Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
Using electron microscopy, the authors report discontinuities in the plasma membrane of C. elegans embryos. They associate these discontinuities with cell division and speculate that membrane rupture and subsequent resealing contribute to cytokinesis. They further discuss the proximity of these sites to vesicles and propose a role for vesicle-mediated membrane extension.
Weaknesses:
(1) The possibility that the membrane discontinuity is an artifact
Although the authors focus on discontinuities in the plasma membrane, similar discontinuities are also observed in mitochondria, the nuclear envelope, and yolk granules. This raises concerns about whether the electron micrographs presented are suitable for assessing membrane continuity.
Electron micrographs result from a lengthy sample preparation process, including high-pressure freezing, freeze substitution in acetone containing OsO4, gradual warming, uranyl acetate staining, resin embedding, and ultrathin sectioning. In general, lipids are soluble in acetone at temperatures above −30 {degree sign}C, and preservation of membrane structures relies heavily on efficient OsO4 fixation.
Insufficient OsO4 treatment would be expected to reduce membrane contrast.
C. elegans embryos are encapsulated by an eggshell that forms at fertilization and gradually develops during the first few cell divisions. It is unclear how efficiently OsO4 in acetone penetrates the eggshell during freeze substitution, raising further concern about plasma membrane preservation under the conditions used.
We thank the reviewer for raising this important technical concern. We have taken this question seriously since first observing membrane discontinuities six years ago, and we have since conducted extensive controls to rule out fixation artifacts. Below, we present multiple lines of evidence—ranging from technical reproducibility to orthogonal imaging approaches—that collectively demonstrate the biological reality of these structures.
(1) Technical expertise and standard protocols
Our laboratory has extensive experience with electron microscopy across diverse biological systems, including neurons, muscle cells, and hypodermis in C. elegans, as well as tissues from Drosophila, mouse, bacteria, and cultured cells (Chen et al., 2013; Ding et al., 2018; Guan et al., 2022; Y. Li et al., 2018; Miao et al., 2024; Qin et al., 2014; Wang et al., 2026; J. Xu et al., 2022; M. Xu et al., 2021; L. Yang et al., 2020; X. Yang et al., 2019; Zhu et al., 2022). Importantly, we did not introduce any novel or unconventional steps in our EM preparation; all protocols were standard and well-established. Thus, the observed membrane discontinuities are unlikely to stem from technical inexperience or idiosyncratic methods.
In addition to membrane discontinuities, we would like to emphasize that a large number of single plasma membranes separating adjacent cytoplasmic domains were also detected under EM (Figure 1, 3 and 4, for instance). This observation is particularly significant because the invagination model cannot generate single plasma membrane barriers between adjacent cytoplasmic domains. Instead, independent extension of detached sister membranes could explain the formation of cytoplasm-enclosed membranes. Furthermore, as the morphology and continuity of these single cytoplasm-immersed membrane structures are well preserved, this indicates successful EM processing and argues against inefficient fixation or other technical issues.
(2) Reproducibility across independent preparations and techniques
To test whether the discontinuities were preparation-specific, we examined four independent sample batches collected in the lab over the years. Membrane discontinuities, as well as cytoplasm-immersed membranes, on embryonic cells were consistently observed across all batches, indicating that the phenomenon is not dependent on a single preparation method. Furthermore, we validated our findings using two EM techniques: transmission electron microscopy (HPF-TEM) and dualbeam scanning electron microscopy (SEM). Membrane discontinuities were clearly identifiable with both techniques, further supporting their robustness.
(3) Validation using an independent public dataset
We examined the publicly available C. elegans embryo EM collection (WormAtlas). In several instances, particularly at the embryonic periphery where plasma membrane discontinuities are more readily visualized (https://www.wormimage.org/image.php?id=140265&page=1), we identified similar structures. The presence of these features in an independent dataset generated by different researchers confirms that they are not artifacts unique to our sample preparation.
(4) Developmental regulation of membrane discontinuities
We analyzed embryos across multiple developmental stages. Membrane discontinuities were observed in both intrauterine and laid embryos at early stages. However, as embryos reached the comma stage—a period marked by the onset of elongation and reduced cell proliferation—the incidence of discontinuities dropped dramatically (0/13, 0/17, and 0/30 cells examined). This developmental specificity argues strongly against a general fixation artifact, which would be expected to occur randomly across stages. Additionally, the eggshell is present throughout the embryonic stage of C. elegans; therefore, the dramatic reduction of membrane discontinuities in comma-stage of embryo argues against the possibility that the eggshell poses a fixation problem.
