Author response:
The following is the authors’ response to the original reviews
Reviewer #1 (Public review):
We thank reviewer 1 for the helpful comments. As indicated in the responses below, we have taken all comments and suggestions into consideration in this revised version of the manuscript.
Weaknesses:
While this study convincingly describes the phenotype seen upon Drp1 loss, my major concern is that the mechanism underlying these defects in zygotes remains unclear. The authors refer to mitochondrial fragmentation as the mechanism ensuring organelle positioning and partitioning into functional daughters during the first embryonic cleavage. However, could Drp1 have a role beyond mitochondrial fission in zygotes? I raise these concerns because, as opposed to other Drp1 KO models (including those in oocytes) which lead to hyperfused/tubular mitochondria, Drp1 loss in zygotes appears to generate enlarged yet not tubular mitochondria. Lastly, while the authors discard the role of mitochondrial transport in the clustering observed, more refined experiments should be performed to reach that conclusion.
It would be difficult to answer from this study whether Drp1 plays a role beyond mitochondrial fission in zygotes. However, the reasons why Drp1 KO zygotes differ from the somatic Drp1 KO model can be discussed as follows.
First, the reviewer mentioned that the loss of Drp1 in oocytes leads to hyperfused/tubular mitochondria, but in fact, unlike in somatic cells, the EM images in Drp1 KO oocytes show enlarged mitochondria rather than tubular structures (Udagawa et al., Curr Biol. 2014, PMID: 25264261, Fig. 2C and Fig. S1B-D), as in the case of zygotes in this study. Mitochondria in oocytes/zygotes have the shape of a small sphere with an irregular cristae located peripherally. These structural features may be the cause of insensitivity or resistance to inner membrane fusion the resultant failure to form tubular mitochondria as seen in somatic cell models. Nonetheless, quantitative analysis of EM images in the revised version confirmed that the mitochondria of Drp1-depleted embryos were not only enlarged but also significantly elongated (Figure 2J-2M). Therefore, in Drp1-depleted embryos, significant structural and functional (e.g., asymmetry between daughters) changes in mitochondria were observed, and these are expected to lead to defects in the embryonic development.
As for mitochondrial transport, we do not fully understand the intent of this question, but we do not entirely rule out mitochondrial transport. At least clustered mitochondria did not disperse again, but how mitochondria behave through the cytoskeleton within clusters will require further study, as the reviewer pointed out.
Reviewer #1 (Recommendations For The Authors):
(1) The authors show no effect of Myo19 Trim-Away, yet it remains unclear whether myo19 is involved in the positioning of mitochondria around the spindle. Judging by their co-localization during that stage, it might be. Therefore, in the absence of myo19, mitochondria might remain evenly distributed throughout mitosis, thus passively resulting in equal partitioning to daughter cells, with no severe developmental defects. Could the authors show a video of the whole process and discuss it?
We have newly performed live imaging of mitochondria and chromosomes in Myo19 Trim-Away zygotes (n=13). As shown in Figure 1-figure supplement 2 and Figure 1-Video 2, there were no obvious changes in mitochondrial (and chromosomal) dynamics throughout the first cleavage and no significant mitochondrial asymmetry was observed, Therefore, we conclude that depletion of Myo19 does not cause mitochondrial asymmetry during embryonic cleavage. These results are described in the revised manuscript (Line 218-221).
(2) Mitochondrial aggregation upon Drp1 depletion should be characterized in more detail: for example, % of mitochondria free, % in small clusters (> X diameter), and % in big clusters (>Y diameter).
In the revised version, mitochondrial aggregation has been quantified by comparing the cluster size and number in control, Drp1 Trim-Away and Drp1 Trim-Away embryos expressing exogenous Drp1 (mCh-Drp1) (Figure 2G, 2H). In control embryos, mitochondria were interspersed in a large number of small clusters, while in Drp1-depleted embryos, mitochondria became highly aggregated into a small number of large clusters that was reversed by expression of mCh-Drp1. These results are described in the revised manuscript (Line 242-245).
(3) The discrepancies with parthenogenetic embryos derived from Drp1 (-/-) parthenotes should be commented on. Quantification of the dimensions of the clusters would help establish the degree of similarity/difference. Could the authors comment on their hypothesis as to why the clusters are remarkably larger in Drp1 depleted zygotes?
