Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B

  1. Manuel Lera-Ramirez
  2. François J Nédélec
  3. Phong T Tran

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    Evaluation Summary:

    This study carefully quantifies microtubule dynamics during anaphase in the fission yeast S. pombe. The high quality data revealed two new observations: that microtubule rescue occurs preferentially at the edge of the midzone and that microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone in late anaphase. This sheds new light on the interplay between the nuclear membrane and the midspindle in closed mitosis, and the study will be of interest to cell biologists studying spindle dynamics and mitosis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeast S. pombe to measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that in S. pombe , which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

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  1. ASAPbio crowd review

    Author response

    • A comment on the overall organization of the paper. Figure 2 has a major location in the paper, but it seems that its main takeaway is that these MAPs aren't really involved in the main process this paper is probing. While these are important findings, it might be more satisfying to move some of the central results earlier.

    We agree that this figure displays mostly negative results. However, most work on anaphase B microtubule dynamics from our group and others has focused on the effect that motors and MAPs may have on microtubule dynamics (EB1 and kinesin-8 in budding yeast, klp9 in fission yeast). Therefore, we consider it is important to clearly show that previously proposed candidates are not required for the observed decrease in microtubule growth speed, prior to introducing the unexpected effect of the membrane.

    *A model schematic might drive home the main finding of the paper, and be particularly useful for readers who are not experts in microtubule or spindle dynamics. That said, the Discussion does an excellent job of summarizing the findings and explaining the takeaway message(s), even for the non-expert.

    We have added a model schematic and we have referred to it in the main text.

    Specific comments

    • ‘In higher eukaryotes’ - Suggest avoiding the terms higher and lower when describing organisms, and instead, directly defining which organisms, for instance in animals/metazoans that would be a better description.

    We have removed this terminology.

    • Figure 1 E-F - It is hard to see the difference in the distribution, maybe a different color could be used instead of stars.

    We have used a different color.

    • Figure 1 Data shown in pink in G comes from 832 midzone length measurements during anaphase, from 60 cells in 10 independent experiments - The pink here does not correspond to the pink coding in D, consider colour choice for clarity across panels.

    We have changed this.

    • Finally, yeasts undergo closed mitosis - How does this relate to the findings in the Dey paper (cited here) which shows it was somewhat semi-closed or semi-open. According to the Dey paper, the membrane disassembles locally twice, at the SPB and the bridge.

    Membrane disassembly at the nuclear membrane bridge occurs at late anaphase, and leads to the disassembly of the spindle, presumably by the action of cytoplasmic factors (Dey et al. 2020). We do not believe the membrane disassembly itself has a role in spindle elongation or microtubule dynamics, as when it happens the spindle is then disassembled. However, the fact that les1D reduces the decrease in microtubule growth speed associated with internalisation of microtubules in the nuclear membrane bridge suggest that the organisation of the nuclear membrane bridge required for its local disassembly at late anaphase might affect microtubule growth (see section “Formation of Les1 stalks […]”).

    • ‘vertical comets in kymographs (Fig. 1C) do not correspond to non-growing microtubules, but rather microtubules that grow at a speed matching the sliding speed’- For clarity, it might be nice to add: "(as the SPB moves away from the plus end in the kymograph)".

    We have included this useful clarification.

    • ‘significantly shorter than in interphase, where growth events last more than 120 seconds on average [42, 43]. Microtubule shrinking speed did not change during anaphase either (Fig. 1-Supplement 1D), and was on average 3.56±1.75 μm/min, also lower than in interphase (~8 min/μm)’ - This comment concerns the comparison of growth and shrinking rate as well as growth duration. The authors did not measure microtubule dynamics in interphase in this manuscript but compared their numbers to literature values. The comparison raises some questions for three reasons: 1) the microscopy method used is different in this paper and the two references provided, 2) the sample is mounted differently compared to the two references provided - 1) and 2) combined could lead to different levels of stress on the cells which could affect MT dynamics-, 3) (probably the most important caveat) the experiments are done at different temperatures: 27C in this paper versus 25C in the references provided. Microtubule dynamics are sensitive to temperature so this could explain part of the differences observed. Also, there are multiple values published for MT dynamics in interphase depending on the strain used and the microscopy method used. Suggest that the authors measure microtubule dynamics in interphase cells at 27C in SIM to ensure that the differences are not due to the technical parameters employed. Small item - should ‘8 min/μm’ read “8 μm/min"?

