A FUCCI sensor reveals complex cell cycle organization of Toxoplasma endodyogeny

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   Dec 19, 2024

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   Reply to the reviewers

   We thank reviewers for their comments and constructive criticisms of our study. We have implemented* corrections** that were suggested for the manuscript, and we have also clarified any concerns that were raised in our responses below. *

   *Reviewer #1 *

   Overall technology development is good though as they claim that they are first is not true as it has been used earlier by https://doi.org/10.1128/msphere.00160-22. Hence may be that they have used to decipher the cell cycle.

   *The cited paper used FUCCI in the host cells and not in the parasites themselves. Our study thus reports the first FUCCI model in a **unicellular **eukaryote. *

   The manuscript is …

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   Reply to the reviewers

   We thank reviewers for their comments and constructive criticisms of our study. We have implemented* corrections** that were suggested for the manuscript, and we have also clarified any concerns that were raised in our responses below. *

   *Reviewer #1 *

   Overall technology development is good though as they claim that they are first is not true as it has been used earlier by https://doi.org/10.1128/msphere.00160-22. Hence may be that they have used to decipher the cell cycle.

   *The cited paper used FUCCI in the host cells and not in the parasites themselves. Our study thus reports the first FUCCI model in a **unicellular **eukaryote. *

   The manuscript is extremely dense and at times very difficult to read and to be clear if they are focussing on the technology or cell cycle. The technology may be a better part of manuscript but the dissection of cell cycle is not very novel and at times very confusing to follow. Many of these aspects has been dissected out previously from their own group and many group in Toxoplasma and Plasmodium and it is quite known about that the cell cycle in Apicomplexa is very complex.

   We adapted FUCCI to the* Toxoplasma model to *help dissect the organization of its cell cycle, which as the reviewer noted, is highly complex. While overlaps between some phases were anticipated based on prior data, these overlaps had not been measured. We were able to determine the extent of these overlaps in the post-G1 period and describe the organization of the non-conventional cell cycle of T. gondii.

   Another aspect that most FUCCI use Geminin and CDT1 factors and since Geminin is not present it would have been better to validate that with CDT1 that is present in Apicomplexa and may be more relevant than PCNA1.

   Unfortunately, the Toxoplasma ortholog of CDT1 (TgiRD1) cannot be used as a FUCCI marker for the reasons stated in lines 116-117; the expression of TgiRD1 is not limited to a specific cell cycle phase (Hawkins et al., 2024). PCNA1 can be (and has been) used as a FUCCI marker, but it was not considered an ideal marker in mammalian cells due to its relatively low expression levels. However, Toxoplasma PCNA1 is highly abundant in tachyzoites, and its expression correlates with the period of DNA replication. Furthermore, Plasmodium ortholog of PCNA1 had been used as a DNA replication sensor in the recent studies (35353560). *Altogether, it validates PCNA1 as an appropriate S-phase FUCCI probe. *

   The first part of the manuscript only deals with first to identify the function and localisation of PCNA1 and then develop FUCCI technology and then go on to study cell cycle. So the focus of the manuscript is not clear. It seems three different results are just assembled together in one manuscript with out clear focus. In order to get clear focus the authors should clear set out the focus as to why they developed FUCCCI and how they decipher either replication, budding, apical or basal complex, centrosome or cytokinesis as well to be used for drug discovery The way it is organised it is not flowing well and confuses the reader who may not be aware of different compartment of Toxoplasma cell or not a molecular parasitologist.

   We believe the reviewer has described the logic of our study. Our goal was to* dissect the cell cycle. Consequently, we adapted a suitable technology, FUCCI. We described the relevant experiments that allowed us to produce a new molecular tool for an apicomplexan model, and illustrated how we used this tool to better understand the complicated processes of its cell division. Therefore, we organized our study accordingly and included our goal, plans, results, and conclusions that support the success of adopted technology and establishment of the cell cycle organization. We hope this brief explanation can provide some clarification for the reviewer.*

   Some of the conclusion on the that Replication starts at centromere region is not novel and has been studied previously.

   *We agree that the centromeric start of DNA replication is not a novel feature, which is stated in the text. This result was shown to demonstrate that Toxoplasma replicates its DNA according to the rules conserved across eukaryotes. *

   The manuscript needs revising by writing precisely eliminating too much literature reference in the result section with clear focus. Some of these references can be elaborated in the introduction and discussion to keep the focus.

