Chromokinesin Klp-19 regulates microtubule overlap and dynamics during anaphase in C. elegans
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Abstract
Recent studies have highlighted the significance of the spindle midzone, the region between the segregating chromosomes, in ensuring proper chromosome segregation. By combining 3D electron tomography, cutting-edge light microscopy and a novel single cell in vitro essay allowing single molecule tracking, we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans by the chromokinesin KLP-19, and its relevance for proper spindle function. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and KLP-19 in C. elegans , the homologs of PRC1 and KIF4a, suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length and thus microtubule-based interactions in the spindle midzone, which affects spindle dynamics and force transmission. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone.
Summary
KLP-19 controls microtubule length in the spindle midzone of C. elegans , affecting spindle dynamics and force transmission during mitosis.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This study by Zimyanin et al. examines the role of the C. elegans chromokinesin KLP-19 in the formation and architecture of the anaphase central spindle in C. elegans zygotes. Through a combination of electron and light microscopy, along with RNAi-mediated perturbations, the authors propose that KLP-19 influences central spindle stiffness by regulating microtubule dynamics.
In Figure 5, the effect of KLP-19 depletion on central spindle microtubules appears unconvincing. The FRAP results show no significant difference with or without KLP-19, and overall microtubule density does not …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This study by Zimyanin et al. examines the role of the C. elegans chromokinesin KLP-19 in the formation and architecture of the anaphase central spindle in C. elegans zygotes. Through a combination of electron and light microscopy, along with RNAi-mediated perturbations, the authors propose that KLP-19 influences central spindle stiffness by regulating microtubule dynamics.
In Figure 5, the effect of KLP-19 depletion on central spindle microtubules appears unconvincing. The FRAP results show no significant difference with or without KLP-19, and overall microtubule density does not consistently respond to its depletion. Additionally, the double klp-19; gpr-1/2 (RNAi) condition does not exhibit a strong increase in microtubule density, though a statistical test is missing for this condition. Furthermore, the spd-1; gpr-1/2 double depletion produces a similar increase in microtubule density to most klp-19 depletion conditions, suggesting that the effect cannot be solely attributed to the absence of KLP-19.
Figure 5A shows that depletion of KLP-19 leads to an increase in tubulin signal in the spindle midzone. The reviewer is correct, that there are differences in the microtubule density between KLP-19 depletion alone and KLP-19 + GPR-1/2 depletion. While depletion of KLP-19 alone leads to a significant increase, co-depletion of GPR-1/2 and KLP-19 leads to a slight, but not significant increase. Along this line, we have added Supplementary Table 1 that contains all p-Values for the different conditions for Figure 5A. However, depletion of GPR-1/2 alone does not affect the microtubule density in the midzone, arguing that changes in pulling forces do not affect the microtubule density in the midzone. It is possible, that the double RNAi leads to a decrease in efficiency and thus a reduced effect on microtubule intensity. We will demonstrate the RNAi efficiency by western blot. Another possibility is that there are some feedback mechanisms that responds to presence/ absence of pulling forces and some of our data (not from this manuscript) hints in this direction, but we have not yet worked out the details of this. We are planning to publish this in a follow up publication.
In response to the spd-1 + gpr-1/2 (RNAi), the reviewer is correct, that the microtubule density in the midzone is not significantly different from klp-19 (RNAi) conditions and we think it is interesting to note that spd-1 + gpr-1/2 (RNAi) leads to an increased microtubule density in the midzone. This could be, as above mentioned caused by some feedback mechanisms that responds to pulling forces, or also due to some functions of SPD-1 that affects microtubules in the midzone. Interestingly, our data also shows that metaphase spindles are significantly shorter in the absence of SPD-1 in comparison to spindles in control embryos, suggesting that SPD-1 plays a role in regulating microtubules or force transmission. We are currently working on understanding SPD-1's role in this process.
We also agree that there is no significant effect on the microtubule turn-over as shown in Figure 5B and we have stated this in the text. Our data does show a trend to a decreased turn-over, but the difference is not significant. This could be due to the low sample number.
Overall, we think our data, the light microscopy and even more so the EM data does show a clear effect on midzone microtubules.
The use of hcp-6 depletion to argue that KLP-19 depletion affects central spindle elongation independently of stretched chromatin is problematic. hcp-6 encodes a component of the Condensin II complex in C. elegans, and its depletion leads to chromatin decompaction rather than the stretched, dense chromatin observed in the midzone during anaphase in klp-19 (RNAi) embryos. These conditions are not equivalent and do not effectively rule out the possibility that chromatin stretching contributes to the observed phenotype.