    We have measured microtubule growth speed and growth event duration using GFP-Mal3 during interphase and anaphase B in the same conditions as proposed (see Figure 1 – Supplement 2). Unfortunately, shrinkage speed cannot be measured using GFP-Mal3, so we cannot confirm that the difference between our measurements and the literature values would be observed.

    • ‘we observed two populations of microtubules (fast and slow growing)’ - Does this statement about thistle fast and slow growing populations refer to the data in Fig. 1C and 2A?

    Yes, we have added reference to this figures in the next sentence (mentioned below).

    • ‘In some cells, all microtubules seemed to switch to the slow growing phase simultaneously (Fig. 1C), while in others fast and slow growing microtubules co-existed (Fig. 2A)’ - This is a very interesting observation, could we know how many cells (%) were detected in each case? Is it that in 90% of the cells the switch is simultaneous, and hence the microtubule growth is somehow synchronized? Or is it more random, e.g. around 50%?

    This was just to point the reader to two kymographs and show that a clear point where all microtubules change speed is not present in all kymographs, as one may think from Fig. 1C. Later in the paper, we show that the change in growth depends on whether the microtubule rescue occurs inside or outside the nuclear membrane bridge, so it is a matter of where microtubules are rescued once the dumbbell transition occurs, which is a stochastic process. We have added another sentence pointing the reader to examples in the kymograph (see line 152, This representation captures…).

    • ‘On such a plot, the data points visibly cluster in two separate clouds and the variation of growth speeds can be fitted by an error function (Fig. 1F)’ - It is unclear that there are two distinct clusters, maybe the assertion should be toned down, or some sort of cluster analysis provided.

    We acknowledge that the data is widely spread across the y axis, and given that the magnitude “distance to the closest pole at rescue” is continuous the transition is not a clear cut. However, we consider the fact that the averaged curve closely matches the error function fit to be sufficient evidence for the existence of two populations of microtubule growth. Additionally, R2 of the fit is ~0.5 indicating that half of the variance is explained by this model. In any case, we show later that these two populations do exist (Fig. 3D), and why plotting microtubule growth against distance to the closest pole at rescue is a good way to segregate them (Fig. 3E).

    • ‘speed of interphase microtubules (~2.3 μm/min)’ - It would be interesting to see the dynamics in a les1 mutant (Dey Nature 2020) paper. Just as a control for presence/absence of the bridge?

    We thank the reviewers for kindly suggesting this interesting experiment. We have included it after the ase1 section. Les1 forms stalks at the edges of the nuclear membrane bridge that restrict nuclear membrane disassembly to the center of the bridge at the end of mitosis (Dey at al. 2020). While les1 deletion does not prevent the formation of the nuclear membrane bridge, it has been proposed that Les1 stalks may constitute sites of close interaction between the nuclear membrane and the spindle. Therefore, these sites may influence microtuble growth. Indeed, we have found that removing these Les1 stalks by either deleting les1 or nem1 leads to a smaller decrease in microtubule growth speed when plus ends enter the nuclear membrane bridge (see section “Formation of Les1 stalks […]”)

    *‘Figure 2, Transition from fast to slow microtubule growth occurs in the absence of known anaphase MAPs’ - It looks like the overlap zone is larger on the mal3 kymograph. Is the size of the midzone changed in some of the mutants? It could be important to report. Related to it, is the spindle length changed in some of the mutants? (It does not look like it from the kymographs displayed).

    The midzone is indeed longer in mal3D strains, now this can be seen in Fig. 2 – Supp. 2 and it is mentioned in the main text in line 272. As for the spindle length, diverse kinds of alterations in spindle length have been previously reported for the mutants that we used in this study. For instance, ase1D /cls1off cells have shorter spindles at anaphase onset (Loiodice et al. 2005 and data not shown), and klp5Dklp6D have longer spindles at anaphase onset (Syrivatkina et al. 2013). klp9D / clp1D / dis1D cells have lower spindle elongation velocity and may not reach the wild-type spindle length by the end of anaphase (Kruger et al. 2019). Despite these differences, the decrease in microtubule growth as a function of distance to the closest pole has a similar tendency across conditions, suggesting that the mentioned differences in spindle length are unlikely to have an important effect.