In the revised version, we have quantified the mitochondrial aggregation in Drp1 KO parthenotes (Figure 2-figure supplement 1; the data for Drp1 KO parthenotes has been reorganized into the supplemental figure, due to lack of space in figure 2 caused by the addition of quantitative data for Drp1 Trim-Away embryos). The size of mitochondrial clusters in Drp1 KO parthenotes was significantly increased compared to controls, but as the reviewer noted, mitochondrial aggregation appears to be moderate compared to that in Drp1-depleted embryos. The phenotypic discrepancies in two Drp1-deficient embryo models is discussed below.
First, it is clear that phenotypic severity of Drp1 KO oocytes is dependent on the age of the female. Indeed, oocytes collected from 8-week-old female arrested meiosis after NEB, mainly due to marked mitochondrial aggregation (Udagawa et al., Curr Biol. 2014, PMID: 25264261), whereas oocytes from juvenile female completed meiosis (Adhikari et al., Sci Adv. 2022, PMID: 35704569), and thus Drp1 KO pathenotes were obtained from juvenile female in the present study. Comparison of mitochondrial morphology in Drp1 KO oocytes in both papers also suggests that mitochondrial aggregation in adult mice is more intense (Udagawa et al., Curr Biol. Fig. 2A) than in juvenile mice (Adhikari et al., Sci Adv. 2022: Fig. 1G, 1H), and appears to be similar to Drp1-depleted embryos in this study (Figure 2E). There may be differences in the level of Drp1 depletion in these Drp1-deficient oocytes/zygotes. Similar results occurring between juvenile and adult KO female have been reported in a previous paper (Yueh et al., Development 2021, PMID: 34935904), as adult-derived Smac3Δ/Δ<?sup> zygotes arrested at the 2-cell stage, whereas juvenile-derived Smac3Δ/Δ<?sup> zygotes have developmental competence comparable to the wild type. Remarkably, the SMC3 protein levels in juvenile Smac3Δ/Δ<?sup> oocytes was also comparable to Smc3fl/fl. The authors surmised that the decline maternal SMC3 between juvenile and sexual maturity is probably due to the continuous induction of the promoter-Cre driver, suggesting that similar induction may also occur in Drp1 KO oocytes. In addition, we also observed not only age differences but also batch differences in Drp1 KO oocytes (and resulting embryos) such that little mitochondrial aggregation was observed in oocytes collected from some juvenile KO colonies. Therefore, for KO models showing age (sexual maturation)-dependent gradual phenotypic changes, Trim-way may be an approach that provides more reproducible results as it induces acute degradation of maternal proteins.
(4) Mitochondrial clusters in Drp1 trim-away zygotes resemble those seen when defects in mitochondrial positioning are obtained by TRAK2 induction (PMID: 38917013), pointing again to a role of actin in the clustering process. Could the authors explore the role of actin further?
TRAK2 and microtubule-dependent mechanisms may also be involved in mitochondrial dynamics during the first cleavage division, possibly in association with migration of two pronuclei. Although the mitochondrial aggregation induced by TRAK2 overexpression is similar to that in Drp1-depleted embryos, it is unlikely that changes at the EM level occurred as seen in Drp1-depleted embryos (enlarged mitochondria, etc.). In addition, in TRAK2-overexpressing embryos, rather than uneven partitioning of mitochondria, the daughter blatomeres themselves were uneven in size after cleavage, making it difficult to precisely assess the similarity between the two models.
Regarding the role of F-actin, we show that the subcellular distribution of cytoplasmic actin overlaps with that of mitochondria throughout the first cleavage and seems to accumulate in aggregated mitochondria, particularly during the mitotic phase, as higher correlation was observed (Figure 1E). Although it was not observed that actin and the myo19 motor regulate mitochondrial partitioning, as reported in somatic cell-based studies, it is possible that actin accumulated in mitochondria may be indirectly involved in mitochondrial dynamics via mitochondrial fission. For example, inverted formin 2 (INF2) enhance actin polymerization and is required for efficient mitochondrial fission as an upstream function of Drp1 (Korobova et al., Science 2013, PMID: 23349293). In the revised manuscript, we have added the description on this point. (Line 452-456)
(5) Electron microscopy images showed indeed aberrant morphology of the mitochondria, yet not a hyperfused morphology. Aspect ratio (long/short axis) quantification should be included, besides the current measurement, since mitochondria in Drp1 trim-away look bigger yet as round as in the control.