We agree with the reviewer that the HCP-6 experiments do not entirely rule out effects from lagging chromosomes. Proving that the reduced spindle and chromosome separation is not due to lagging chromosomes is challenging. Most of the depletions that lead to lagging chromosomes are based on defective kinetochore microtubule connections, such as depletion of KNL-1, NDC-80 or CLS-2 (CLASP). In C. elegans, this leads to the mass of Chromosomes staying behind in anaphase and increased spindle pole separation, which is not comparable to KLP-19 depletion. Perturbations that do not affect kinetochore microtubules but still lead to lagging chromosomes are often targeting cohesin or condensin. Ultimately none of these conditions are directly comparable.
A probably better way to test this would be to deplete KLP-19 only after metaphase to prevent its effect on chromosome alignment. However, this is currently not possible as the time window is about 1 minute or less. We currently do not have the tools to conduct this type of experiment. As other reviewers also criticized this experiment and its significance for the paper, we have removed this entirely and have added the following part to the discussion about the potential effect of lagging chromosomes:
" *We can not unambiguously rule out that failure to properly align chromosomes and the resulting lagging chromosomal material could also lead to some of the observed effects on spindle dynamics, such as slow chromosome segregation and pole separation rates as well as preventing spindle rupture in absence of SPD-1. However, several observations argue in favor of KLP-19 actively changing the midzone cytoskeleton network and thus affecting spindle dynamics. *
Most of the protein depletions in C. elegans that lead to lagging chromosomes are based on defective kinetochore microtubule connections, such as depletion of CeCENP-A, CeCENP-C, KNL-1 or NDC-80 (70-72). This mostly leads to the Chromosome mass staying behind in anaphase and increased spindle pole separation (70-72), which is not comparable to KLP-19 depletion. Perturbations that do not affect kinetochore microtubules but still lead to lagging chromosomes are often targeting cohesin or condensin, which depletion leads to chromatin decompaction (73-74) rather than the stretched, dense chromatin as observed in the midzone during anaphase in klp-19 (RNAi) embryos. Ultimately none of these conditions are directly comparable, making it difficult to completely rule out an effect of lagging chromosomes. A better way to test this would be to deplete KLP-19 only after metaphase to prevent its effect on chromosome alignment. However, this is currently not possible as the time window is about 1 minute or less and we do not have the tools to conduct this type of experiment.
*Based on our results we hypothesize that the observed spindle dynamics in absence of KLP-19 are not only caused by lagging chromosomes. Instead, KLP-19 RNAi results in a global rearrangement of the spindle and leads to a significant reduction of the spindle size, microtubule overlap, growth rate, and stability. Furthermore, the increase of microtubule interactions after klp-19 (RNAi) could also contribute to lagging of chromosomes and exacerbation of fragmented extrachromosomal material." *
Additionally, the authors report that KLP-19 influences astral microtubule dynamics (Figure 5E), yet in Figure 3E, they show that KLP-19 localizes exclusively to kinetochores and spindle microtubules, excluding astral microtubules and spindle poles. How do they reconcile this apparent contradiction?
We think that KLP-19 localizes also to astral Microtubules. Our KLP-19 GFP CRISPR line is very dim and this makes it hard to see. We are proposing to use a TIRF approach to image KLP-19 GFP on the C. elegans cortex, which we will include in the revised version. In addition, in support of our hypothesis of KLP-19 binding to astral Microtubules as well we would like to note that there is a PhD thesis available from Jack Martin* in Josana Rodriguez Sanchez's Lab in Newcastle (LINK, will lead to a download of the thesis! ) that has reported KLP-19s localization to cortical Microtubules in C. elegans. In this thesis the author also reports an effect on astral microtubule growth.*
Figure legends lack consistency and do not adhere to standard C. elegans nomenclature conventions (e.g., protein names should not be capitalized, and genetic perturbations should be italicized). Standardizing these elements would improve clarity and readability.
We have checked our figure legend and to our best knowledge the legends adhere to the C. elegans nomenclature. All RNAi conditions are lower case italicized and Protein names are capitalized as it is standard in other C. elegans publications. We have however noticed some variation in our Figures, i.e. EB-2 instead of EBP-2 and have corrected this in all figures.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Zimyanin et al, Chromokinesin Klp-19 regulates microtubule overlap and dynamics during anaphase in C. elegans.