(5) Rigorous criteria for identifying membrane discontinuities
To ensure unbiased analysis, we systematically collected images from early embryonic cells using the following criteria:
(1) Random section selection: For each sample, we randomly selected one section containing the largest number of embryos or cells (Sup Figure 2) for initial analysis. We found membrane discontinuities in 159 cells distributed across 57 embryos, representing 95% of the total sampled embryos This portion of the data is summarized in Figure 1.
(2) Whole-membrane examination: Each putative membrane discontinuity was identified only after examining the entire plasma membrane of the cell on a given section. Importantly, aside from the discontinuity, the remainder of the plasma membrane remained intact. Moreover, in most cells, only a single discontinuity was present per section, arguing against random, widespread membrane tearing during preparation.
(3) Neighboring section verification: Because EM preparation yields serial sections, we verified nearly all membrane discontinuities by examining adjacent sections. Again, the same membrane discontinuity was confirmed only after inspecting the entire plasma membrane on those neighboring sections as well. We will include this verification protocol in the revised Methods and additional imaging of consecutive sections would be provided if needed.
(4) Serial section reconstruction: To further determine whether a dividing cell indeed contains one membrane rupture, we performed two serial reconstruction experiments.
First, we used HPF-TEM to analyze 105 consecutive sections of a metaphase cell, reconstructing the entire plasma membrane and chromosome configuration. We found that one membrane rupture largely encircled the chromosomal disc (Figure 2 and Video S1), spatially aligning with the future segregation zone. Second, we used AutoCUTS-SEM to collect approximately 600 sections covering ~95% of a telophase cell containing three nuclei sharing a common cytoplasm. This tri-nucleated cell was enclosed by three distinct plasma membranes, each harboring a single rupture site. These three ruptures converged to form a Y-shaped exposed cytoplasmic region spanning >351 sections (Figure 5). Collectively, these reconstructions demonstrate that each cell contains only one discontinuity from a 3D point of view, further supporting that the phenomenon is not due to random sample preparation damage.
(6) Orthogonal validation by live imaging: In addition to EM, we performed live imaging of plasma membrane dynamics. While live imaging provides important temporal context, we recognize its limitations in resolving membrane ultrastructure. The rapid kinetics of membrane extension (approximately 20–30 seconds for metaphase and less than 3 minutes for cytokinesis), combined with embryo motility, introduces spatiotemporal ambiguities. To capture dynamic membrane events, our live imaging using the GFP::PH membrane marker was performed at 4-second intervals, approaching the practical limit for single-section scanning of the embryo. With single-plane live imaging, nevertheless, both membrane ruptures and free-ended sister membrane structures could be detected (Figures 6), providing additional evidence that membrane rupture and independent extension of detached sister membranes underlie cytokinesis in C. elegans embryos. Notably, 3D membrane dynamics analysis using light-sheet microscopy (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016. DOI:10.1038/ncomms11088) revealed membrane ruptures in dividing early C. elegans embryonic cells, including during telophase or metaphase. Therefore, live imaging further validates the membrane rupture phenomena in dividing embryonic cells in C. elegans.
While future advances in imaging technology may enable real-time visualization at near-EM resolution, our extensive, multi-year effort to test the artifact hypothesis has convinced us that these membrane discontinuities are genuine biological features of dividing C. elegans embryonic cells.
We are confident that the cumulative evidence presented here addresses the reviewer's concerns and demonstrates that the observed membrane discontinuities, as well as cytoplasm-immersed membranes, are not procedural artifacts but rather reflect a previously underappreciated aspect of plasma membrane dynamics during embryonic cell division.
(2) Lack of evidence linking membrane discontinuity to cell division
The reported plasma membrane discontinuities are not specific to mitotic cells. If this were a physiological process playing an important role in cytokinesis, it should occur in a temporally and spatially coordinated manner with nuclear division. However, it remains unclear at what stage of the cell cycle the membrane rupture occurs and where it is located relative to chromosomes and the mitotic spindle.
Thank you for this insightful comment. We agree that establishing a direct link between plasma membrane discontinuities and mitotic progression is critical, and we appreciate the opportunity to clarify this point.
In C. elegans embryos, the early stages of development are characterized by rapid and extensive cell division. Within approximately 100 minutes, a two-cell embryo develops into an embryo containing nearly 30 cells. The majority of the electron microscopy analyses in our study were performed on embryos at stages with fewer than 30 cells, where most cells are actively dividing. Thus, it is reasonable to infer that the cells exhibiting membrane discontinuities are predominantly mitotic cells.
