research is interesting and provides some novel insight into the role of ZYG-8. Inm particular the observed spindle elongation and subsequent spindle fragmentation are novel and had not yet been reported. Also, the observation that degradation of ZYG-8 in monopolar klp-18(RNAi) spindles can restore bipolarity is novel and interesting, as well as the observation that this is somewhat dependent on the presence of BMK-1. This will be of interest to a broad audience and provides some new insight into the role of importance of ZYG-8 and BMK-1. The limitation of the study is the interpretation of the results and the lack of solid evidence that the observed phenotypes are due to ZYG-8 regulation motor activity, as the title claims. To support this some more experiments would be required. In addition, ZYG-8 has been reported to affect microtubule dynamics, which can certainly affect the action of motors on microtubules. This line of research is not explored in the paper but would certainly add to its value.

    Field of expertise: Research in cell division

  5. Version published to 10.1101/2023.11.22.568242v1 on bioRxiv