   We examined the results section, and as much as we wanted to comply with this reviewer, we found no references that could be eliminated or transferred to the introduction. We believe that to aid the reader, some foundational knowledge needs to be presented together with obtained results to support those findings.

   Some points with respect to figures: Generally with image panels, arrows don't stand out well

   We* have adjusted the images.

   Fig1: no scale bars and the green arrow do not stand out. So may be to make white.

   *The scale bar can be found in the bottom right image, which applies to every image in the panel. We changed the color of the arrows. *

   Fig 2E: state the time point in the fig without IAA treatment (-IAA)

   The requested information was added to the figure legend.

   Fig4: no bell shaped curve

   We rephrased the description. The” bell-shape” analogy applies to the temporal dynamics of DNA replication, which starts with a single aggregate, expands to numerous replication foci, and is reduced to a few aggregates at the end of replication. We attempted to quantify aggregates, but their irregular shape makes this task impossible. Our statement is supported by steady-state images and real-time microscopy of the DNA replication included in the manuscript.

   Fig 5D: it isn't obvious what the numbers on the right hand side of the graph mean. If it is size, there should be a unit given

   We provided an explanation in the figure legend*.

   Figure 6 - how do they determine that the tachyzoites have progressed through 61% of S phase? Make this clearer here.

   *We examined only DNA replicating parasites (S-phase) and determined the fraction of BCC0-positive (39%) and BCC0-negative (61%) tachyzoites. Quantifications can be found in Table S4, in the S-C worksheet. *

   Fig7: it a strange way of ordering the figure as FigE is after Fig F hence no logical order. Thank you, we have corrected the order of these panels*. *

   Fig 8H is not mentioned in the text

   *Thank you, we referenced the wrong panel. Fig. 8H is now included in the text. *

   Figure 9 is nice and useful but the arrows could be made proportional of time spent in each cell cycle phase. They're a little off in the conventional cell cycle at the minute

   • *These schematics are intended to illustrate the dramatic difference in cell cycle organization rather than to directly describe cell cycle organization, the latter of which can be found in Figure 6.

   Some comment on the text in the manuscript: Line 137: describing the expression pattern: the following papers first described the expression pattern of PCNA1 and 2 can be cited in the result. https://doi.org/10.1016/j.molbiopara.2005.03.020 We added the reference.

   Line 154: Provide schematic for AID HA cloning and confirmation.

   The schematics and PCR confirmations* can be found in the supplemental figure S2.

   Line 157: Fig 2 after 4 h treatment FACS analysis shows more than 1 and less than 2n genomic content. Does this study have any -IAA treated control for 4h and 7h to compare as what should the standard genomic content to be there at this time point of development. At 4 h of development can the authors provide any statistical analysis with their 3 experiments to prove their point that the replication is actually stalled. Downregulation of TgPCNA1 as shown is western blot is still basal protein left to carry the genomic replication in 7 mins. Can authors also state that TgPCNA 2 which is although non-essential but has no redundant role in the replication machinery.

   The -IAA control is indicated as 0h and is shown in blue. The statistical analysis of three independent experiments showing the increase of the S-phase population is included in Table S3. The Fig. 2 WB shows over 99% TgPCNA1 degradation, and the residual >1% would be insufficient to carry out full DNA replication. This residual signal is likely due to PCNA1 remaining* in complex, which would resist **proteolysis. Unfortunately, we do not feel comfortable to make the final statement suggested. We believe that the lack of TgPCNA2 complementation with yeast PCNA1 (Guerini et al, 2005) is insufficient to draw the conclusion that TgPCNA2 plays a non-redundant role in Toxoplasma replication machinery. *

   Line 178 : typing error "that that

   Thank you, this has been corrected*.

   Line 179: states the role of TgPCNA 1 in DNA1 replication, however line 159 and 160 states the TgPCNA1 deficient can fulfil DNA replication. Can author confirm this contrast in the results. Results trying to illustrate the same fact TgFUCCIs or TgPCNA1ng that TgPCNA1 first aggregates at centromeres and then distributed on many replication forks and disappears late during cytokinesis. The part of the result can be merged.

   *We apologize for the **confusion. We rephrased our statements and supported them by corresponding references. Although it may seem repetitive, but it was our intention to emphasize a consistent spatial-temporal expression of TgPCNA1-HA and TgPCNA1-NG. *

   Line 189: Typing error, should say "such as nucleus", currently as is missing

   Thank you*, this has **been corrected.

   Line 346-349: basically explaining the same thing twice.