In the revised version, detailed quantitative data on EM images has been added (Figure 2J-2M). In Drp1 depleted embryos, significant increases were observed in both the major and minor axes of mitochondria. As the reviewer noted, we also assumed that mitochondria in depleted embryos were enlarged rather than elongated, but the quantification of aspect ratio shows that significant elongation occurred. These results has been described in the revised manuscript (Line 252-256).
(6) Why are mitochondria in golgi-mcherry-expressing cells showing a different morphology of the clusters?
As noted by the reviewer, compared to other mitochondrial images, Drp1-depleted embryos expressing Golgi-mCherry appear to have less mitochondrial aggregation. The exact reason is not known, but may be due to inter-lot variation of Trim21 mRNA used in this experiment. Nevertheless, substantial mitochondrial aggregation was observed compared to the control, which does not seem to affect the conclusion.
(7) Authors comment on ROS being enriched (highly accumulated) in mitochondria. However, while quantification is missing, it might seem that ROS are equally distributed in control or Drp1 Trim-Away embryos. Could the authors quantify ROS signal inside and outside of the mitochondria, perhaps using a mask drawn by mitotracker? Furthermore, it would make these data more convincing to artificially induce/deplete ROS to validate the sensitivity of the technique to variations. Also, why is ROS pattern referred to as ectopic?
Thank you for your useful suggestions. In the revised version, masked binary images were created from mitochondrial images to quantify ROS levels inside and outside mitochondria (Line 734-741). The result shows the accumulation of ROS to mitochondria in Drp1-depleted embryos (Figure 4-figure supplement 1E). The term ectopic was used to mean excessive accumulation of ROS in the mitochondria compared to normal embryos, but has been deleted as it is not very accurate.
Minor comments:
(A) Video 1: images at t=-00:20 and t=00:00 of the mtGFP are actually the same images as H2B-mCherry.
Probably a faulty filter/shutter control failed to capture GFP fluorescence at these times. It appears that the autocontrast function detected a small amount of mCherry fluorescence leakage. It would be possible to replace it with another video, but as the relevant frame were unrelated to the analysis, the previous video was used as is. The same problem also occurs in the newly added Myo19-depleted zygote movie (Figure 1-Video 2, 03:15).
(B) Could you calculate the degree of colocalization between mt-GFP and ER-mCherry in ctrl and Drp1 trim-away? While it is apparent that ER is somehow more associated with mitochondrial clusters, it would be informative to quantify it.
Since the ER is partially confined to the mitochondrial aggregation site, it was difficult to calculate correlation coefficients from fluorescence images of mt-GFP and ER-mCherry to quantitatively assess colocalization. Instead, line scan analysis of whole mitochondrial clumps showed that the peak of the ER-mCherry signal overlaps with that of mt-GFP, but this is not the case for Golgi-mCherry or peroxisome-mCherry (Figure 2-figure supplement 2A-2C).
(C) Regarding the developmental arrest: The quantification of the different stages at each developmental time could be more informative. For example, at E4.5 how many embryos are at each stage (2-cell, 4-cell, ... blastocyst)? Also, could the authors comment on the reduction in developmental competence in Figure 4C, regarding the blastocyst stage?
Many arrested embryos do not maintain their morphologies and undergo a unique degenerative process over time, known as cell fragmentation. Therefore, it is difficult to accurately determine the number of each developmental stage at, for example, E4.5 days. In this study, the 2-cell stage was observed at E1.5, the 4-8 cell at E2.5-E3.0, morula at E3.5 and the blastocyst at E4.5.
Although the rate of embryos reaching the blastocyst stage was reduced compared to that of normal embryos, the overexpression of mCh-Drp1 may explain the failure of complete restoration of developmental competence, since embryos injected solely with mCh-Drp1 mRNA also showed reduced developmental competence. For rescue experiments, the comparison with internal controls is more important and therefore we described below. This is a specific effect of Drp1 deletion because none of the internal control conditions increased arrest at the 2-cell stage and arrest was completely reversed by microinjecting Trim-away insensitive exogenous mCh-Drp1 mRNA (Line 337-340).
