Reduction of cortical pulling at mitotic entry facilitates aster centration
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Abstract
Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry, we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested that a wavelike loss of cortical pulling at mitotic entry leads to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from ∼300pN/µm to ∼100pN/µm. Following the loss of cortical force generators at mitotic entry, long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.
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Referee #1
- The authors should provide more information when...
Responses
- The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A).
- Though this is not stated in the MS
- Figure 6: Why has only...
Response: We expanded the comparison
Minor comments:
- The text contains several...
Response: We added...
Referee #2
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Referee #2
Evidence, reproducibility and clarity
Summary:
This manuscript addresses how the first mitotic spindle is centered in the ascidian zygote to promote a symmetric cell division. This is a universal problem in animals because the sperm centrosome, which will form the poles of the mitotic spindle, is initially at the edge of the zygote because it comes in from the outside. The authors hypothesize that cortical pulling is turned off so that a combination of microtubule pushing and cytoplasmic pulling can center the mitotic spindle. Experimental methods include live imaging of ascidian zygotes injected with mRNAs and proteins as well as computational modeling. Results …
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Referee #2
Evidence, reproducibility and clarity
Summary:
This manuscript addresses how the first mitotic spindle is centered in the ascidian zygote to promote a symmetric cell division. This is a universal problem in animals because the sperm centrosome, which will form the poles of the mitotic spindle, is initially at the edge of the zygote because it comes in from the outside. The authors hypothesize that cortical pulling is turned off so that a combination of microtubule pushing and cytoplasmic pulling can center the mitotic spindle. Experimental methods include live imaging of ascidian zygotes injected with mRNAs and proteins as well as computational modeling. Results support the hypothesis that cortical pulling is turned off at the same time that the spindle centers.
Major comments:
Inhibition of entry into mitosis by p21 injection prevents centrosome centering. This results supports the idea that CDK activity is required for centrosome centering but does not specifically support the inhibition of cortical pulling model.
Data in figure 3 is used to support the conclusion that cytoplasmic pulling does not change between interphase and mitosis and therefore an increase in cytoplasmic pulling during mitosis cannot be responsible for the centering of the spindle. This interpretation needs to be more carefully qualified in the text of the results and discussion. The only "pulling proxy" that is quantitatively compared between interphase and mitosis is movement of Cell Mask Red-labeled endosomes toward or away from the centrosome. (1) Only endosomes are tracked and it is possible that yolk granules or mitochondria could exhibit different results.
(2) If endosomes were the only contributors to cytoplasmic pulling, the ratio of anterograde vs retrograde transport would be significant. Data in Fig. S2C looks like this might change between interphase and mitosis but no statistical result is shown testing for a change in this ratio.
(3) Single plane imaging is justifiably used for this high speed analysis which means that vesicles are likely to leave the focal plane frequently. Leaving the focal plane would artificially reduce "track persistence" which is strangely reported in "n.u." units with very small values. N.u. units are not defined. The units should by um like total transport. A more accurate statement might be: "We could not detect a significant change in centrosome-direct endosome transport in our experiments but we cannot exclude the possibility that a significant difference would be detected with different cargo or different methods." The data presented in figure 4 provides very strong evidence that cortical pulling is reduced in mitosis relative to interphase, supporting the overall conclusion of the paper.
The interpretation of fig. 5 is not strongly supported. Latrunculin is not necessarily going to inhibit cortical pulling and cortical pushing without affecting cytoplasmic pulling. In C. elegans, depletion of GPR/LGN would be the appropriate experiment. The interpretation should be qualified. The presentation of figure 6 could be improved. The protrusions indicative of cortical pushing are not quantified interphase vs mitosis. Qualitatively, there are more protrusions during mitosis than interphase which could support an increase in cortical pushing as a mechanism promoting centration. The interpretation of this result should be clarified. Whether cortical pushing is regulated in the model should also be clarified. The cytosim results presented in figure 7 lend support to he overall conclusions of the manuscript. The legend for figure 7B should state the number of simulations run for each condition.Minor comments:
The presentation of figure 1 could be improved. 1A and 1D are redundant, and C and D are cited in the text before B. The most important data is figure 1B (quantification of the distance of the paternal DNA from the cell center) but no images of DNA are shown. Only a cartoon of DNA localization is shown. The results text states that the data in fig. 1B was derived from time-lapse sequences of zygotes
(1) expressing histone h2b::tomato,
(2) histone h2b::venus, or
(3) stained with Hoechst. If the zygotes all had microtubules and DNA labelled, Fig. 1A could be 2 color time-lapse images showing both DNA and microtubules. 1A could have the larger number of time points currently in 1D, then 1D could be deleted. The authors should then take care to cite the sub figures in order in the text. Given the number of DNA labeling methods, it would also be appropriate in the methods to state how the authors know that the labeling methods are non-toxic (especially the live cell Hoechst labeling). Fig. S1B needs statistically significant differences marked since the text states that the significant differences in Fig. 1B were reproduced in fixed images in S1B.
The legend for fig. 2B needs to be clarified. It states that the dark shaded bar is "mitosis entry" but the p21 injected zygotes are not entering mitosis at this timepoint.
The legend to figure 4B could spell out Cell Mask Orange.
Referees cross-commenting
I agree with most of reviewer 1's comments. The author's should validate their membrane ingression assay for cortical pulling by providing quantification of cortical actin after latrunculin treatment in each condition. Regarding the expectation that the sperm aster should move toward the cortex during interphase when cortical pulling is active, Fig. 1B shows significant movement toward the cortex during meiosis but movement away (with no statistical significance marked) during interphase. This might be due to a balance with microtubule pushing on the membrane. However, this question raises the need for better presentation of the data in figure 1B. The sperm DNA should start at 0 um from the cortex at fertilization. Is the huge variability in sperm DNA position at the first time point due to variability in egg diameter? If so, an additional plot of distance from the cortex or better a plot with %radius instead of um would be helpful. Or, is the huge variability due to significant movement of the sperm DNA during meiosis before the first time point? If this is the cases, the authors might present a scatter plot of the net displacement toward or away from the cortex for each individual zygote. This improved analysis might address some of reviewer 1's concerns.