   *We apologize for the confusion, the first sentence describes compartments where MORN1 is located. The second sentence describes how MORN1 localization changes during cell cycle progression, information which is used later in our **quantitative IFA of cell cycle phases. *

   Line 347 - immunfluorescent should be immunofluorescence

   Thank you, this has been corrected*.

   Line 395-399: does this study has any non-inhibited (-IAA control) at 4h and 7 h. for fig 7C & 7G. Can the authors provide any statistical analysis for the significance with their 3 experiments.

   *The untreated control (7h mock) is shown as 0h treatment (first bar in each panel). The figure also shows the results of the statistical analysis (t-test, numbers above) that can also be found in Table S7.

   Line 415: Why the authors have not used the TgFUUCI sc lines which expresses the TgPCNAng and IMCmch both. This could have helped to understand the real time dynamics of DNA replication and budding initiation (cytokenesis), rather then fixing and staining with TgIMC.

   *The recent study by Gubbels lab identified the earliest known budding marker BCC0. Unfortunately, BCC0 is a low abundant factor and cannot be used in FUCCI. IMC3 emerges in the midst of budding when the daughter conoid and polar rings are assembled and thus does not signify either the beginning or the end of cytokinesis. We added IMC3 as a supporting budding marker, while our primary focus remains on the DNA replication marker PCNA1. *

   Overall good technology development as FUCCI but the rest of the manuscript is extremely dense and the focus of the study is not clear after technology part. The complexity of the cell cycle is known and hence not much novelty here and extremely descriptive and hard read. Science can be simplified.

   The reviewer agrees the apicomplexan cell cycle is highly complex, and the field has worked diligently to piece together what we can about it, which contributes to the density of the manuscript. We hope that the targeted audience will find it thoughtful, and we strove to provide sufficient information for those outside our field. We also respectfully disagree that our study offers little novelty; while it is known how complex the apicomplexan cell cycle is, there is still much to uncover. While overlapping cell cycle phases exist in other eukaryotes, there were no such studies that showed the degree of these overlaps across the entire T. gondii cell cycle. We believe there are valuable insights to be gained from the identification of the composite cell cycle phase, which in turn could help draw attention to other understudied features of the cell cycle in non-conventional eukaryotes*. *

   *Reviewer #2 *

   1. It is not always clear where apical and basal ends of the parasite are. E.g. in Fig 3F are the two parasites on the right facing down with their apical end? In Fig 4 it is even harder to see. Might be helpful to turn these images with their apical end up to make comparative interpretation of figures easier. In the text it mentions that PCNA1 concludes at the 'proximal' end of the nucleus (or with the nucleus proximal, which is not clear either??). Please define clearly where the proximal site is, as it is not clear in the figures or in the movie (the 'last focus' marker in Fig 4D??). *Thank you for the suggestion. We rotated images in Fig. 3 and marked the parasite ends in Fig. 4. *We also indicated parasites’ polarity in the movies.

   Synchrony of replication cycle. Tight synchronization depends on the retention of the cytoplasmic bridge, as mentioned by the authors. In larger vacuoles, it is very conceivable not all parasites are connected with each other (notably in large cysts with bradyzoites), which could lead to loss of tight synchrony. The results section states "One plausible explanation is that the rosette split shortens the communication path between tachyzoites". This is somewhat cryptic language: does a 'rosette split' imply the rupture of the cytoplasmic bridge? This statement should be clarified. Another factor could be centrosome maturation, with the mother centrosome ready sooner than the daughter, which is a model proposed in schizogony, where the nuclear cycles in a shared cytoplasm are even more asynchronous/independent.

   Yes, by ‘rosette split’, we refer to the break of the connection, or a cytoplasmic bridge. The model based on centrosome maturation is interesting, however, it does not explain the synchronization of a vacuole of 16, unless centrosome age resets at that point*. *

   Centrosome duplication. This has been documented to occur at the basal side of the nucleus (the whole nucleus rotates for centrosome duplication). The images as depicted in Fig 6 do not seem to indicate this event, possibly because it is not easy to track apical and basal end of the cell (#1 above). Please comment, as this could be an additional spatial cue to the specific stage of the cycle.

   This is a very interesting suggestion, thank you. Indeed, the centrosome often duplicates away from the apical end (disconnects from the Golgi), sometimes on the side or the basal end, but quickly rotates back to the apical position to reconnect with co-segregating organelles. Centrosome traveling is an interesting feature, and it is possible that this reorientation back to the apical end signifies budding initiation. We will explore this hypothesis in future studies.