Supporting this notion, as embryos reached the comma stage—a period marked by the onset of elongation and reduced cell proliferation—the incidence of membrane discontinuities dropped dramatically (0/13, 0/17, and 0/30 cells examined). This developmental specificity strongly suggests that membrane discontinuities are tightly linked to cell division.
Importantly, mitotic features such as metaphase chromosomes aligned at the equatorial plane or two (or more) nuclei sharing common cytoplasm can be identified in EM images. In our single random EM section analysis, we captured membrane discontinuities in cells at metaphase, anaphase (characterized by fewer than 10 chromosomal clumps), and telophase (defined by two nuclei sharing cytoplasm). Hence, membrane discontinuities are indeed present on mitotic cells. In addition, a published work by Fu et al (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016. DOI:10.1038/ncomms11088) using light-sheet microscopy captured similar membrane discontinuities in cells displaying classical mitotic features, including anaphase or telophase.
To further investigate the spatial relationship between membrane ruptures and chromosome organization, we performed three-dimensional reconstructions on a metaphase cell. As shown in Figure 2 and Video S1, the membrane discontinuities largely encircled the condensed chromosome disc and were spatially aligned with the future segregation zone, further revealing the relative location of membrane discontinuities to chromosomes, at least at metaphase.
We further collected 3D information for a telophase cell containing three nuclei. This tri-nucleated cell was enclosed by three distinct plasma membranes, each harboring a single rupture site that merged to form a single rupture. The observation that membrane ruptures are present in a tri-nucleated cell is particularly informative. The tri-nucleated feature indicates that this cell underwent two rounds of cell division and that both divisions were at telophase. The presence of a single membrane rupture suggests that membrane discontinuities may persist throughout the cell cycle, as the second cell cycle began from a mother cell that still shared cytoplasm with its sister cell and already had one membrane rupture. Therefore, in addition to the mitotic phase, membrane discontinuities—at least in this context—also exist during the DNA synthesis stage.
(3) Lack of evidence for extension of the separated membrane
Although the authors speculate that resealing of the ruptured membrane occurs via extension of the separated membrane, no direct evidence supporting this mechanism is presented. Proximity to vesicles alone does not demonstrate that membrane extension occurs through vesicle fusion. More direct evidence is required to support this claim.
Thank you for raising this important point. We appreciate the opportunity to clarify our conclusion.
In our study, EM analysis revealed the presence of cellular vesicles in close proximity to both free membrane edges and the already separated sister plasma membranes (Figure 4). However, we acknowledge that without advanced live-cell imaging, it is not possible to conclusively determine whether the extension of these separated sister membranes occurs through vesicle fusion.
We realize that a statement in the Discussion section—“The expansion of the plasma membrane is generally driven by vesicle fusion”(page 16)—may have inadvertently led the reviewer to interpret this as our own conclusion regarding the mechanism of membrane extension in this context. In fact, that statement was intended to reflect the current general understanding of membrane expansion, not to imply that we had demonstrated such a mechanism for the free-ended sister membranes. As we subsequently noted, “However, this remains speculative and requires further experimental validation.”
To avoid any misunderstanding, we will revise this section to clearly state that the mechanism by which the separated sister membranes extend remains unknown and that further investigation is needed to determine how existing models of membrane expansion may apply to or be adapted for this novel context.
We thank the reviewer again for their thoughtful comment, which has helped us improve the clarity of our manuscript
(4) Inconsistency with published work
Numerous studies have examined cell division in developing C. elegans embryos using the GFP::PH(PLC1δ1) marker expressed from the ltIs38 transgene [pAA1; pie-1::GFP::PH(PLC1δ1) + unc-119(+)], generated by the Oegema lab (https://wormbase.org/species/c_elegans/transgene/WBTransgene00000911#01--10 ). To date, no study has reported membrane ruptures of the magnitude described here. The complexity of cell surface morphology from the 8- to 12-cell stages onward has been well documented, for example, by Fu et al. (2016) using light-sheet microscopy and 3D reconstruction (doi:10.1038/ncomms11088).
Supplementary Movies 5, 6, and 10 of this paper illustrate how single-plane images can easily produce apparent membrane discontinuities, for example, due to membrane orientations nearly parallel to the imaging plane.
The three single-plane images from only three embryos presented in Figure 6 are insufficient to support the authors' strong conclusions. Raw 3D data should be provided.
Thank you for this important comment. We fully agree that the GFP::PH(PLC1δ1) marker, generated by the Oegema lab, has been widely and effectively used to study various aspects of C. elegans embryonic development. In fact, we also employed this same marker in our study to assess membrane integrity.