Significance
Because inhibition of cortical pulling during M phase has been reported in multiple species before, to have a really high impact, the authors would need to identify the relevant phosphorylation sites on dynein/dynactin/LGN and show that non-phosphorlatable mutants retain invaginations in M phase and the aster fails to center. The work could have a moderate impact on the field with just the controls already suggested.
Significance
Significance
Centering of the first mitotic spindle is an important biological process that has been previously addressed in C. elegans and mammalian zygotes. This manuscript provides a high quality description of centering in ascidian zygotes with appropriate comparisons to mammals and C. elegans. While the quality of the data is strong, no new molecular mechanisms are identified which limits the significance. Because the authors cite primate papers showing that ascidian centering follows the pattern of primates, it is not clear how the current study adds new medical significance to what is already known. Perhaps the authors could highlight what was not shown in the primate papers that is shown in the current manuscript.
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Referee #1
Evidence, reproducibility and clarity
Summary:
Proper mitotic spindle orientation is decisive for asymmetric cell division and cell volume regulation after cell division. Different cellular systems utilize many ways to orient the spindle in space and time. In this work, the authors investigate the mechanisms regulating sperm aster centration in the zygote of ascidian Phallusia mammillata. The authors show that the sperm aster stays close to the cell cortex at the vegetal pole during interphase and migrates to the cell centre during mitosis. Their data reveal that cortical pulling forces are active during interphase, and these forces are off during mitosis. The strength …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
Summary:
Proper mitotic spindle orientation is decisive for asymmetric cell division and cell volume regulation after cell division. Different cellular systems utilize many ways to orient the spindle in space and time. In this work, the authors investigate the mechanisms regulating sperm aster centration in the zygote of ascidian Phallusia mammillata. The authors show that the sperm aster stays close to the cell cortex at the vegetal pole during interphase and migrates to the cell centre during mitosis. Their data reveal that cortical pulling forces are active during interphase, and these forces are off during mitosis. The strength of the work is that the authors are analyzing the aster positioning in eggs of ascidians, where the aster behaviour is similar to primates. The work's limitation is that most important conclusions are made using inhibitors that can impact multiple processes in the zygote of Phallusia mammillata (please see below).
Major Comments:
In Fig. 4, the authors claim that the cortical forces are strong in interphase and weak in mitosis. This experiment was performed in conditions that disturb the actin cytoskeleton, and the cell membrane invaginations were monitored as a proxy for cortical force generation. Here the authors observed that the number of invaginations are decreased in mitosis, compared to the interphase. This led them to conclude cortical pulling forces are higher in interphase than in mitosis. The change in behaviour of the cell membrane invaginations could be because of a difference in actomyosin-based cytoskeleton thickness/dynamics between interphase and mitosis. Do the authors know if the cortical actomyosin meshwork between interphase and mitosis remains the same?
Similarly, did the authors test if the injection of p21 or cyclinBdelta90 does not change the actin cytoskeleton in the injected cell versus the non-injected cell (Fig. 4)?
If the cortical pulling forces are acting on the sperm asters in interphase, as the authors concluded, I wonder why do the authors not observe a significantly more number of invaginations close to the sperm asters because of the high density of microtubules in that region?
The authors mentioned that these membrane invaginations are microtubule-dependent and cited Godard at al., 2021. This point is vital; thus, the authors should include the nocodazole experiment in their data. Since the dynamic nature of microtubules is critical for aster positioning in C. elegans zygote, the authors should further test if dynamic microtubules regulate sperm aster position in interphase by treating these cells with taxol.
Also, the authors should analyze if the membrane invaginations during interphase are dynein/dynactin-dependent.
If the cortical pulling forces are the chief reason to keep sperm asters close to the cell membrane during interphase, then over time, the sperm aster distance from its geometric centre to the cell membrane should decrease. Do the authors observe this?
The authors should quantify the number of invaginations at the two-cell stage in p21 or cyclinBdelta90 injected cells.
In Fig. 5, the authors inhibit the actin cytoskeleton for characterizing if the cortical pulling forces are critical to prevent aster migration. The impact of the actin cytoskeleton on aster migration is quite indirect and does not affirmatively support their conclusions that it is via impacting cortical pulling forces. Can the authors show that cortical force generators (dynein/dynactin complexes) are localized at the membrane in their system, and if the actin inhibitors impact their localization?
It would be important for the readers to see both the DNA and the asters in Fig. 1 as the authors have injected Ensconsin-GFP and H2B-Tom mRNAs. Also, why did the authors choose to measure the distance between male DNA and the cell centre? They could measure the geometric centre of the male aster to the cell centre before the meeting and the centre of the mitotic spindle to the cell centre after spindle assembly, which would be more appropriate for studying spindle behaviour.
Minor points:
Do the authors know if the microtubule dynamics remain unaltered in p21 injected zygote (Fig.2)? It could simply be that the impact of p21 injection on aster migration is because of the change in microtubule dynamics
In line 149, the authors write, 'During meiosis I, the aster is in the egg cortex' I guess that the authors would like to say that it is juxtaposed to the cell cortex.
Significance
The strength of the work is that the authors are analyzing the aster positioning in eggs of ascidians, where the aster behaviour is similar to primates. The work's limitation is that most important conclusions are made using inhibitors that can impact multiple processes in the zygote of Phallusia mammillata (please see the comments).
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