   • Specific experimental issues that are easily addressable.

   1. The term "Apicomplexan" should be spelled with a lower case "apicomplexan", which is not consistently applied throughout the manuscript. Thank you, we have corrected the spelling*. *

   1. Line 567 the term used in 2008 was "tightly knit" not "closely woven". We wanted to avoid the exact citation and rephrased the title of the review.

   *Reviewer #3 *

   -The authors choose to describe PCNA1 and IMC3 as FUCCI markers. The efficiency of this system in mammalian cells is based on the proof that the markers are regulated through a rapid proteolysis process. However, the data available for these markers point toward a transcriptional regulation of these markers (Toxodb and (1)). In contrast, the authors do not provide any data indicating that these proteins are true FUCCI markers. Consequently, they should not use the term FUCCI throughout the paper unless they prove that the cell cycle expression depends on proteolysis. For example, the authors could express these genes with a promoter that is not cell cycle regulated.

   PCNA1 was one of the original FUCCI markers for* mammalian cells, later replaced by the more abundant geminin. PCNA1 ubiquitination is well supported across all eukaryotes, and we believe there is much data to support this same turnover mechanism acts to regulate PCNA1 in Toxoplasma. Transcriptional profiles show that TgPCNA1 mRNA is constantly present in cells, never dropping below 80%, making this mRNA is among the most abundant in the cell. It also indicates that proteolysis, rather than halted transcription, controls TgPCNA1** protein levels, since TgPCNA1 protein expression drops to nearly undetectable levels in early G1 and budding (Fig. 1). In addition, TgPCNA1 is highly conserved in** structure** (Fig. S1) and in function (TgPCNA1 interactome, Fig. 1). The TgPCNA1 Ub sites were detected in global ubiquitome analyses (ToxoDB), supporting the fact that TgPCNA1 protein abundance is regulated by ubiquitin-dependent degradation. Furthermore, PCNA1 as a FUCCI marker in model eukaryotes was not tested for proteolysis because it was **unquestionable **that PCNA1 is regulated by proteolysis. In addition, Plasmodium ortholog of PCNA1 had been used as a DNA replication sensor in the recent studies (35353560), which validates PCNA1 as an appropriate S-phase FUCCI probe. **The modern FUCCI probes are fragments of CDT1 and Geminin mimicking the spatiotemporal expression of the corresponding cell cycle regulators. **The transcriptional profile of TgIMC3 is also largely unchanged across the cell cycle, which heavily implies that proteolysis controls its dynamic protein expression. Therefore, we believe that the term FUCCI applies to TgPCNA1 and TgIMC3. *

   -The authors show that the localization of PCNA1 change during the cell cycle and indicate that the PCNA1 aggregates observed are replication forks. They do not provide data supporting this. They should co-localize these aggregates with other markers such as ORC, MCM proteins or DNA polymerase to better characterize these aggregates. There are number of techniques that could be used to localize the origin(s) of replication. Similarly, ExM should be used to characterize the colocalization between PCNA1 aggregates and the centromeres. As such, the images provided are of poor quality and do not support the author conclusions. The few PCNA1 aggregates toward the end of the S phase are also not characterized. Are they telomeres?

   Although this is an important point, such detailed analyses of the DNA replication machinery is out of the scope of the current study and will be examined in a follow-up study. Data that suggest the aggregates correspond to replication forks include proteomics analyses of chromatin-bound PCNA1 that identified replisome components such as the MCM, high conservation of TgPCNA1 sequence and structure (Fig. S1), and its conserved* interactions (Fig. 1)**. **Recent studies used Plasmodium ortholog of PCNA1 to trace DNA replication dynamics during schizogony (35353560), **Therefore, we doubt that TgPCNA1 would perform functions outside of its role as a DNA replication factor, which has been extensively studied in other eukaryotes. *

   • The authors characterized the proteins associated with PCNA1. All the proteins found to potentially interact are chromatin-bound and are not naturally found in other localization (2). It is unclear why the authors insist on the fact that there are two PCNA1 complexes (one chromatin-bound and one non-chromatin bound). More concerning is the lack of verification of this dataset through reciprocal IP for example.