However, while live imaging provides invaluable temporal resolution, its limitations in resolving membrane ultrastructure are substantial. In C. elegans embryos, early development is marked by rapid and extensive cell divisions. Within approximately 100 minutes, a two-cell embryo develops into one containing nearly 30 cells. During this fast-dividing stage, the rapid kinetics of membrane extension—approximately 20–30 seconds during metaphase and less than 3 minutes during cytokinesis— combined with embryo motility, introduce considerable spatiotemporal ambiguities. Furthermore, the longstanding invagination model of cytokinesis has shaped interpretations in the field, which may have led to ambiguous structures such as free-ended extensions being dismissed as potential artifacts rather than recognized as alternative morphological features. Theoretical and computational models have largely been built upon invagination-centric assumptions, which may have further constrained conceptual frameworks. Therefore, fluorescence protein-based live imaging analysis alone could not serve as a convincing approach to challenge the current dogma of cell division, nor did we intend it to.
However, when reexamined in light of our findings, previous studies using this same GFP marker have in fact revealed membrane discontinuities that went unnoticed. For example, Fu et al (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016. DOI:10.1038/ncomms11088) using light-sheet microscopy and 3D reconstruction, captured membrane discontinuities in cells undergoing mitotic phases such as anaphase or telophase. Similarly, an earlier study by Harrell and Goldstein (Harrell and Goldstein. 2011. Internalization of multiple cells during C. elegans gastrulation depends on common cytoskeletal mechanisms but different cell polarity and cell fate regulators. Developmental Biology. DOI:10.1016/j.ydbio.2010.09.012) showed regions where the GFP::PH signal appeared fuzzy and discontinuous.
Nevertheless, given the inherent limitations of fluorescence microscopy in resolving membrane ultrastructure, high-resolution electron microscopy—supported by rigorous controls and serial section analysis—remains the gold standard for definitively identifying such membrane discontinuities.
We acknowledge that our findings are surprising. We did not set out to challenge the long-held view of membrane integrity during cell division. In fact, this study began when our dedicated EM technician, Jingjing Liang, first observed membrane discontinuity phenomena in control samples—wild-type embryos. Had she not come across this observation, we likely would never have pursued this line of inquiry.
We appreciate the opportunity to clarify these points and thank the reviewer for thoughtful engagement with our work.
Reviewer #2 (Public review):
Summary:
Liang et al. explore an unusual observation of membrane discontinuities in dividing C. elegans embryonic cells. This report is the first to demonstrate that, instead of the classical invagination of membranes during cytokinesis, cells in the early embryos of C. elegans exhibit separation of sister membranes that extend independently. TEM images of high-pressure-frozen samples provide strong evidence for the presence of Membrane Openings (MOs) in cells at various stages of the cell cycle, predominantly during mitosis. High-resolution images (x 30,000) clearly show the wrinkled plasma membrane and smooth MOs.
The electron microscopy data are supported by the live cell imaging of strains with fluorescently tagged membrane markers. This study opens up the possibility of tracking MOs at other stages of C. elegans development, and also asks if it might be a common phenomenon in other species that exhibit rapid embryonic growth and divisions.
Strengths:
(1) Thorough verification of Membrane Openings (MO) by several methods:
(a) 4 independent sample batches.
(b) Examined historical collections.
(c) Analysed embryos at different stages of development. The absence of MOs in later stages (comma) serves as a negative control and gives confidence that MOs are genuine and not technical artifacts.
(2) Live cell imaging of strain with fluorescently labelled membranes provides realtime dynamics of membrane rupture.
(3) After observing the membrane rupture, the next obvious question is - what prevents the cytosol from leaking out? The EM images showing PBL and PEL - extracellular matrix serving as barriers for the cytosol are convincing.
Thanks to the reviewer for the encouragement. Highly appreciated.
Weakness:
(1) The association of membrane discontinuities with cell division is not convincing, as there are 159 cells out of 425 showing MOs, but it is not mentioned clearly how many of these are undergoing cell division. Also, it's not clear whether the 20 dividing cells analysed for MOs are a part of the 159 cells or a separate dataset. A graphical representation of the number of samples and observed frequencies would be helpful to understand the data collection workflow.
We sincerely thank the reviewer for raising this important question and appreciate the opportunity to clarify these points.
(1) Relationship between membrane discontinuities and cell division
In C. elegans embryos, early development is characterized by rapid and extensive cell division: within approximately 100 minutes, a two-cell embryo develops into one containing nearly 30 cells. Most of our electron microscopy (EM) analyses were performed on embryos at stages with fewer than 30 cells, in which the majority of cells are actively dividing. Therefore, it is reasonable to infer that the cells exhibiting membrane discontinuities (MOs) are predominantly mitotic. Supporting this, as embryos reached the comma stage—when cell proliferation declines and elongation begins—the incidence of MOs dropped sharply (0/13, 0/17, and 0/30 cells examined. This developmental specificity strongly links MOs to cell division.