    • Additionally, adding the data about rescue localization in the mutant (equivalent of Fig 1 G) would be interesting to better describe the role of these different proteins. Figure 2, Panel G to L - Could the authors indicate the value for the average +/- error in each bin for the WT and the mutants? Also, it is hard to say from the plots, but it looks like the WT average speed in the first bin is different in every panel, that would be good to know to have an idea of the reproducibility/variability.

    We have added a figure with the rescue distribution (see Fig. 2 – Supp. 2). This apparent difference in the wt speed in different experiments might have come from looking at normalised data. The new way of representing the data in fig. 2H and J shows that the microtubule growth velocity in the wild-type is very consistent across experiments. We have added a table with microtubule growth velocity values (Table 1), and the source data is available.

    • The dots making up the "thick lines" are centered on 1.5/2.5/etc.. in some panels (G and K) and centered on 1/2/3/etc.. the others (I,J,L). Could the authors provide some clarification?

    We have fixed this inconsistency across the paper.

    • Figure 3 - Can the authors indicate the average values +/- error for each of the distributions in Fig. 3D? Maybe on the plot itself, in the legend or as a table. This would make them easily available without having to infer them from the Y axis. This comment is also valid for Fig 4I and 4J.

    We have added tables with average values and confidence intervals in the appendix.

    • Figure 3E ‘Distance from the plus-end to the nuclear membrane bridge edge at rescue as a function of distance from the plus-end to the closest pole at rescue’ - The Y axis reads as "distance to the bridge edge" but it shows negative values, could this be "position to the bridge edge" instead? (same item throughout the text).

    We have fixed this.

    • Figure 3 ‘Number of events: 442 (30 cells) wt, 260 (27 cells) klp9OE, 401 (35 cells) cdc25-22, from 3 independent experiments’ - P values this small raise a concern. Presumably the number of degrees of freedom in the regression analysis should not exceed the number of independent experiments. Instead, the DoF listed under "error" in the analysis output is hundreds or thousands instead of 3. To address this, the regression analysis should use either the "Error" function in R or a linear mixed-effects model to account for the nesting of the repeated measurements within each independent experiment. Alternatively, it is also possible to just calculate summary means for each independent experiment, and calculate p values based on that N=3. See: Lazic. Experimental Design for Laboratory Biologists. p. 157. and the supplemental file of: https://doi.org/10.1371/journal.pbio.2005282 and the additional file 1 of: https://doi.org/10.1186/s12868-015-0228-5 and this for an alternative plotting approach: https://doi.org/10.1083/jcb.202001064 Recommend either recalculating the p values by one of the methods above or removing the reported p values from the paper. The large effects observed in many cases are self-evident without a significance metric, so eliminating the p values would be acceptable here. (This comment applies to other figures through the paper that report p values based on number of cells or number of measurements instead of number of independent samples/experiments.)

    We thank the reviewers for suggesting the improvements to the statistical analysis, as well as for pointing us to useful resources that described the statistical methods and their implementation in detail. We have followed Aarts et al. 2015 and used a linear mixed effects model (see Methods>Statistical Analysis)

    Due to the change in statistical analysis method, to show that some of the differences we had reported previously were significant, we included more cells in the analysis from our existing data. We did this for klp5Dklp6D kymographs (Fig. 2I and Fig.2 – Supp. 1). Spindle dynamics in ase1D (Fig. 5D and Fig. 5 – Supp. 1) and klp9D (Fig. 2 – Supp. 3 A, C). Cell length (Fig. 3 – Supp. 1A).

    For the same reason, we measured anaphase spindle elongation velocity (Fig. 3 – Supp. 1C) from kymographs instead of measuring them from the 1 minute interval movies that we had used previously (from Fig. 3 – Supp 1B). We have reflected this in the methods (see added text in line 800 and deleted text in line 809 in the document with changes highlighted).

    None of these changes has altered our conclusions.

    • Figure 4 - Nice experiment. It brings the question of how cell-shape affects all these dynamics (probably out of the scope of this work). But a for3 mutant for example?

    This is an interesting suggestion, to be tested in the future. Furthermore, we believe that nuclear shape should also have an important effect, since the spindle is confined inside the nuclear membrane. We would expect that mutants that perturb nuclear shape might have effects on microtubule growth. We have observed that the decrease in growth speed associated with internalisation of microtubules in the nuclear membrane bridge is reduced upon nem1 deletion, which increases nuclear membrane surface, and produces membrane ruffling (Fig. 4-Supplement 2). However, nem1 deletion also removes les1 stalks from the nuclear bridge (Dey et al. 2020). It would be interesting to find a perturbation of the nuclear membrane that does not remove the les1 stalks.