The authors used a myriad of techniques, including confocal live-cell imaging, 2-photon microscopy, second harmonic generation imaging, FRAP, microfluidic-coupled TIRF, EM-tomography, to study spindle midzone assembly dynamics in C. elegans one-cell stage embryos. In particular, they illuminated the role of kinesin-4 KLP-19 in maintaining proper midzone length and organization. Inhibition of KLP-19 results in longer more stable midzones, implying KLP-19 functions in depolymerizing microtubules.
Indeed, much of the results in the current study are consistent with previously published results elsewhere. Nevertheless, the current work represents a tour-de-force showcase of diverse and state-of-the-art technology application to address spindle assembly dynamics. How KLP-19 functions to define microtubule length at the midzone is still not known. But the current work, with diverse and solid data, serves to highlight where future work should focus.
Minor comments:
Fig 3E / There is an unusual diagonal line bisecting the embryo. Visually this does not affect viewing of the His::GFP and KLP-19::GFP signals. However, when these signals are quantified and normalized (as in Fig 3F), the diagonal bisect displaying different background signal may impact the measurements.
We are very sorry about this line in the images. The line is due to a defect in the camera chip of the spinning disc. We will acquire new images for this Figure using our new spinning disc microscope.
Fig 4B / While the kymographs clearly show KLP-19::GFP motility on microtubules, they also show that the majority of KLP(-::GFP do not move. Perhaps some quantification and discussion of this result is appropriate?
The reviewer is correct that only a small fraction small fraction of molecules, maybe ~10%, moves. We will add this quantification to the paper and discussion. This could be due to several reasons: Many of the non-moving particles are not visibly colocalized with microtubules, which could mean they are sticking non-specifically to the surface (or sticking to small tubulin aggregates that aren't long enough to support movement). In addition, as this experiment is done in a lysate it is hard to interpret if the immobile KLP-19 is not moving because other proteins are bound along the microtubule blocking its way or if the KLP-19 requires some activation (i.e. phosphorylations) to become mobiles. We think this could be very interesting and will follow up on this in the future.
Reviewer #2 (Significance (Required)):
Indeed, much of the results in the current study are consistent with previously published results elsewhere. Nevertheless, the current work represents a tour-de-force showcase of diverse and state-of-the-art technology application to address spindle assembly dynamics. How KLP-19 functions to define microtubule length at the midzone is still not known. But the current work, with diverse and solid data, serves to highlight where future work should focus.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary:
The anaphase spindle midzone is an essential structure for cell division. It consists of antiparallel overlapping microtubules organized by the antiparallel microtubule bundler PRC1, molecular motors and other regulatory proteins. This manuscript investigates the role of KLP-19 (C. elegans ortholog of human kinesin-4 KIF4A) and SPD-1 (C. elegans ortholog of PRC1) for spindle midzone organization in the C. elegans embryo and its relevance for proper spindle function. Advanced fluorescence microscopy, 3D electron tomography, and a fluorescence microscopy-based single molecule assay in embryo lysate are used in a unique combination. The authors confirm several aspects of PRC1 and KIF4A function in anaphase, as reported in previous work, mostly in human cells and Drosophila embryos and also in C. elegans embryos. Measurements are mostly very quantitative and to a high quality standard. The main difference to previous conclusions is that here, the authors propose that KLP-19 does not interact with SPD-1, in contrast to what has been established for other animal kinesin-4s and PRC1, and instead localizes to the spindle midzone independently of PRC1 by a mechanism that remains unknown. The authors provide evidence that KLP-19 nevertheless controls microtubule overlap length as in other species and that it produces outward forces sliding midzone microtubules apart a movement that SPD-1 counteracts (presumably by friction). The manuscript presents a rich resource of carefully measured quantitative structural and dynamic C. elegans anaphase spindle data.
Major comments:
Key conclusions convincing?
(1) The key conclusions that the length of the central anaphase spindle microtubule overlap remains constant as the C.elegans spindle elongates (Fig. 1), that PRC1 indeed localizes quite precisely to these overlaps as previously assumed based on its in vitro (purified protein) behavior (Fig. 3B) and that the kinesin-4 KLP-19 controls overlap length as in other species (Fig. 3B) are all convincingly shown. What's missing are quantitative KLP-19 together with microtubule polarity profiles in the presence and absence of SPD-1, leaving it unclear to which extent this kinesin localizes to microtubule overlaps in the two situations. Such data seem crucial, given the authors' claim that KLP-19 localizes to the midzone and that this localization of KLP-19 is mostly unaffected by the absence of SPD-1.