Moreover, in single random EM sections, we observed MOs in cells displaying clear mitotic features, such as metaphase chromosomes aligned at the equatorial plate, or anaphase/telophase configurations (fewer than 10 chromosomal clumps or two nuclei sharing common cytoplasm). Thus, MOs are indeed present in mitotic cells.
From our 3D reconstruction (Figure 5), we identified a telophase cell containing three nuclei, each enclosed by its own plasma membrane, with each membrane harboring a single rupture that converged into a single opening. This tri-nucleated configuration indicates that the cell had undergone two rounds of division and was at telophase in both. The presence of a single membrane rupture in this context suggests that MOs can persist beyond mitosis, as the second cell cycle initiated from a mother cell that already shared cytoplasm with its sister and already contained a rupture. Thus, in this case, MOs were also present during DNA synthesis stage.
(2) Clarification of sample numbers and datasets
In Figure 1, we present results from a single EM section per embryonic cell, with sections randomly selected per embryo as detailed in Sup Figure 2. This initial dataset (425 cells) forms the basis of Figure 1.
From the same pool of 425 cells, we used additional EM sections—distinct from those shown in Sup Figure 2—to locate 20 dividing cells for analysis of membrane discontinuities. Thus, while these 20 cells originated from the same set of embryos, they were not derived from the sections used in Figure 1 or Sup Figure 2.
A graphical summary of sample numbers from the single-section analysis is already provided in Figure 1. Notably, cells with two clearly visible nuclei are more likely to be sectioned through or near their maximal diameter. In contrast, the randomly selected sections used for Figure 1 captured cells at variable planes, reducing the likelihood of observing MOs. Consistent with this, in the three embryos where no MOs were detected (one example is Sup Figure 2N), the sections likely passed through peripheral regions of the cells. Consequently, the frequency of MOs in randomly sectioned cells (Figure 1) is not directly comparable to that observed in the 20 dividing cells, which were analyzed using sections more likely to capture cells near their maximal diameter. These 20 dividing cells should therefore be considered a separate analysis. We will add detailed explanations in the Methods section to ensure this distinction is clearly understood.
We are grateful for the reviewer’s thoughtful feedback and believe these clarifications will improve the clarity and rigor of the manuscript.
(2) In Figures 3A and 3B, the resolution of the images is not enough to verify 3A as classical membrane invagination and 3B as detached sister membranes.
Thank you for your valuable comment. In the revised manuscript, we will provide additional images at higher magnification to better illustrate the classical membrane invagination in Figure 3A and the detached sister membranes in Figure 3B.
(3) Figure 6 lacks controls. How does the classical invagination look in this strain? Also, adding nuclear dye would be informative, in order to correlate the nuclear division with membrane rupture, as claimed.
Thank you for this important comment. As we addressed how we correlated nuclear division with membrane rupture in response to weakness (1), below we will focus on how we may distinguish classical invagination from membrane rupture.
While live imaging provides invaluable temporal resolution, its limitations in resolving membrane ultrastructure are substantial. In C. elegans embryos, early development is marked by rapid and extensive cell divisions. Within approximately 100 minutes, a two-cell embryo develops into one containing nearly 30 cells. During this fast-dividing stage, the rapid kinetics of membrane extension—approximately 20–30 seconds during metaphase and less than 3 minutes during cytokinesis— combined with embryo motility, introduce considerable spatiotemporal ambiguities. Furthermore, the longstanding invagination model of cytokinesis has shaped interpretations in the field, which may have led to ambiguous structures such as free-ended extensions being dismissed as potential artifacts rather than recognized as alternative morphological features. Theoretical and computational models have largely been built upon invagination-centric assumptions, which may have further constrained conceptual frameworks. Therefore, fluorescence protein-based live imaging analysis alone could not serve as a convincing approach to challenge the current dogma of cell division, nor did we intend it to.
However, when reexamined in light of our findings, previous studies using GFP::PH or similar markers have in fact revealed membrane discontinuities that went unnoticed. For example, using light-sheet microscopy and 3D reconstruction, Fu et al captured membrane discontinuities in cells undergoing division such as anaphase or telophase (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016.DOI:10.1038/ncomms11088)
Similarly, an earlier study by Goldstein et al. (Harrell and Goldstein. 2011. Internalization of multiple cells during C. elegans gastrulation depends on common cytoskeletal mechanisms but different cell polarity and cell fate regulators. Developmental Biology. DOI:10.1016/j.ydbio.2010.09.012) showed regions where the GFP::PH signal appeared fuzzy and discontinuous.