   The PCNA IP was used to confirm its conserved function as a DNA replication factor; similarly to what was observed in other eukaryotes, we detected PCNA in both a chromatin-bound and unbound state. PCNA1 is produced in late G1 (diffuse nuclear stain) but is engaged in the replisome only upon DNA replication initiation (aggregated form). Rather than characterize the function of the highly conserved PCNA1, our primary goal was to determine the Toxoplasma cell cycle organization, which explains our choice of the experimental design.

   • Quantification of some of the phenotypes is lacking. For example, the DNA content analysis are shown but the change in number are not. Similarly, there is no quantification of the PCNA1 mutant phenotypes observed by ExM. Quantification of the bell shape observed by video-microscopy in figure 4 should also be provided.

   *The quantifications supporting the main claims of our study are included in the five supplemental **Tables S3-S8, including DNA content and microscopy analysis of the phenotype. **The U-ExM microscopy has been solely used to visualize details of the phenotype. *

   • The PCNA1 mutant phenotypes are not sufficiently explored by ExM. What happen to the mitotic spindle? What happens to kinetochore (CenH3 is a centromere protein and does not represent kinetochores)? Many markers for these structures have been described, see (3).

   *The primary goal of our study was to examine and map out the organization of the tachyzoite cell cycle. PCNA1 deficiency was used to demonstrate that Toxoplasma PCNA1 is a conserved DNA replication factor and can be used as an S-phase marker in FUCCI. Thus, we focused on the mutant-induced changes in the dynamics of DNA replication (DNA content) and arrest prior to mitosis (unresolved centrocone). *

   • TgPCNA1NG strain has a number of concerns. The localization to the daughter cells conoids seems artificial since not observed in the HA-AID mutant and the expression pattern seems different as well than the previous mutant suggesting the mNG tag is affecting the localization and expression dynamics. Did the authors explore other fluorescent proteins to verify that these discrepancies where not due to this tag ?

   The conoidal PCNA1 accumulation was detected only with NeonGreen-tagged PCNA1. We also built and examined tdTomato- and mCherry-tagged versions and detected minor accumulations in the conoid of tdTomato-tagged PCNA1, but not with the mCherry-tagged variant. We believe these aggregations could be attributed to the partially degraded PCNA1-NeonGreen having an affinity to conoidal proteins, thus producing this unexpected signal. Although not included* in the manuscript, our quantifications, based on both PCNA1-HA and PCNA1-NeonGreen, showed similar cell cycle organization (G1, S and budding phases) of tachyzoites. The FUCCI probe is an** indicator of the cell cycle phase. It does not have to be a functional protein. As we mentioned before, many FUCCI probes are fragments of the factors that mimic the spatiotemporal expression of the corresponding cell cycle regulators*.

   -Cytokinesis seems to be only defined by the presence of IMC3. The marker appears early during the budding process and it is not normally considered as a cytokinesis marker. The author should the text to reflect this.

   *We agree with the reviewer that IMC3 is not a true budding marker, which is why we used BCC0 in our *quantifications. IMC3 is proven to broadly define the mid-budding stage, making it a convenient supplemental marker. We are currently exploring and testing alternative and additional FUCCI markers. It is not an easy task, since these markers are required to have high expression levels and to be localized into large organelles. For instance, BCC0 was eliminated due to low abundance.

   • Throughout the manuscript, the authors seems to ignore an essential characteristic of the tachyzoite cell cycle: the nuclear cycle and the budding cycle are independently regulated. It is therefore normal they overlap as it has been shown by the authors themselves in previous studies. This should be better described and discussed in the paper to understand the peculiarities of the parasite cell cycle.

   We apologize for the confusion, but the tachyzoite cell cycle does not contain a nuclear cycle, it consists of a single budding cycle. The nuclear cycle is only a feature in multinuclear cell cycles such as schizogony and endopolygeny. This is the main reason why the overlap between phases is so surprising.

   • l196: "The surface of the growing buds": could the authors rephrase?

   We rephrased the statement.

   -L217: proximal end of the nucleus rather than "parasite ".

   *We clarified the statement. It is, in fact, the shift of the nucleus to the proximal end of the parasite.