If we understand this correctly the reviewer is asking for second harmonic imaging (SHG) together with imaging of KLP-19 GFP. This is currently not possible due to the way this imaging must be done (2-photon of GFP-Tubulin followed by the SHG). The only thing we can do is provide KLP-19 GFP profiles for control and SPD-1 depleted embryos. We can also use the line co-expressing SPD-1 Halo-tag and KLP-19 GFP to plot their respective localizations in control conditions. We are happy to provide such plots. Generally, we see KLP-19 going to the midzone in absence of SPD-1 and the SHG data does show that the overlap is increased. If KLP-19 specifically localizes to microtubule overlap (rather to i.e. microtubule ends) can currently not be distinguished in the spindle midzone. In vitro data from other labs and our in vitro assay suggests that KLP-19 does not specifically bind to antiparallel overlaps but rather microtubules in general.
(2) 'Normalized KLP-19 intensities' are used to demonstrate that the total amount of this kinesin localizing to the spindle midzone does not depend on the presence of SPD-1 (Fig. 3F). Given that this claim represents a major novelty of the study, the efficiency of the SPD-1 knock-down should be documented, ideally by western blot and fluorescence microscopy.
We agree with the reviewer and will provide western blots.
(3) The authors show convincingly that the kinesin KLP-19 contributes to outward microtubule sliding (and can contribute to spindle rupture in the absence of SPD-1) (Fig. 2), which is interesting and in line with the author's main claim.
(4) The interaction between KIF4a and PRC1 is well established in other species and has been clearly demonstrated both in cells and in vitro (with purified proteins). The authors claim that this concept does not apply to the C. elegans orthologs. To show 'in vitro' (outside of the spindle) that the C. elegans homologs KLP-19 and SPD-1 do not interact, the authors use a novel microfluidic fluorescence-based single-molecule assay in lysate (Fig. 4). Although very original, these experiments do not reach the biochemical standard of previous experiments with purified proteins without appropriate controls. Given that the lysate setup is fairly novel, it's advisable to present at least one positive control demonstrating that interactions between soluble proteins can indeed be detected using this assay. It would also be useful to show the absence of interaction between KLP-19 and SPD-1 by a more conventional method like co-IP, again with a positive control, to support the authors' claim. Eventually, experiments with purified proteins will have to unequivocally demonstrate whether KLP-19 and SPD-1 indeed do not interact - something which appears, however, to be beyond the scope of this study. Without additional experimental proof, the authors may want to indicate that these results are of more preliminary nature.
*We agree with the reviewer, and we will conduct co-IPs of SPD-1 and KLP-19. We will also add CYK-4 as a positive control as previous publications have shown the interaction of CYK-4 with SPD-1. We are now generating lines co-expressing CYK-4 GFP and SPD-1 Halo-tag for the co-IP experiments. *
(5) Unfortunately, the authors do not propose an alternative mechanism for KLP-19 localization to the midzone in SPD-1 depleted embryos, limiting the conceptual advance. Does KLP-19 bind directly to antiparallel microtubules, for example in the assay presented in Fig. 4 (where signs of microtubule crosslinking are shown for SPD-1)? If not, how would it accumulate in the midzone (if it does) in the C. elegans embryo anaphase spindle? The authors do also not propose a mechanism explaining why central antiparallel microtubule overlap length does not change as the spindle elongates in anaphase. Moreover, there is no discussion regarding the potential mechanism leading to KLP-19 controlling microtubule dynamics globally instead of locally where the motor accumulates, indicating limitations in mechanistic insight.
*We agree with the reviewer and will add these points to the discussion. *
*To address some of the points: *
*How does KLP-19 end up in the midzone? : Our data shows that localization of KLP-19 does depend on AIR-2 and BUB-1 as previously reported. However, those proteins primarily affect the formation of the midzone. The in vitro assay does not suggest that KLP-19 has a preference for overlaps, unlike SPD-1, but rather binds microtubules in general. One possible mechanism of midzone localization could be microtubule end-tagging, as has been suggested for PRC1 (SPD-1 homolog). This could lead to an accumulation of KLP-19 in the midzone. *
Why does the central overlap stay constant? : This can be explained by constant microtubule growth at the plus-ends why maintaining the overlap length. Alternatively, this could be explained by some (sophisticated) rearrangements of microtubules that ensure the overlap length stays the same. Generally, this is a very interesting question, as each of this scenario still requires that the overlap length is tightly regulated. Our data suggests that this is correlated with microtubule length in the midzone, as KLP-19 depletion leads to longer microtubules and overlap. This suggests that the regulation of microtubule dynamics might be an important factor in this process. We will add this to the discussion.