Here, to capture dynamic membrane events, our live imaging using the GFP::PH membrane marker was performed at 4-second intervals, approaching the practical limit for single-section scanning of the embryo. With single-plane live imaging, both membrane ruptures and free-ended sister membrane structures (Figures 6) could be detected, providing additional evidence that membrane rupture and independent extension of detached sister membranes underlie cytokinesis in C. elegans embryos.
However, given the inherent limitations of fluorescence microscopy in resolving membrane ultrastructure, high-resolution electron microscopy—supported by rigorous controls and serial section analysis—remains the gold standard for definitively distinguishing invagination from membrane discontinuities.
While future advances in imaging technology may enable real-time visualization at near-EM resolution, our extensive, multi-year effort to test the artifact hypothesis has convinced us that these membrane discontinuities are genuine biological features of dividing C. elegans embryonic cells.
Reviewer #3 (Public review):
Summary:
In this manuscript, the authors challenge a dogma in cell biology, namely that cells are at any time point engulfed by a continuous plasma membrane. Liang et al. find that during C elegans embryogenesis, a high number of cells are not entirely surrounded by a plasma membrane but show membrane openings (MOs). These openings are enriched at the embryo's periphery, towards the eggshell. The authors propose that plasma membrane discontinuities emerge during metaphase of mitosis and that independent extension of "sister membranes" engulfs the daughter cells.
Strengths:
On the positive side, the authors find plasma membrane discontinuities not only by electron microscopy but also by fluorescence microscopy and provide information about the dynamics of membrane openings and their emergence. While this is assuring, the authors conclude that MOs emerge during metaphase. From what the authors show, this particular information cannot be deduced, as there is no dynamic capture of a membrane scission event together with a chromatin marker that would indicate mitosis. The authors could, however, attempt to find such events in live movies, given the high incidence of MOs reported from their EM data.
Thanks to the reviewer for the encouragement. Highly appreciated.
Weaknesses:
In order to convincingly demonstrate the absence of any plasma membrane in the respective regions of the embryonic periphery or between cells of the embryo, the authors would have to show consecutive serial TEM sections where MOs are detected over more z-planes, beyond the mere 3D reconstructions. Although the authors state in the methods section that continuous ultrathin sections were cut for the metaphase sample (page 21, line 472), consecutive sections are never shown in TEM. While we do see the 3D reconstructions, better documentation of the underlying TEM data is missing. It would be necessary to show a membrane opening in consecutive z sections. Alternatively, the authors could seek the possibility to convincingly back up their claims with volume imaging by focused ion beam scanning EM (FIBSEM), where cellular volumes can be sectioned in almost isotropic resolution
We Thank the reviewer for raising these important technical concerns. We have taken this question seriously since first observing membrane discontinuities six years ago, and we have since conducted extensive controls to rule out fixation artifacts.
First of all, in addition to membrane discontinuities, we would like to highlight that a large number of single plasma membranes separating adjacent cytoplasmic domains were detected by EM (Figure 1, 3 and 4). This observation is particularly significant because the invagination model cannot account for the formation of single plasma membrane barriers between adjacent cytoplasmic domains. Instead, independent extension of detached sister membranes offers a plausible explanation for the generation of cytoplasm-immersed membranes. Furthermore, the morphology and continuity of these single cytoplasm-immersed membrane structures are well preserved, indicating successful EM processing and arguing against potential issues such as inadequate fixation or other technical limitations.
Second, we applied rigorous criteria for identifying membrane discontinuities:
(1) To test whether the discontinuities were preparation specific, we examined four independent sample batches and validated our findings using two EM techniques: transmission electron microscopy (HPF-TEM) and dual-beam scanning electron microscopy (SEM).
(2) We analyzed embryos across multiple developmental stages. Membrane discontinuities were observed in both intrauterine and laid embryos at early stages. However, as embryos reached the comma stage—a period marked by the onset of elongation and reduced cell proliferation—the incidence of discontinuities dropped dramatically (0/13, 0/17, and 0/30 cells examined). This developmental specificity argues strongly against a general fixation artifact, which would be expected to occur randomly across stages. Additionally, the eggshell is present throughout the embryonic stage of C. elegans; therefore, the dramatic reduction of membrane discontinuities in comma-stage of embryo argues against the possibility that the eggshell poses a fixation problem.