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   Referee #3

   Evidence, reproducibility and clarity

   This is a manuscript from Batra et al. entitled "A FUCCI sensor reveals complex cell cycle organization of Toxoplasma endodyogeny ". It describes the characterization of PCNA1 as cell cycle marker in the parasite Toxoplasma gondii. Tachyzoite endodyogeny is a simplified division process that is crucial for the proliferation of the parasite. Some studies have used fluorescent markers to describe the segregation of organelles and the nuclear division during endodyogeny but the production of more tools to dissect the cell cycle and better characterize mutants is timely. Most of the experiments are based on characterization of PCNA1 …

   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 #3

   Evidence, reproducibility and clarity

   This is a manuscript from Batra et al. entitled "A FUCCI sensor reveals complex cell cycle organization of Toxoplasma endodyogeny ". It describes the characterization of PCNA1 as cell cycle marker in the parasite Toxoplasma gondii. Tachyzoite endodyogeny is a simplified division process that is crucial for the proliferation of the parasite. Some studies have used fluorescent markers to describe the segregation of organelles and the nuclear division during endodyogeny but the production of more tools to dissect the cell cycle and better characterize mutants is timely. Most of the experiments are based on characterization of PCNA1 mutant and the use of a strain expressing a PCNA1-mNG construct. Unfortunately, there are a number of concerns in this study that need to be addressed.

   Major concerns:

   • The authors choose to describe PCNA1 and IMC3 as FUCCI markers. The efficiency of this system in mammalian cells is based on the proof that the markers are regulated through a rapid proteolysis process. However, the data available for these markers point toward a transcriptional regulation of these markers (Toxodb and (1)). In contrast, the authors do not provide any data indicating that these proteins are true FUCCI markers. Consequently, they should not use the term FUCCI throughout the paper unless they prove that the cell cycle expression depends on proteolysis. For example, the authors could express these genes with a promoter that is not cell cycle regulated.
   • The authors show that the localization of PCNA1 change during the cell cycle and indicate that the PCNA1 aggregates observed are replication forks. They do not provide data supporting this. They should co-localize these aggregates with other markers such as ORC, MCM proteins or DNA polymerase to better characterize these aggregates. There are number of techniques that could be used to localize the origin(s) of replication. Similarly, ExM should be used to characterize the colocalization between PCNA1 aggregates and the centromeres. As such, the images provided are of poor quality and do not support the author conclusions. The few PCNA1 aggregates toward the end of the S phase are also not characterized. Are they telomeres?
   • The authors characterized the proteins associated with PCNA1. All the proteins found to potentially interact are chromatin-bound and are not naturally found in other localization (2). It is unclear why the authors insist on the fact that there are two PCNA1 complexes (one chromatin-bound and one non-chromatin bound). More concerning is the lack of verification of this dataset through reciprocal IP for example.
   • Quantification of some of the phenotypes is lacking. For example, the DNA content analysis are shown but the change in number are not. Similarly, there is no quantification of the PCNA1 mutant phenotypes observed by ExM. Quantification of the bell shape observed by video-microscopy in figure 4 should also be provided.
   • The PCNA1 mutant phenotypes are not sufficiently explored by ExM. What happen to the mitotic spindle? What happens to kinetochore (CenH3 is a centromere protein and does not represent kinetochores)? Many markers for these structures have been described, see (3).
   • TgPCNA1NG strain has a number of concerns. The localization to the daughter cells conoids seems artificial since not observed in the HA-AID mutant and the expression pattern seems different as well than the previous mutant suggesting the mNG tag is affecting the localization and expression dynamics. Did the authors explore other fluorescent proteins to verify that these discrepancies where not due to this tag ? -Cytokinesis seems to be only defined by the presence of IMC3. The marker appears early during the budding process and it is not normally considered as a cytokinesis marker. The author should the text to reflect this.
   • Throughout the manuscript, the authors seems to ignore an essential characteristic of the tachyzoite cell cycle: the nuclear cycle and the budding cycle are independently regulated. It is therefore normal they overlap as it has been shown by the authors themselves in previous studies. This should be better described and discussed in the paper to understand the peculiarities of the parasite cell cycle.

   Minor

   • l196: "The surface of the growing buds": could the authors rephrase?
   • L217: proximal end of the nucleus rather than "parasite ".

   1. Behnke,M.S., Wootton,J.C., Lehmann,M.M., Radke,J.B., Lucas,O., Nawas,J., Sibley,L.D. and White,M.W. (2010) Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PloS One, 5, e12354.
   2. Barylyuk,K., Koreny,L., Ke,H., Butterworth,S., Crook,O.M., Lassadi,I., Gupta,V., Tromer,E., Mourier,T., Stevens,T.J., et al. (2020) A Comprehensive Subcellular Atlas of the Toxoplasma Proteome via hyperLOPIT Provides Spatial Context for Protein Functions. Cell Host Microbe, 28, 752-766.e9.
   3. L,B., N,D.S.P., Ec,T., D,S.-F. and M,B. (2022) Composition and organization of kinetochores show plasticity in apicomplexan chromosome segregation. J. Cell Biol., 221.