    • ‘Ase1 is required for microtubule growth speed to decrease during anaphase B, this is unlikely to be a direct effect’ - If it is unlikely to be a direct Ase1 effect is the title of the section accurate? "Ase1 is required for normal rescue distribution and for microtubule growth speed to decrease in anaphase B"

    Ase1 recruits multiple proteins to the spindle midzone, so the fact that ase1 deletion produces a given phenotype does not necessarily mean that this phenotype results from the absence of Ase1 protein activity. For instance, deleting ase1 perturbs rescue distribution, but it does not mean that Ase1 acts as a rescue factor itself, or at least to a relevant extent, given that deletion of cls1 completely prevents rescue, but ase1 deletion does not. In the discussion we propose some indirect effects of ase1 deletion that may produce this effect. In any case, upon more careful analysis we have found that ase1 deletion does not prevent the decrease in microtubule growth speed during anaphase B, but rather makes it smaller (see section “The decrease in growth speed associated with internalisation of microtubules in the nuclear membrane bridge is reduced upon ase1 deletion”).

    • Figure 5 - What about an ase1 lem1 double mutant?

    We suppose that the intended gene is les1. We have studied the effects of les1 deletion in the new version of the manuscript. However, we do not see the information we would obtain from a double deletion ase1D les1D.

    • ‘In summary, Ase1 is required for rescue organisation and for microtubule growth speed to decrease during anaphase B ‘- In this context it could make sense to discuss the observations from this paper (doi:10.1371/journal.pone.0056808) about the role of Ase1 ortholog's MAP65-1 in coordinating MT dynamics within bundles.

    In the mentioned paper, the authors showed that the presence of PRC1 (ase1 orthologue) in bundles increases microtubule rescue rate, and that it slightly reduces microtubule growth speed.

    We observe a small increase in microtubule growth speed throughout anaphase upon ase1 deletion (Fig. 5), which is consistent with the in vitro observation that PRC1 decreases microtubule growth. However, once more this might not be a direct effect of Ase1, since less Cls1 is recruited if ase1 is deleted, and Cls1 reduces microtubule growth speed (Fig. 2). In addition, this can also be a result of higher concentration of tubulin / MAPs resulting from less polymerised tubulin in ase1 deleted cells, which have less spindle microtubules on average.

    Regarding the increase in rescue rate produced by PRC1 in vitro, it is possible that Ase1 contributes to microtubule rescue in the spindle. However, given that no rescues occur upon inactivation of cls1 (Bratman et al. 2007), we believe Cls1 is the dominant factor, and Ase1 contribution is likely negligible.

    • ‘We initially set the microtubule growth velocity to 1.6 μm/min (early anaphase speed, Fig. 1F), and aimed to reproduce the experimental distribution of positions of rescue and catastrophe at early anaphase (spindle length < 6 μm’ - Kudos to the authors for detailing the model and its parameters in a way that even non-modelling experts can understand.

    Discussion - ‘Our data suggests that microtubule growth speed is mainly governed by spatial cues’ - Is it right to assume that in the cases where fast and slow growing microtubules were simultaneously observed, the fast microtubules were not/had not yet reached the midzone?

    Our data suggests that it’s not about being inside the midzone, but rather inside the nuclear membrane bridge formed after the dumbbell transition. We have elaborated more on this in the main text, pointing the reader to examples in the kymograph, and giving a quantitative argument for distance to the closest pole being a better predictor than anaphase progression or position with respect to the center (which is equivalent to distance to the midzone), see line 152.

    • Methods - ‘PIFOC module (perfect image focus), and sCMOS camera’ - Is this Nikon's "Perfect Focus" autofocus, or some other manufacturer's system? And back-thinned sCMOS.

    We have clarified this in the Methods section.