(3) Each putative membrane discontinuity was identified only after examining the entire plasma membrane of the cell on a given section. Importantly, aside from the discontinuity, the remainder of the plasma membrane remained intact. Moreover, in most cells, only a single discontinuity was present per section, arguing against random, widespread membrane tearing during preparation. Because EM preparation yields serial sections, we verified nearly all membrane discontinuities by examining adjacent sections. Again, the same membrane discontinuity was confirmed only after inspecting the entire plasma membrane on those neighboring sections as well. We will include this verification protocol in the revised Methods and additional imaging of consecutive sections would be provided if needed.
To further determine whether a dividing cell indeed contains one membrane rupture, we performed two serial reconstruction experiments using consecutive sections, as the reviewer suggested. First, we used HPF-TEM to analyze 105 consecutive sections of a metaphase cell, reconstructing the entire plasma membrane and chromosome configuration. We found that one membrane rupture largely encircled the chromosomal disc (Figure 2 and Video S1), spatially aligning with the future segregation zone. Second, we used AutoCUTS-SEM to collect approximately 600 sections covering ~95% of a telophase cell containing three nuclei sharing a common cytoplasm. This tri-nucleated cell was enclosed by three distinct plasma membranes, each harboring a single rupture site. These three ruptures converged to form a Yshaped exposed cytoplasmic region spanning >351 sections (Figure 5). Collectively, these reconstructions demonstrate that each cell contains only one discontinuity from a 3D point of view, further supporting that the phenomenon is not due to random sample preparation damage.
(4) In addition to EM, we performed live imaging of plasma membrane dynamics. While live imaging provides important temporal context, we recognize its limitations in resolving membrane ultrastructure. The rapid kinetics of membrane extension (approximately 20–30 seconds for metaphase and less than 3 minutes for cytokinesis), combined with embryo motility, introduces spatiotemporal ambiguities. To capture dynamic membrane events, our live imaging using the GFP::PH membrane marker was performed at 4-second intervals, approaching the practical limit for single-section scanning of the embryo. With single-plane live imaging, nevertheless, both putative membrane ruptures (Figure 6A) and free-ended sister membrane structures could be detected (Figures 6B and 6C), providing additional evidence that membrane rupture and independent extension of detached sister membranes underlie cytokinesis in C. elegans embryos. Notably, 3D membrane dynamics analysis using light-sheet microscopy (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016. DOI:10.1038/ncomms11088). revealed membrane ruptures in dividing early C. elegans embryonic cells, including during telophase and metaphase. Therefore, live imaging further validates the membrane rupture phenomena in dividing embryonic cells in C. elegans.
We are confident that the cumulative evidence presented here addresses the reviewer's concerns and demonstrates that the observed membrane discontinuities, as well as cytoplasm-immersed membranes, are not procedural artifacts but rather reflect a previously underappreciated aspect of plasma membrane dynamics during embryonic cell division.
Another critical issue concerns the detection of the membrane discontinuities in electron micrographs, which, in my opinion, is ambiguous. How do the authors reliably discriminate in their TEM images whether there is a plasma membrane or not? The absence - or weak appearance - of the stain of the electron dense material at membranes, which seems to be their criterion for MOs, is also apparent at other, intracellular membranes, like at the NE or at the ER (for example, see Figure 1C). Also, the plasma membrane itself appears unevenly stained in regions that the authors delineate as intact (for example, Figure 1C, 2B/1).
We thank the reviewer for raising this important concern.
First, our laboratory has extensive experience with electron microscopy across diverse biological systems, including neurons, muscle cells, and hypodermis in C. elegans, as well as tissues from Drosophila, mouse, bacteria, and cultured cells (Chen et al., 2013; Ding et al., 2018; Guan et al., 2022; Y. Li et al., 2018; Miao et al., 2024; Qin et al., 2014; Wang et al., 2026; J. Xu et al., 2022; M. Xu et al., 2021; L. Yang et al., 2020; X. Yang et al., 2019; Zhu et al., 2022). Importantly, we did not introduce any novel or unconventional steps in our EM preparation; all protocols were standard and well established. Thus, the observed membrane discontinuities are unlikely to result from technical inexperience or idiosyncratic methods.
Second, because EM preparation yields serial sections, we verified nearly all membrane discontinuities by examining adjacent sections. Specifically, a membrane discontinuity was confirmed only after inspecting the entirety of the plasma membrane in neighboring sections. We will include this verification protocol in the revised Methods section, and additional images of consecutive sections can be provided if needed.