Potential mechanism leading to KLP-19 controlling microtubule dynamics globally: We think that KLP-19 localizes to spindle and astral microtubules and regulates the dynamics on all of those, leading to a global regulation. By increasing it's concentration locally, microtubule dynamics can be regulated in the midzone. We will add data showing the localization of KLP-19 to astral microtubules.
Claims justified/preliminary and clearly presented?
The observation that the spindle length remains constant throughout anaphase in C. elegans is based on elegant, but unconventional fluorescence microscopy data (Fig. 1A & B). It would be helpful to add images of SHG and two-photon microscopy to help the reader understand the graphs. Measurements are presented based on distances between the poles. It is unclear why the distances between 15-20 µm were chosen and how they translate to anaphase progression. Can measurements be carried out across the entire duration of cell division to demonstrate that the overlap's 'constant length' property is unique to anaphase? (This could demonstrate already in Fig. 1 that the method in principle is capable of measuring different overlap lengths.)
We agree with the reviewer and have moved the SHG images from supplementary Fig. 6 to the main Figure 1A for better visibility. In addition, we have added a plot as an inset in (now) Figure 1B and C explanation of how the used spindle pole distances related to the progression through anaphase. Unfortunately, we can only acquire a single timepoint and not a live movie during the SHG.
Even though the manuscript contains an impressive amount of data, it appears to be lengthy, the motivation for several experiments is not clearly described, and the order of data presentation can probably be improved. For example, it is unclear why SPD-1 profiles are presented late and why KLP-19 profiles are missing - one would expect to see them early on as an essential characterization of the system under study. The motivation of the paragraph investigating the relation of KLP-19 and SPD-1 to HCP-6 is especially unclear (more than 1 page of text describing supplementary material).
We will go through our text again and will revise the order of presented experiments. As stated above, we have removed the HCP-6 data.
The absence of interaction between KLP-19 and SPD-1 is not demonstrated to the same quality standard as the presence of interaction between the orthologs in the literature, which should at least be mentioned.
Additional experiments essential to support the claims of the paper?
KLP-19 profiles in the presence and absence of SPD-1 seem to be essential.
We agree with the reviewer and will add this.
A co-IP of KLP-19 and SPD-1 (including positive control) to prove that the proteins are not interacting would help to support the claim.
We agree with the reviewer and will add this
Data and methods presented so that they can be reproduced? Yes.
Experiments adequately replicated and statistical analysis adequate? Yes.
Minor comments:
Generating cellular electron tomography data is very laborious. It is a pity that no raw data is provided; for example, a slice of a reconstructed tomogram or a video of whole volumes without segmentation would be an informative addition and allow assessment of the data quality.
We agree with the reviewer and will add movies of the raw electron microscopy data.
The clear evidence for direct interaction between PRC1 and kinesin-4 in other species should be correctly acknowledged throughout the text.
We agree with the reviewer and have corrected this
The average (mean or median?) values and STDs reported in the text do not appear to match those in Fig. 1D.
*We thank the reviewer for pointing this out and have corrected the figure. The violin lot showed the mean and percentiles, we have now changed the plot to show mean and STD. *
The kymograph of spd-1 RNAi in Fig. 2A seems stretched, and the size based on the scale bar does not fit the values stated in the text.
We thank the reviewer for pointing this out and have corrected the figure.
The figure numbering, as stated in the text, does not seem to agree with those in Supplementary Figure 8.
*We thank the reviewer for pointing this out and have corrected the text. *
Page numbers and/or line numbers and figure numbers on the figures would help the reader to navigate the manuscript more easily.
We agree with the reviewer and have added this.
Reviewer #3 (Significance (Required)):
The manuscript is a rich resource of quantitative measurements of C.elegans' structural and dynamic spindle properties, using advanced light microscopy and 3D electron microscopy imaging. In large parts, the work confirms previous conclusions of the function of PRC1 and kinesin-4 in the anaphase spindle, but also reports some interesting differences, namely that the C.elegans proteins differ from their orthologs in that they do not interact with each other, raising the question of how the kinesin-4 KLP-19 localizes to the central spindle in this organism. This work is of interest for researchers studying cell division, and specifically spindle architecture, dynamics, and function.