(D) In lines 103 to 105, proliferation should be changed to division or development.
In the revised version, proliferation has been changed to division (Line 103).
(E) Could the authors reference the statement in lines 168-169?
The following 3 references have been added (Hardy et al., 1993, PMID: 8410824; Meriano et al., 2004, PMID: 15588469; Seikkula et al., 2018, PMID: 29525505).
(F) Line 448: "Cells lacking Drp1 have highly elongated mitochondria that cannot be divided into transportable units,..." This is clearly not the case for zygotes, so why are then these mitochondria still clustering and not transported elsewhere?
Although it is difficult to answer this reviewer's question precisely, EM images of Drp1-depleted embryos suggest that individual mitochondria appear not only to be enlarged but also to have increased outer membrane attachment due to excessive aggregation. Thus, these large mitochondrial clumps may therefore be preventing transport.
Reviewer #2 (Public review):
We thank reviewer 2 for the helpful comments. As indicated in the responses below, we have taken all comments and suggestions into consideration in this revised version of the manuscript.
Weaknesses:
The authors first describe the redistribution of mitochondria during normal development, followed by alterations induced by Drp1 depletion. It would be useful to indicate the time post-hCG for imaging of fertilised zygotes (first paragraph of the results/Figure 1) to compare with subsequent Drp1 depletion experiments.
In the revised version, the time after hCG has been indicated (Line 176-182). In subsequent Drp1 depletion experiments, the revised version notes that “no significant delay in cell cycle progression was observed following Drp1 depletion (data not shown) compared to control embryos (Figure 1A)” (Line 291-193). There was a slight discrepancy in the time post-hCG between live imaging and immunofluorescence analysis (Figure 1-figure supplement 1A), which may be due to manipulation of zygotes outside incubator during the microinjection of mRNA.
It is noted that Drp1 protein levels were undetectable 5h post-injection, suggesting earlier times were not examined, yet in Figure 3A it would seem that aggregation has occurred within 2 hours (relative to Figure 1).
As the reviewer pointed out, the depletion of Drp1 is likely to have occurred at an earlier stage. In this study, due to the injection of various mRNAs to visualize organelles such as mitochondria and chromosomes, observations were started after about 5 h of incubation for their fluorescent proteins to be sufficiently expressed. Therefore, for the Western blot analysis, samples were prepared according to the time of the start of the observation.
Mitochondria appear to be slightly more aggregated in Drp1 fl/fl embryos than in control, though comparison with untreated controls does not appear to have been undertaken. There also appears to be some variability in mitochondrial aggregation patterns following Drp1 depletion (Figure 2-suppl 1 B) which are not discussed.
In the revised version, mitochondrial aggregation has been quantified by comparing the cluster size and number in control, Drp1 Trim-Away and Drp1 Trim-Away embryos expressing exogenous Drp1 (mCh-Drp1) (Figure 2G, 2H). We have also quantified the mitochondrial aggregation in Drp1fl/fl and Drp1Δ/Δ parhenotes (Figure 2-figure supplement 1; note that the data for Drp1 KO parthenotes has been reorganized into the supplemental figure, due to lack of space in figure 2 caused by the addition of quantitative data for Drp1 Trim-Away embryos). Mitochondria appear to be slightly more aggregated in Drp1fl/fl embryos than in control, but no significant differences in cluster size or number were observed (data not shown). On the other hand, mitochondrial clusters in Drp1 Trim-Away embryos were remarkably larger than Drp1Δ/Δ parhenotes, Please refer to the response to reviewer 1's comment (3) for discussion of this discrepancy.
As noted by the reviewer, compared to other mitochondrial images, Drp1-depleted embryos expressing Golgi-mCherry appear to have less mitochondrial aggregation. The exact reason is not known, but may be due to inter-lot variation of Trim21 mRNA used in this experiment. Nevertheless, substantial mitochondrial aggregation was observed compared to the control, which does not seem to affect the conclusion.