  2. Version published to 10.7554/elife.72630 on eLife
  3. eLife

    Evaluation Summary:

    This study carefully quantifies microtubule dynamics during anaphase in the fission yeast S. pombe. The high quality data revealed two new observations: that microtubule rescue occurs preferentially at the edge of the midzone and that microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone in late anaphase. This sheds new light on the interplay between the nuclear membrane and the midspindle in closed mitosis, and the study will be of interest to cell biologists studying spindle dynamics and mitosis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The authors investigated spindle growth dynamics during anaphase B in S. pombe, a unicellular eukaryote which undergoes closed mitosis. In order to accurately quantify spindle growth speeds, the authors tagged Alp7, a protein that localizes to plus ends of microtubules, with 3xGFP, and tracked its position in the dividing cell during mitosis, resulting in precise measurements of both the duration and velocity of microtubule growth events for the first time. By performing these experiments across a set of different genotypes (e.g. in strains with knock-outs of specific microtubule-associated proteins (MAPs)) the authors conclude that (1) microtubule rescues preferentially occur at the midzone edges, (2) microtubule growth speed decreases throughout anaphase B, independent of several known anaphase MAPs and (3) wrapping of the nuclear membrane around the spindle is responsible for this reduction in MT growth speed.

    The data in this study is of very high quality and the conclusions are largely well supported by the data, but some changes could be made in the interest of simplicity and clarity, and some additional experiments should ideally be performed to strengthen the third claim.

    Many changes occur in the nuclear bridge in late anaphase, including the disassembly of nuclear pore complexes and the fenestration of the nuclear envelope (Lucena et al. 2015, Exposito-Serrano et al. 2020, Dey et al. 2020). This opens up the possibility for a different interpretation of some of the authors' data - for example, that local alterations in the permeability barrier directly alter microtubule polymerisation dynamics, rather than the wrapping of the nuclear envelope in the bridge per se. This could help explain the ark1-as3 data, for example, in which (non-physiological) membrane tubes wrap around the spindle but local NEB is prevented (Dey et al. 2020).

    The authors state that 'transition from fast to slow microtubule growth occurs in the absence of known anaphase MAPs' (heading paragraph 3 (239-240) and Figure 2), yet two paragraphs later, they show that the MAP Ase1 does in fact impact this transition. This distinction might prove confusing for readers, especially those unfamiliar with the S. pombe spindle.
    The authors state that Ase1 is required for the decrease in growth speed during anaphase. While the example kymograph in Figure 5B looks convincing, there seem to be too few points at the right side of Figure 5F to properly see the two distributions. Without any statistics to compare wt to ase1∆, it is difficult to tell if the apparent absence of decrease is real. In addition, as the authors also show in Figure 1C-E, the spindle collapses, causing the final spindle length to be shorter than wt. It could be that the spindle collapses before the characteristic drop in growth rate could be observed.
    One of Ase1's direct interactors, Cls1, was shown to not have an impact on the transition of fast to slow microtubule growth (Figure 2). In Ase1's case, a full deletion of the gene was necessary to reveal loss of the transition. The ase1off strain with reduced Ase1 expression showed a similar transition to wt, similar to the cls1off strain. cls1 is an essential gene and cannot be deleted, but similar to its interactor, its phenotype might be hidden even at low expression values.

    Some observations, such as Figure 2G-L, lack associated statistics. In addition, since Alp7 tagged with 3xGFP is used throughout the paper, it seems important to test whether this tag influences microtubule dynamics.

  5. eLife

    Reviewer #2 (Public Review):

    Lera-Ramirez et al. investigated how interpolar microtubules are regulated during anaphase B to support sustained spindle elongation. This is a perplexing question, since spindle elongation is known to require sliding between anti-parallel microtubules in the spindle midzone, and these microtubules must therefore grow to maintain the midzone as the spindle poles move further and further apart. Whereas previous studies have identified proteins that are necessary for spindle elongation and midzone function, this study focuses on understanding the changes in midzone microtubule dynamics and how those changes impact spindle stability during elongation. They report two major findings. First they use a clever combination of high-resolution imaging of living yeast cells and with genetic manipulation of spindle and nuclear membrane dynamics to show that the decrease in microtubule polymerization rate when the spindle reaches long lengths requires the formation of a compact nuclear membrane bridge that surrounds the midzone microtubules. Second, they combine their live-cell imaging approach with computational simulations to establish that the promotion of microtubule rescues near the midzone edges prevents spindle collapse during elongation. Overall, this is a well-written manuscript that advances our understanding of anaphase spindle elongation and will likely spur future research to extend the findings.