   Significance

   This study provides the characterization of a new cell cycle marker to decipher the tachyzoite cell cycle of the apicomplexan parasite Toxoplasma gondii. A better understanding of the cell cycle is needed to prevent the proliferation of this parasite. This study builds on previous works characterizing organellar segregation in T. gondii. It provides data about the overlap of each cell cycle phase and the synchronicity of the cell cycle in a single vacuole. However, it is limited by the use of a single marker and more data are needed to support the conclusions of this study. This study can be of interest to a broad audience.

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   Referee #2

   Evidence, reproducibility and clarity

   • Are the key conclusions convincing?

   The data support the new model put forward in the final figure: a composite cell cycle phase

   There are couple of points that need attention:

   1. It is not always clear where apical and basal ends of the parasite are. E.g. in Fig 3F are the two parasites on the right facing down with their apical end? In Fig 4 it is even harder to see. Might be helpful to turn these images with their apical end up to make comparative interpretation of figures easier. In the text it mentions that PCNA1 concludes at the 'proximal' end of the nucleus (or with the nucleus proximal, which is not clear either??). Please …

   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 #2

   Evidence, reproducibility and clarity

   • Are the key conclusions convincing?

   The data support the new model put forward in the final figure: a composite cell cycle phase

   There are couple of points that need attention:

   1. It is not always clear where apical and basal ends of the parasite are. E.g. in Fig 3F are the two parasites on the right facing down with their apical end? In Fig 4 it is even harder to see. Might be helpful to turn these images with their apical end up to make comparative interpretation of figures easier. In the text it mentions that PCNA1 concludes at the 'proximal' end of the nucleus (or with the nucleus proximal, which is not clear either??). Please define clearly where the proximal site is, as it is not clear in the figures or in the movie (the 'last focus' marker in Fig 4D??).
   2. Synchrony of replication cycle. Tight synchronization depends on the retention of the cytoplasmic bridge, as mentioned by the authors. In larger vacuoles, it is very conceivable not all parasites are connected with each other (notably in large cysts with bradyzoites), which could lead to loss of tight synchrony. The results section states "One plausible explanation is that the rosette split shortens the communication path between tachyzoites". This is somewhat cryptic language: does a 'rosette split' imply the rupture of the cytoplasmic bridge? This statement should be clarified. Another factor could be centrosome maturation, with the mother centrosome ready sooner than the daughter, which is a model proposed in schizogony, where the nuclear cycles in a shared cytoplasm are even more asynchronous/independent.
   3. Centrosome duplication. This has been documented to occur at the basal side of the nucleus (the whole nucleus rotates for centrosome duplication). The images as depicted in Fig 6 do not seem to indicate this event, possibly because it is not easy to track apical and basal end of the cell (#1 above). Please comment, as this could be an additional spatial cue to the specific stage of the cycle.

   • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

   The authors are on the conservative end of interpretations and clearly outline the limitations of their approaches and observations, while discussing alternative interpretations.

   • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

   No, the presented experiments and data are very complete

   • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

   n/a

   • Are the data and the methods presented in such a way that they can be reproduced?

   yes

   • Are the experiments adequately replicated and statistical analysis adequate?

   yes

   Minor comments:

   • Specific experimental issues that are easily addressable.

   1. The term "Apicomplexan" should be spelled with a lower case "apicomplexan", which is not consistently applied throughout the manuscript.
   2. Line 567 the term used in 2008 was "tightly knit" not "closely woven".

   • Are prior studies referenced appropriately?

   Yes

   • Are the text and figures clear and accurate?

   Yes, exceptional

   • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

   See major point #1 above.

   Referees cross-commenting

   Comment to Rev 1: https://doi.org/10.1128/msphere.00160-22. reports on use of FUCCI in the host cell, not in the parasite itself. This comment therefore does not apply.

   Comment to Rev 3: the technicality on FUCCI acting on the protein level. That is a legit concern that needs attention, and could be fixed by avoiding the term FUCCI, or putting the term in the exact context.

   Looks like a shared general concern is that it is not always clear where apical and basal ends are in the presented data. This should be addressed in revision.