The authors use western blotting to validate the depletion of Drp1, however do not quantify band intensity. It is also unclear whether pooled embryo samples were used for western blot analysis.
In the revised version, the band intensities in Western blot analysis were quantified and validated the previous results (Figure 1H for Myo19 depletion, Figure 2B for Drp1 expression during preimplantation development, Figure 2D for Drp1 depletion). The number of embryos analyzed was described in Figure legends (Pooled samples ranging from 20 to 100 were used).
Likewise, intracellular ROS levels are examined however quantification is not provided. It is therefore unclear whether 'highly accumulated levels' are of significance or related to Drp1 depletion.
In the revised version, masked binary images were created from mitochondrial images to quantify ROS levels inside and outside mitochondria (Line 734-741). The result shows the accumulation of ROS to mitochondria in Drp1-depleted embryos (Figure 4-figure supplement 1E).
In previous work, Drp1 was found to have a role as a spindle assembly checkpoint (SAC) protein. It is therefore unclear from the experiments performed whether aggregation of mitochondria separating the pronuclei physically (or other aspects of mitochondrial function) prevents appropriate chromosome segregation or whether Drp1 is acting directly on the SAC.
In the revised manuscript, we have discussed this reference (Zhou et al., Nature Communications, PMID: 36513638) (Line 482-483).
Reviewer #2 (Recommendations For The Authors):
The authors report that disruption of F-actin organization led to asymmetry in mitochondrial inheritance, however depletion of Myo19 does not impact inheritance. The authors note in the discussion that loss of another mitochondrial motor protein, Miro, has been shown to affect mitochondrial inheritance. They suggest this may be due to reduced levels of Myo19, despite data from the present study suggesting a lack of involvement of Myo19. Given that Miro1 also interacts with microtubules, and crosstalk between actin filaments and microtubules has been reported, have the authors considered whether other motor proteins, such as KIF5, may be involved in mitochondrial movement in the zygote and therefore inheritance? Myo19 also plays a role in mitochondrial architecture. Were any differences noted at the EM level?
During oocyte meiosis and early embryonic cleavage, kinesin-5 has been reported to be important for the formation of bipolar spindles (Fitzharris, Curr Biol., 2009, PMID: 19465601) and may have some involvement in mitochondrial dynamics. Given that the migration of two pronuclei towards the zygotic centre is dynein-dependent manner (Scheffler Nat Commun. 2021PMID: 33547291), dynein may also be involved in the process of mitochondrial accumulation around the pronuclei. Nevertheless, whether microtubule-dependent mechanisms regulate mitochondrial partitioning remains controversial. Mitochondria basically diverge from microtubules at the onset of mitosis, and indeed Miro1-deleted zygotes did not show the asymmetric mitochondrial partitioning (Lee et al., Front Cell Dev Biol. 2022, PMID: 36325364). More recently, it was reported that overexpression of TRAK2 causes significant mitochondrial aggregation in embryos (Lee et al., Proc Natl Acad Sci U S A. 2024, PMID: 36325364), but since overexpression might disrupt a regulatory balance by other motors/adaptor complexes, further investigation using TRAK2-deficient embryos is expected.
As noted by the reviewer, myo19 seems to be important for the maintenance of mitochondrial cristae architecture and, consequently, for the regulation of mitochondrial function (Shi et al., Nat Commun. 2022, PMID: 35562374). We have not observed the EM images in myo19-depleted embryos, but we examined their membrane potential and ROS by TMRM and H2DCF staining, respectively, and confirmed that they were comparable to control embryos (data not shown). The loss of myo19 in zygotes/embryos did not cause any functional changes in mitochondria, suggesting that mitochondrial architecture may not be substantially affected either.
Transcriptomic analysis would be useful to identify alterations in cell cycle checkpoint regulators, as well as immunofluorescence to identify changes in spindle assembly checkpoint protein recruitment.
The present results showed that the majority of Drp1-depleted embryos arrest at the G2 stage, possibly due to cell cycle checkpoint mechanisms. Transcriptome analysis would certainly be beneficial, but eventually more detailed analysis of proteins and their phosphorylation modifications, etc. is needed for accurate assessment. These studies will be the subject of future work.
Minor comments:
There are many instances where the English could be improved, particularly the overuse of the word 'the'.