    Strengths:

    The results supporting the two major conclusions are generally clear and convincing. Although the study does not define the molecular mechanisms that control polymerization decrease and rescue at the midzone edges (more on that below), the major findings are nevertheless important. A key strength of the study is the use of structured illumination microscopy to measure the dynamics of microtubule ends in the midzone during spindle elongation. Whereas previous studies used FRAP experiments to infer microtubule dynamics, the direct approach used here enables a new level of insight. This method, combined with approaches to prevent bridge formation by either inhibiting Aurora B or inhibiting membrane biosynthesis with cerulenin, lead to the proposed model that microtubule polymerization slows when the nuclear membrane bridge surrounds the midzone in late anaphase. The resulting model that the "spatial cue" of the encroaching nuclear membrane drives the slowing of microtubule polymerization is an exciting idea and may connect to recent studies from the Gatlin and Rodinov labs on how cytoplasmic volume impacts microtubules dynamics.

    Another strength is the use of computational simulations to complement the experimental measurements and explore the impact of rescue enrichment on spindle stability. This is important, because the authors show that ase1∆ mutants severely alter rescue frequency in the midzone, but the computational approach allows the authors to explore how a range of perturbations to rescue frequency and position impact the larger process of spindle stability. This is a nice integration of experiment and computation.

    Weaknesses:

    1. The changes in microtubule growth rate and regulation of rescues at microtubule ends are dealt with as separate processes, leaving the reader to wonder how they might be related. For example, the experiments in Figure 2 examine polymerization rate in the context of a variety of mutant or overexpression conditions that alter known microtubule regulators in the midzone. These results are largely negative with respect to polymerization, but what about rescue? Since the mechanism for regulating rescue at the midzone edges is unresolved, this data set seems like a valuable resource for identifying potential regulators.
      Similarly, the authors state that "we also observed that in ase1Δ cells microtubule growth velocity no longer decreased during anaphase B (Fig. 5F, Fig. 5-Supplement 1B)." Given the previous figures, and the model invoking the nuclear membrane, it seems important to examine whether Ase1 has a role in the formation or function of the nuclear membrane bridge. An alternative possibility is that ase1∆ spindles do not reach long enough lengths to pull the nuclear membrane into the bridge.

    2. Throughout the manuscript, the authors analyze microtubule dynamics by labeling ends with Alp7-3GFP and Sid4-GFP. Since only the ends are labeled, the authors must make some assumptions about which spindle pole each plus end is connected to. This is essential for determining whether a feature in their kymographs represents a growing microtubule from the distal pole or a shrinking microtubule from the proximal pole. Which criteria were used could be more clearly stated.

    3. Recent papers from the Kapoor and Moore labs have investigated the role of Ase1/PRC1 in controlling anaphase spindle elongation and the regulation of midzone microtubule dynamics. These papers are relevant to the discussion here and should be included.
      The authors state that "... depletion of PRC1/Ase1 does not perturb anaphase spindle elongation either [4], suggesting that if microtubule rescue organisation is required for spindle stability, it may rely on a mechanism other than recruitment of CLASP by PRC1." This statement contradicts the study from the Kapoor lab (PMID: 31248912), which showed that cells depleted for PRC1 do exhibit faster anaphase spindle elongation. Given the contradictory results from the Tolic and Kapoor labs, whether PRC1 depletion perturbs anaphase spindle elongation in mammalian cells may be an open and somewhat complicated question.
      The paper from the Moore lab (PMID: 32997572) shows that Ase1 in budding yeast recruits the EB homologue Bim1 to the midzone and this stabilizes microtubule dynamics during spindle elongation. Mutants that disrupt this recruitment show phenotypes that are reminiscent of those reported here for ase1∆ mutants in fission yeast. A preprint from the Surrey lab indicates that a similar recruitment mechanism may exist in mammalian cells (https://doi.org/10.1101/2020.07.09.195347). This model involving Ase1/PRC1 and Bim1/EB seems very relevant for the discussion.

  6. eLife

    Reviewer #3 (Public Review):

    The authors report that during mitosis, the spindle midzone keeps a constant length throughout anaphase by promoting microtubule rescues at midzone edges. Interestingly, they also observed that wrapping of the nuclear membrane around the spindle midzone reduces microtubule growth speed in an Ase1/PRC1 dependent manner.

    The strengths of this manuscript lie in the quality of the experiments carried out, not only from the technological point of view, but also from the quality of their interpretation. The mechanisms of coordination of microtubule sliding and growth events at the interdigitated zone are still poorly understood and this study brings us a better understanding of the key actors involved in these processes. The finding that the wrapping of the nuclear membrane around the spindle midzone reduces the rate of microtubule growth is in line with recent studies showing an interplay between the nuclear membrane and the central spindle. The authors suggest that such a cross-talk between the nuclear membrane and the central spindle might affect microtubule dynamics.