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Referee #3
Evidence, reproducibility and clarity
Summary:
The anaphase spindle midzone is an essential structure for cell division. It consists of antiparallel overlapping microtubules organized by the antiparallel microtubule bundler PRC1, molecular motors and other regulatory proteins. This manuscript investigates the role of KLP-19 (C. elegans ortholog of human kinesin-4 KIF4A) and SPD-1 (C. elegans ortholog of PRC1) for spindle midzone organization in the C. elegans embryo and its relevance for proper spindle function. Advanced fluorescence microscopy, 3D electron tomography, and a fluorescence microscopy-based single molecule assay in embryo lysate are used in a unique …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary:
The anaphase spindle midzone is an essential structure for cell division. It consists of antiparallel overlapping microtubules organized by the antiparallel microtubule bundler PRC1, molecular motors and other regulatory proteins. This manuscript investigates the role of KLP-19 (C. elegans ortholog of human kinesin-4 KIF4A) and SPD-1 (C. elegans ortholog of PRC1) for spindle midzone organization in the C. elegans embryo and its relevance for proper spindle function. Advanced fluorescence microscopy, 3D electron tomography, and a fluorescence microscopy-based single molecule assay in embryo lysate are used in a unique combination. The authors confirm several aspects of PRC1 and KIF4A function in anaphase, as reported in previous work, mostly in human cells and Drosophila embryos and also in C. elegans embryos. Measurements are mostly very quantitative and to a high quality standard. The main difference to previous conclusions is that here, the authors propose that KLP-19 does not interact with SPD-1, in contrast to what has been established for other animal kinesin-4s and PRC1, and instead localizes to the spindle midzone independently of PRC1 by a mechanism that remains unknown. The authors provide evidence that KLP-19 nevertheless controls microtubule overlap length as in other species and that it produces outward forces sliding midzone microtubules apart a movement that SPD-1 counteracts (presumably by friction). The manuscript presents a rich resource of carefully measured quantitative structural and dynamic C. elegans anaphase spindle data.
Major comments:
Key conclusions convincing?
- The key conclusions that the length of the central anaphase spindle microtubule overlap remains constant as the C.elegans spindle elongates (Fig. 1), that PRC1 indeed localizes quite precisely to these overlaps as previously assumed based on its in vitro (purified protein) behavior (Fig. 3B) and that the kinesin-4 KLP-19 controls overlap length as in other species (Fig. 3B) are all convincingly shown. What's missing are quantitative KLP-19 together with microtubule polarity profiles in the presence and absence of SPD-1, leaving it unclear to which extent this kinesin localizes to microtubule overlaps in the two situations. Such data seem crucial, given the authors' claim that KLP-19 localizes to the midzone and that this localization of KLP-19 is mostly unaffected by the absence of SPD-1.
- 'Normalized KLP-19 intensities' are used to demonstrate that the total amount of this kinesin localizing to the spindle midzone does not depend on the presence of SPD-1 (Fig. 3F). Given that this claim represents a major novelty of the study, the efficiency of the SPD-1 knock-down should be documented, ideally by western blot and fluorescence microscopy.
- The authors show convincingly that the kinesin KLP-19 contributes to outward microtubule sliding (and can contribute to spindle rupture in the absence of SPD-1) (Fig. 2), which is interesting and in line with the author's main claim.
- The interaction between KIF4a and PRC1 is well established in other species and has been clearly demonstrated both in cells and in vitro (with purified proteins). The authors claim that this concept does not apply to the C. elegans orthologs. To show 'in vitro' (outside of the spindle) that the C. elegans homologs KLP-19 and SPD-1 do not interact, the authors use a novel microfluidic fluorescence-based single-molecule assay in lysate (Fig. 4). Although very original, these experiments do not reach the biochemical standard of previous experiments with purified proteins without appropriate controls. Given that the lysate setup is fairly novel, it's advisable to present at least one positive control demonstrating that interactions between soluble proteins can indeed be detected using this assay. It would also be useful to show the absence of interaction between KLP-19 and SPD-1 by a more conventional method like co-IP, again with a positive control, to support the authors' claim. Eventually, experiments with purified proteins will have to unequivocally demonstrate whether KLP-19 and SPD-1 indeed do not interact - something which appears, however, to be beyond the scope of this study. Without additional experimental proof, the authors may want to indicate that these results are of more preliminary nature.
- Unfortunately, the authors do not propose an alternative mechanism for KLP-19 localization to the midzone in SPD-1 depleted embryos, limiting the conceptual advance. Does KLP-19 bind directly to antiparallel microtubules, for example in the assay presented in Fig. 4 (where signs of microtubule crosslinking are shown for SPD-1)? If not, how would it accumulate in the midzone (if it does) in the C. elegans embryo anaphase spindle? The authors do also not propose a mechanism explaining why central antiparallel microtubule overlap length does not change as the spindle elongates in anaphase. Moreover, there is no discussion regarding the potential mechanism leading to KLP-19 controlling microtubule dynamics globally instead of locally where the motor accumulates, indicating limitations in mechanistic insight.