We have checked the manuscript again carefully and hopefully it has been improved some.
Line 144: replace 'took' with 'take'.
We have corrected this in the revised version (Line 140).
Line 157: it is unclear what is meant by 'hinders the functional importance of Drp1 in mature oocytes and embryos'.
This description has been corrected to “complicates the functional analysis of Drp1 in mature oocytes and embryos” (Line 152-153)
Line 198: replace with 'displayed a mitochondrial distribution pattern closely associated with'
We have corrected this in the revised version (Line 195-196).
Line 200: provide a time to clarify when the cytoplasmic meshwork was 'subsequently reorganized'
In the revised version, “at the metaphase” has been added (Line 198).
Line 204: replace 'to' with 'for'
We have corrected this in the revised version (Line 203).
Lines 285-87: consider rearranging the text to improve the flow.
To improve the flow of text before and after, the following sentence has been added; We postulated that this asymmetry was due to non-uniformity in the distribution of mitochondria around the spindle (Line 295-297)
Line 418: replace 'central' with 'centre'
We have corrected this in the revised version (Line 430).
Line 427: replace 'pertaining' with 'partitioning'
We have corrected this in the revised version (Line 438).
Line 574: clarify to what '1-5% of that of the oocytes' refers
We have corrected it to “1-5% of the total volume of the zygote.” (Line 587-588).
Line 619: indicate the dilution used
We apologize for the previous incorrect description. We used a part of the extract as the template, not a dilution, and have corrected it to be accurate (Line 631-632).
Line 634: replace 'on' with 'in' and detail in which medium embryos were mounted.
We have corrected this in the revised version (Line 647).
Please check all spelling in the figures.
Figure 1J - inheritance is spelt incorrectly.
Figure-Suppl 1, D: Interphase (PN) and (2-cell) is spelt incorrectly. G: inheritance is spelt incorrectly.
Figure 5F - bottom section prior to cytokinesis, spindle is spelt 'spincle'
Ensure consistency in abbreviation use (e.g. use of NEB and NEBD).
Thank you for your careful correction of typographical errors. In the revised version, all points raised by the reviewers have been corrected.
Reviewer #3 (Public review):
We thank reviewer 2 for the helpful comments. As indicated in the responses below, we have taken all comments and suggestions into consideration in this revised version of the manuscript.
Seemingly, there are few apparent shortcomings. Following are the specific comments to activate the further open discussion.
Line 246: Comments on cristae morphology of mitochondria in Drp1-depleted embryos would better be added.
In the revised manuscript, we have added the following comment; swollen or partially elongated mitochondria with lamella cristae structures in the inner membrane were observed in Drp1 depleted embryos. In addition, the quantification of aspect ratio (long/short axis) shows that significant mitochondrial elongation was occurred (Figure 2M). These results has been described in the revised manuscript (Line 251-256).
- Regarding Figure 2H: If possible, a representative picture of Ateam would better be included in the figure. As the authors discussed in line 458, Ateam may be able to detect whether any alterations of local energy demand occurred in the Drp1-depleted embryos.
Thank you for your very useful comments. Although it would be interesting to investigate whether alterations in ATP levels occurred in localized areas (e.g., around the spindle), the present study used conventional fluorescence microscope instead of confocal laser microscopy to observe ATeam fluorescence in order to quantify the fluorescence intensity in the whole embryo (or whole blastomere) and thus we currently cannot provide the images that reviewer expected. As shown in Figure-figure supplement 1C, the ATP levels tend to be higher at the cell periphery in control and at the mitochondrial aggregation areas in Drp1-depleted embryos, but it would need high resolution images using confocal microscopy to show it clearly.
- Line 282: In Figure 3-Video 1, mitochondria were seemingly more aggregated around female pronucleus. Is it OK to understand that there is no gender preference of pronuclei being encircled by more aggregated mitochondria?
Review of multiple videos shows that aggregated mitochondria were localized toward the cell center, but did not exhibit the behavior of preferentially concentrating near the female pronucleus.
- Line 317: A little more explanation of the "variability" would be fine. Does that basically mean that the Ca2+ response in both Drp1-depleted blastomeres were lower than control and blastomere with more highly aggregated mitochondria show severer phenotype compared to the other blastomere with fewer mito?