    The weaknesses of the manuscript are mainly the lack of a clear mechanism to explain how the nuclear membrane around the spindle midzone reduces microtubule growth speed. Furthermore, it is not clear from the manuscript that this reduction in microtubule growth speed at anaphase B is an essential/important event for cell division and not just phenomenological. So, at this stage, the manuscript is very descriptive.

  7. ASAPbio crowd review

    This review reflects comments and contributions by Ricardo Carvalho, Omaya Dudin, Sónia Gomes Pereira, Samuel Lord, and Arthur Molines

    The work by Lera-Ramirez_et al._ sheds light on the growth and sliding dynamics of microtubules during mitosis in pombe. The kymographs are beautiful and the figures are well laid-out. The data is generally convincing.

    A comment on the overall organization of the paper. Figure 2 has a major location in the paper, but it seems that its main takeaway is that these MAPs aren't really involved in the main process this paper is probing. While these are important findings, it might be more satisfying to move some of the central results earlier.

    A model schematic might drive home the main finding of the paper, and be particularly useful for readers who are not experts in microtubule or spindle dynamics. That said, the Discussion does an excellent job of summarizing the findings and explaining the takeaway message(s), even for the non-expert.

    Specific comments

    ‘In higher eukaryotes’ - Suggest avoiding the terms higher and lower when describing organisms, and instead, directly defining which organisms, for instance in animals/metazoans that would be a better description.

    Figure 1 E-F - It is hard to see the difference in the distribution, maybe a different color could be used instead of stars.

    Figure 1 - Data shown in pink in G comes from 832 midzone length measurements during anaphase, from 60 cells in 10 independent experiments - The pink here does not correspond to the pink coding in D, consider colour choice for clarity across panels.

    ‘Finally, yeasts undergo closed mitosis’ - How does this relate to the findings in the Dey paper (cited here) which shows it was somewhat semi-closed or semi-open. According to the Dey paper, the membrane disassembles locally twice, at the SPB and the bridge.

    Figure 1C - vertical comets in kymographs (Fig. 1C) do not correspond to non-growing microtubules, but rather microtubules that grow at a speed matching the sliding speed’ - For clarity, it might be nice to add: "(as the SPB moves away from the plus end in the kymograph)".

    ‘significantly shorter than in interphase, where growth events last more than 120 seconds on average [42,43]. Microtubule shrinking speed did not change during anaphase either (Fig. 1-Supplement 1D), and was on average 3.56±1.75 μm/min, also lower than in interphase (~8 min/μm)’ - This comment concerns the comparison of growth and shrinking rate as well as growth duration. The authors did not measure microtubule dynamics in interphase in this manuscript but compared their numbers to literature values. The comparison raises some questions for three reasons: 1) the microscopy method used is different in this paper and the two references provided, 2) the sample is mounted differently compared to the two references provided - 1) and 2) combined could lead to different levels of stress on the cells which could affect MT dynamics-, 3) (probably the most important caveat) the experiments are done at different temperatures: 27C in this paper versus 25C in the references provided. Microtubule dynamics are sensitive to temperature so this could explain part of the differences observed. Also, there are multiple values published for MT dynamics in interphase depending on the strain used and the microscopy method used. Suggest that the authors measure microtubule dynamics in interphase cells at 27C in SIM to ensure that the differences are not due to the technical parameters employed.

    Small item - should ‘8 min/μm’ read “8 μm/min"?

    ‘we observed two populations of microtubules (fast and slow growing)’ - Does this statement about thistle fast and slow growing populations refer to the data in Fig. 1C and 2A?

    ‘In some cells, all microtubules seemed to switch to the slow growing phase simultaneously (Fig. 1C), while in others fast and slow growing microtubules co-existed (Fig. 2A)’ - This is a very interesting observation, could we know how many cells (%) were detected in each case? Is it that in 90% of the cells the switch is simultaneous, and hence the microtubule growth is somehow synchronized? Or is it more random, e.g. around 50%?

    ‘On such a plot, the data points visibly cluster in two separate clouds and the variation of growth speeds can be fitted by an error function (Fig. 1F)’ - It is unclear that there are two distinct clusters, maybe the assertion should be toned down, or some sort of cluster analysis provided.

    ‘speed of interphase microtubules (~2.3 μm/min)’ - It would be interesting to see the dynamics in a les1 mutant (Dey Nature 2020) paper. Just as a control for presence/absence of the bridge?