Claims justified/preliminary and clearly presented?
The observation that the spindle length remains constant throughout anaphase in C. elegans is based on elegant, but unconventional fluorescence microscopy data (Fig. 1A & B). It would be helpful to add images of SHG and two-photon microscopy to help the reader understand the graphs. Measurements are presented based on distances between the poles. It is unclear why the distances between 15-20 µm were chosen and how they translate to anaphase progression. Can measurements be carried out across the entire duration of cell division to demonstrate that the overlap's 'constant length' property is unique to anaphase? (This could demonstrate already in Fig. 1 that the method in principle is capable of measuring different overlap lengths.)
Even though the manuscript contains an impressive amount of data, it appears to be lengthy, the motivation for several experiments is not clearly described, and the order of data presentation can probably be improved. For example, it is unclear why SPD-1 profiles are presented late and why KLP-19 profiles are missing - one would expect to see them early on as an essential characterization of the system under study. The motivation of the paragraph investigating the relation of KLP-19 and SPD-1 to HCP-6 is especially unclear (more than 1 page of text describing supplementary material).
The absence of interaction between KLP-19 and SPD-1 is not demonstrated to the same quality standard as the presence of interaction between the orthologs in the literature, which should at least be mentioned.
Additional experiments essential to support the claims of the paper?
KLP-19 profiles in the presence and absence of SPD-1 seem to be essential.
A co-IP of KLP-19 and SPD-1 (including positive control) to prove that the proteins are not interacting would help to support the claim.
Data and methods presented so that they can be reproduced? Yes.
Experiments adequately replicated and statistical analysis adequate? Yes.
Minor comments:
Generating cellular electron tomography data is very laborious. It is a pity that no raw data is provided; for example, a slice of a reconstructed tomogram or a video of whole volumes without segmentation would be an informative addition and allow assessment of the data quality.
The clear evidence for direct interaction between PRC1 and kinesin-4 in other species should be correctly acknowledged throughout the text.
The average (mean or median?) values and STDs reported in the text do not appear to match those in Fig. 1D.
The kymograph of spd-1 RNAi in Fig. 2A seems stretched, and the size based on the scale bar does not fit the values stated in the text.
The figure numbering, as stated in the text, does not seem to agree with those in Supplementary Figure 8.
Page numbers and/or line numbers and figure numbers on the figures would help the reader to navigate the manuscript more easily.
Significance
The manuscript is a rich resource of quantitative measurements of C.elegans' structural and dynamic spindle properties, using advanced light microscopy and 3D electron microscopy imaging. In large parts, the work confirms previous conclusions of the function of PRC1 and kinesin-4 in the anaphase spindle, but also reports some interesting differences, namely that the C.elegans proteins differ from their orthologs in that they do not interact with each other, raising the question of how the kinesin-4 KLP-19 localizes to the central spindle in this organism. This work is of interest for researchers studying cell division, and specifically spindle architecture, dynamics, and function.
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Referee #2
Evidence, reproducibility and clarity
Zimyanin et al, Chromokinesin Klp-19 regulates microtubule overlap and dynamics during anaphase in C. elegans.
The authors used a myriad of techniques, including confocal live-cell imaging, 2-photon microscopy, second harmonic generation imaging, FRAP, microfluidic-coupled TIRF, EM-tomography, to study spindle midzone assembly dynamics in C. elegans one-cell stage embryos. In particular, they illuminated the role of kinesin-4 KLP-19 in maintaining proper midzone length and organization. Inhibition of KLP-19 results in longer more stable midzones, implying KLP-19 functions in depolymerizing microtubules.
Indeed, much of the results …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Zimyanin et al, Chromokinesin Klp-19 regulates microtubule overlap and dynamics during anaphase in C. elegans.
The authors used a myriad of techniques, including confocal live-cell imaging, 2-photon microscopy, second harmonic generation imaging, FRAP, microfluidic-coupled TIRF, EM-tomography, to study spindle midzone assembly dynamics in C. elegans one-cell stage embryos. In particular, they illuminated the role of kinesin-4 KLP-19 in maintaining proper midzone length and organization. Inhibition of KLP-19 results in longer more stable midzones, implying KLP-19 functions in depolymerizing microtubules.