We think that the reviewer's comments are mostly correct. It is clear that there is a bias in Ca2+ store levels between blastomeres of Drp1 depleted embryos, However, since mitochondria were not stained simultaneously in this experiment, we cannot draw conclusions in detail, such that daughter blastomere that inherit more mitochondria have higher Ca2+ stores, or that blastomere with more aggregated mitochondria have lower Ca2+ stores.
- Regarding Figure 5B (& Figure 1-figure supplement 1B): Do authors think that there would be less abnormalities in the embryos if Drp1 is trim-awayed after 2-cell or 4-cell, in which mitochondria are less involved in the spindle?
The marked centration of mitochondrial clusters in Drp1-depleted embryos appears to be associated with migration of the pronuclei toward the cell center, which is unique to the first embryonic cleavage. Since the assembly of the male and female pronuclei at the cell center is also unique to the first cleavage, binucleation due to mitochondrial misplacement was observed only in the first cleavage. Therefore, if Drp1 is depleted at the 2-cell or 4-cell stage, chromosome segregation errors may be less frequent. However, since unequal partitioning of mitochondria is thought to occur, some abnormalities in embryonic development is likely to be observed.
Reviewer #3 (Recommendations For The Authors):
Specific comments
- Line 262: "Since mitochondrial dynamics are spatially coordinated at the ER-mitochondria MCSs," adequate ref. would better be added.
We have added an adequate reference to the revised manuscript (Friedman et al., 2011, PMID: 21885730).
- Line 333-336: "...as assessed by the presence of the nuclear envelope." Do authors show the data? In Figure 4-figure supplement 1A, the difference of the phosphoH3-ser10 signal between control and Trim-Away group might be weak. For clarity, it would be helpful if authors indicate the different points to note in the figure.
Although the data is not shown, nuclear staining of arrested 2-cell stage embryos exhibited clear nuclear membranes, similar to the DAPI image in Figure 4-figure supplement 1A. We have indicated that the data is not shown in the revised version (Line 345). Based on a report that phosphorylated histone H3 (Ser10) localizes in pericentromeric heterochromatin that hat can be visualized by DAPI staining in late G2 interphase cell (Hendzel et al., 1997, Chromosoma, PMID: 9362543), this study qualitatively estimated the G2 phase from the phosphorylated histone H3 signal and the DAPI counterstained images. We have noted this point in the revised figure legend (Line 1012-1014).
Typos or points for reword/rephrase
- Line 149: "molecular identification" may better be " molecular characteristics".
We have corrected this in the revised version (Line 145).
- Line 157: "hinders the functional importance" would be "implies the functional importance" or "complicates the functional analysis".
We have corrected this in the revised version (Line 152-153).
- Line 208: "Since the role of F-actin in many cellular events, such as cytokinesis, preclude them as targets for experimentally manipulating mitochondrial distribution, " may better be "Given many cellular roles, disruption of F-actin per se was unsuitable as a strategy for manipulating mitochondrial distribution", for example.
We have corrected this in the revised version (Line 207-208).
- Line 260: "with MCSs with the plasma.." may better be "with MCSs such as with the plasma..".
We have corrected this in the revised version (Line 267-268).
- Line 312: "distribution and segregation" may better be "distribution and the resulting segregation of the inter-organelle contacts".
We have corrected this in the revised version (Line 324-325).
- Line 427: "pertaining" might be "partitioning".
We have corrected this in the revised version (Line 438).
Line 463: "loss of Drp1 induced mitochondrial aggregation disturbs" may better be "mitochondrial aggregation induced by the loss of Drp1 disturbs".
We have corrected this in the revised version (Line 478-479).
- Line 752: "endoplasmic reticulum (pink) " would be " endoplasmic reticulum (aqua) ".
We have corrected this in the revised version (Line 780).
- Figure 5E: "(Noma 2-cell embryos)" would be "(Nomal 2-cell embryos)".
- Figure 5F: "Mitochondrial centration prevents dual spincle assembly" would be "Mitochondrial centration prevents dual spindle assembly".
Thank you for your careful correction of typographical errors. We have corrected all the words/expressions the reviewer pointed out in the revised version.