    ‘Figure 2, Transition from fast to slow microtubule growth occurs in the absence of known anaphase MAPs’ - It looks like the overlap zone is larger on the mal3 kymograph. Is the size of the midzone changed in some of the mutants? It could be important to report. Related to it, is the spindle length changed in some of the mutants? (It does not look like it from the kymographs displayed).

    Additionally, adding the data about rescue localization in the mutant (equivalent of Fig 1 G) would be interesting to better describe the role of these different proteins.

    Figure 2, Panel G to L - Could the authors indicate the value for the average +/- error in each bin for the WT and the mutants? Also, it is hard to say from the plots, but it looks like the WT average speed in the first bin is different in every panel, that would be good to know to have an idea of the reproducibility/variability.

    The dots making up the "thick lines" are centered on 1.5/2.5/etc.. in some panels (G and K) and centered on 1/2/3/etc.. the others (I,J,L). Could the authors provide some clarification?

    Figure 3 - Can the authors indicate the average values +/- error for each of the distributions in Fig. 3D? Maybe on the plot itself, in the legend or as a table. This would make them easily available without having to infer them from the Y axis. This comment is also valid for Fig 4I and 4J.

    Figure 3E ‘Distance from the plus-end to the nuclear membrane bridge edge at rescue as a function of distance from the plus-end to the closest pole at rescue’ - The Y axis reads as "distance to the bridge edge" but it shows negative values, could this be "position to the bridge edge" instead? (same item throughout the text).

    Figure 3 ‘Number of events: 442 (30 cells) wt, 260 (27 cells) klp9OE, 401 (35 cells) cdc25-22, from 3 independent experiments’ - P values this small raise a concern.

    Presumably the number of degrees of freedom in the regression analysis should not exceed the number of independent experiments. Instead, the DoF listed under "error" in the analysis output is hundreds or thousands instead of 3. To address this, the regression analysis should use either the "Error" function in R or a linear mixed-effects model to account for the nesting of the repeated measurements within each independent experiment. Alternatively, it is also possible to just calculate summary means for each independent experiment, and calculate p values based on that N=3.

    See: Lazic. Experimental Design for Laboratory Biologists. p. 157.

    and the supplemental file of:https://doi.org/10.1371/journal.pbio.2005282

    and the additional file 1 of:https://doi.org/10.1186/s12868-015-0228-5

    and this for an alternative plotting approach:https://doi.org/10.1083/jcb.202001064

    Recommend either recalculating the p values by one of the methods above or removing the reported p values from the paper. The large effects observed in many cases are self-evident without a significance metric, so eliminating the p values would be acceptable here.

    (This comment applies to other figures through the paper that report p values based on number of cells or number of measurements instead of number of independent samples/experiments.)

    Figure 4 - Nice experiment. It brings the question of how cell-shape affects all these dynamics (probably out of the scope of this work). But a for3 mutant for example?

    ‘Ase1 is required for microtubule growth speed to decrease during anaphase B, this is unlikely to be a direct effect’ - If it is unlikely to be a direct Ase1 effect is the title of the section accurate? "Ase1 is required for normal rescue distribution and for microtubule growth speed to decrease in anaphase B"

    Figure 5 - What about an ase1 lem1 double mutant?

    ‘In summary, Ase1 is required for rescue organisation and for microtubule growth speed to decrease during anaphase B’- In this context it could make sense to discuss the observations from this paper (doi:10.1371/journal.pone.0056808) about the role of Ase1 ortholog's MAP65-1 in coordinating MT dynamics within bundles.

    ‘We initially set the microtubule growth velocity to 1.6 μm/min (early anaphase speed, Fig. 1F), and aimed to reproduce the experimental distribution of positions of rescue and catastrophe at early anaphase (spindle length < 6 μm’ - Kudos to the authors for detailing the model and its parameters in a way that even non-modelling experts can understand.

    Discussion - ‘Our data suggests that microtubule growth speed is mainly governed by spatial cues’- Is it right to assume that in the cases where fast and slow growing microtubules were simultaneously observed, the fast microtubules were not/had not yet reached the midzone?

    Methods - ‘PIFOC module (perfect image focus), and sCMOS camera’ - Is this Nikon's "Perfect Focus" autofocus, or some other manufacturer's system? And back-thinned sCMOS.

  8. Version published to 10.1101/2021.08.06.455369v1 on bioRxiv