Indeed, much of the results in the current study are consistent with previously published results elsewhere. Nevertheless, the current work represents a tour-de-force showcase of diverse and state-of-the-art technology application to address spindle assembly dynamics. How KLP-19 functions to define microtubule length at the midzone is still not known. But the current work, with diverse and solid data, serves to highlight where future work should focus.
Minor comments:
Fig 3E / There is an unusual diagonal line bisecting the embryo. Visually this does not affect viewing of the His::GFP and KLP-19::GFP signals. However, when these signals are quantified and normalized (as in Fig 3F), the diagonal bisect displaying different background signal may impact the measurements.
Fig 4B / While the kymographs clearly show KLP-19::GFP motility on microtubules, they also show that the majority of KLP(-::GFP do not move. Perhaps some quantification and discussion of this result is appropriate?
Significance
Indeed, much of the results in the current study are consistent with previously published results elsewhere. Nevertheless, the current work represents a tour-de-force showcase of diverse and state-of-the-art technology application to address spindle assembly dynamics. How KLP-19 functions to define microtubule length at the midzone is still not known. But the current work, with diverse and solid data, serves to highlight where future work should focus.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This study by Zimyanin et al. examines the role of the C. elegans chromokinesin KLP-19 in the formation and architecture of the anaphase central spindle in C. elegans zygotes. Through a combination of electron and light microscopy, along with RNAi-mediated perturbations, the authors propose that KLP-19 influences central spindle stiffness by regulating microtubule dynamics.
In Figure 5, the effect of KLP-19 depletion on central spindle microtubules appears unconvincing. The FRAP results show no significant difference with or without KLP-19, and overall microtubule density does not consistently respond to its depletion. Additionally, …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This study by Zimyanin et al. examines the role of the C. elegans chromokinesin KLP-19 in the formation and architecture of the anaphase central spindle in C. elegans zygotes. Through a combination of electron and light microscopy, along with RNAi-mediated perturbations, the authors propose that KLP-19 influences central spindle stiffness by regulating microtubule dynamics.
In Figure 5, the effect of KLP-19 depletion on central spindle microtubules appears unconvincing. The FRAP results show no significant difference with or without KLP-19, and overall microtubule density does not consistently respond to its depletion. Additionally, the double klp-19; gpr-1/2 (RNAi) condition does not exhibit a strong increase in microtubule density, though a statistical test is missing for this condition. Furthermore, the spd-1; gpr-1/2 double depletion produces a similar increase in microtubule density to most klp-19 depletion conditions, suggesting that the effect cannot be solely attributed to the absence of KLP-19.
The use of hcp-6 depletion to argue that KLP-19 depletion affects central spindle elongation independently of stretched chromatin is problematic. hcp-6 encodes a component of the Condensin II complex in C. elegans, and its depletion leads to chromatin decompaction rather than the stretched, dense chromatin observed in the midzone during anaphase in klp-19 (RNAi) embryos. These conditions are not equivalent and do not effectively rule out the possibility that chromatin stretching contributes to the observed phenotype.
Additionally, the authors report that KLP-19 influences astral microtubule dynamics (Figure 5E), yet in Figure 3E, they show that KLP-19 localizes exclusively to kinetochores and spindle microtubules, excluding astral microtubules and spindle poles. How do they reconcile this apparent contradiction?
Edit: In the sentence: "Similar, 60s after anaphase onset, spindles of klp-19 (RNAi) (19.2 μm {plus minus} 0.5 μm) and klp-19/spd-1 (RNAi) treated spindles (16.2 μm {plus minus} 0.6 μm) were significantly shorter in comparison to control (20.6 μm {plus minus} 0.2 μm),".
Figure legends lack consistency and do not adhere to standard C. elegans nomenclature conventions (e.g., protein names should not be capitalized, and genetic perturbations should be italicized). Standardizing these elements would improve clarity and readability.
Significance
The experiments are generally well executed and provide convincing data. However, a key concern is that the role of chromokinesins-particularly Kif4, the vertebrate homolog of KLP-19-in central spindle assembly and microtubule regulation has already been demonstrated (Hu et al., CB 2011). Additionally, the function of KLP-12, a C. elegans paralog of KLP-19, in inhibiting microtubule dynamics was more recently reported and the structural details of this inhibition have been dissected (Taguchi et al., eLife 2022, this prior work should be cited and discussed). Given these considerations, and despite the extensive array of approaches used in this paper, the novelty of the current study appears rather limited and may be of interest for C. elegans researchers mainly.
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