Third, in addition to membrane discontinuities, a large number of single plasma membranes separating adjacent cytoplasmic domains were detected by EM (Figure 1, 3 and 4). This observation is particularly significant because the invagination model cannot account for the formation of single plasma membrane barriers between adjacent cytoplasmic domains. Instead, independent extension of detached sister membranes offers a plausible explanation for the generation of cytoplasm-immersed membranes. Furthermore, the morphology and continuity of these single cytoplasm-immersed membrane structures are well preserved, indicating successful EM processing and arguing against potential issues such as inadequate fixation or other technical limitations.
EM-related publications by Jingjing Liang:
Chen D, Jian Y, Liu X, Zhang Y, Liang J, Qi X, Du H, Zou W, Chen L, Chai Y, Ou G, Miao L, Wang Y, Yang C. 2013. Clathrin and AP2 Are Required for Phagocytic Receptor-Mediated Apoptotic Cell Clearance in Caenorhabditis elegans. PLoS Genetics 9:e1003517. DOI: https://doi.org/10.1371/journal.pgen.1003517
Ding L, Yang X, Tian H, Liang J, Zhang F, Wang G, Wang Y, Ding M, Shui G, Huang X. 2018. Seipin regulates lipid homeostasis by ensuring calcium‐dependent mitochondrial metabolism. The EMBO Journal 37:e97572. DOI: https://doi.org/10.15252/embj.201797572
Guan L, Yang Y, Liang J, Miao Y, Shang A, Wang B, Wang Y, Ding M. 2022. ERGIC2 and ERGIC3 regulate the ER‐to‐Golgi transport of gap junction proteins in metazoans. Traffic 23:140–157. DOI: https://doi.org/10.1111/tra.12830
Li Y, Zhang Y, Gan Q, Xu M, Ding X, Tang G, Liang J, Liu K, Liu X, Wang X, Guo L, Gao Z, Hao X, Yang C. 2018. C . elegans -based screen identifies lysosome-damaging alkaloids that induce STAT3-dependent lysosomal cell death. Protein & Cell 9:1013–1026. DOI: https://doi.org/10.1007/s13238-018-0520-0
Miao Y, Du Y, Wang B, Liang J, Liang Y, Dang S, Liu J, Li D, He K, Ding M. 2024. Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation. eLife 13:RP96353. DOI: https://doi.org/10.7554/eLife.96353
Qin J, Liang J, Ding M. 2014. Perlecan Antagonizes Collagen IV and ADAMTS9/GON-1 in Restricting the Growth of Presynaptic Boutons. Journal of Neuroscience 34:10311–10324. DOI: https://doi.org/10.1523/JNEUROSCI.5128-13.2014
Wang Z, Zhang L, Zhou B, Liang J, Tian Y, Jiang Z, Tao J, Yin C, Chen S, Zhang W, Zhang J, Wei W. 2026. A single MYB transcription factor GmMYB331 regulates seed oil accumulation and seed size/weight in soybean. Journal of Integrative Plant Biology 68:470– 485. DOI: https://doi.org/10.1111/jipb.70101
Xu J, Chen S, Wang W, Man Lam S, Xu Y, Zhang S, Pan H, Liang J, Huang Xiahe, Wang Yu, Li T, Jiang Y, Wang Yingchun, Ding M, Shui G, Yang H, Huang Xun. 2022. Hepatic CDP-diacylglycerol synthase 2 deficiency causes mitochondrial dysfunction and promotes rapid progression of NASH and fibrosis. Science Bulletin 67:299–314. DOI: https://doi.org/10.1016/j.scib.2021.10.014
Xu M, Ding L, Liang J, Yang X, Liu Y, Wang Y, Ding M, Huang X. 2021. NAD kinase sustains lipogenesis and mitochondrial metabolism through fatty acid synthesis. Cell Reports 37:110157. DOI: https://doi.org/10.1016/j.celrep.2021.110157
Yang L, Liang J, Lam SM, Yavuz A, Shui G, Ding M, Huang X. 2020. Neuronal lipolysis participates in PUFA‐mediated neural function and neurodegeneration. EMBO reports 21:e50214. DOI: https://doi.org/10.15252/embr.202050214
Yang X, Liang J, Ding L, Li X, Lam S-M, Shui G, Ding M, Huang X. 2019. Phosphatidylserine synthase regulates cellular homeostasis through distinct metabolic mechanisms. PLOS Genetics 15:e1008548. DOI: https://doi.org/10.1371/journal.pgen.1008548
Zhu J, Lam SM, Yang L, Liang J, Ding M, Shui G, Huang X. 2022. Reduced phosphatidylcholine synthesis suppresses the embryonic lethality of seipin deficiency. Life Metabolism 1:175–189. DOI: https://doi.org/10.1093/lifemeta/loac02