   Significance

   • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

   The presented manuscript reports on a technical innovation in Apicomplexa: establishing a FUCCI system. However they did not stop there and added additional markers to unravel the timing and nature of S/M/G2/C overlaps that illuminated previously underappreciated or unknow details. The tools assembled here will be of great value for understanding not only T. gondii endodyogeny checkpoints and sequence of events, but also paves the way for similar studies in more complex apicomplexan cell division modes, like schizogony and endopolygeny.

   • Place the work in the context of the existing literature (provide references, where appropriate).

   The authors very appropriately provide the wider context and completely cover where the field stands. E.g. this protein microscopy-based work fills in the fine grain details where recent advances in transcriptional profiles by single cell experiments cannot provide resolution. The authors do also an outstanding job in providing the background on the general understanding of molecular players, structures and process controls across eukaryotes that pinpoint where the Apicomplexa are different.

   • State what audience might be interested in and influenced by the reported findings.

   The audience comprises a wide array of people with interests in cell cycle regulation, cell cycle checkpoints, DNA replication, nuclear organization across biological systems, and Apicomplexa in particular

   • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

   Toxoplasma gondii cell biology - sufficient expertise across the board

   Read the original source
4. 

   Review Commons

   Dec 19, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

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   Referee #1

   Evidence, reproducibility and clarity

   The manuscript by Batra et al have tried to dissect out two aspect to understand the complex cell cycle of Toxoplasma endodyogeny. One is to development of Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) technology for Toxoplasma gondii and then to use that for understanding the complex cell cycle. The authors have created ToxoFUCCIs and ToxoFUCCIsc probes using TgPCNA1 tagged with NeonGreen and TgIMC3 tagged with mCherry and used to dissect the different phases of cell cycle like S, G2, G1 and cytokinesis. Overall technology development is good though as they claim that they are first is not true as it has been used …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   The manuscript by Batra et al have tried to dissect out two aspect to understand the complex cell cycle of Toxoplasma endodyogeny. One is to development of Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) technology for Toxoplasma gondii and then to use that for understanding the complex cell cycle. The authors have created ToxoFUCCIs and ToxoFUCCIsc probes using TgPCNA1 tagged with NeonGreen and TgIMC3 tagged with mCherry and used to dissect the different phases of cell cycle like S, G2, G1 and cytokinesis. Overall technology development is good though as they claim that they are first is not true as it has been used earlier by https://doi.org/10.1128/msphere.00160-22. Hence may be that they have used to decipher the cell cycle.

   The manuscript is extremely dense and at times very difficult to read and to be clear if they are focussing on the technology or cell cycle. The technology may be a better part of manuscript but the dissection of cell cycle is not very novel and at times very confusing to follow. Many of these aspects has been dissected out previously from their own group and many group in Toxoplasma and Plasmodium and it is quite known about that the cell cycle in Apicomplexa is very complex. Another aspect that most FUCCI use Geminin and CDT1 factors and since Geminin is not present it would have been better to validate that with CDT1 that is present in Apicomplexa and may be more relevant than PCNA1. The first part of the manuscript only deals with first to identify the function and localisation of PCNA1 and then develop FUCCI technology and then go on to study cell cycle. So the focus of the manuscript is not clear. It seems three different results are just assembled together in one manuscript with out clear focus. Some of the conclusion on the that Replication starts at centromere region is not novel and has been studied previously.

   In order to get clear focus the authors should clear set out the focus as to why they developed FUCCCI and how they decipher either replication, budding, apical or basal complex, centrosome or cytokinesisas well to be used for drug discovery The way it is organised it is not flowing well and confuses the reader who may not be aware of different compartment of Toxoplasma cell or not a molecular parasitologist.
   The manuscript needs revising by writing precisely eliminating too much literature reference in the result section with clear focus. Some of these references can be elaborated in the introduction and discussion to keep the focus.

   Some points with respect to figures:

   Generally with image panels, arrows don't stand out well

   Fig1: no scale bars and the green arrow do not stand out. So may be to make white.

   Fig 2E: state the time point in the fig without IAA treatment (-IAA)

   Fig4: no bell shaped curve

   Fig 5D: it isn't obvious what the numbers on the right hand side of the graph mean. If it is size, there should be a unit given

   Figure 6 - how do they determine that the tachyzoites have progressed through 61% of S phase? Make this clearer here.

   Fig7: it a strange way of ordering the figure as FigE is after Fig F hence no logical order.

   Fig 8H is not mentioned in the text

   Figure 9 is nice and useful but the arrows could be made proportional of time spent in each cell cycle phase. They're a little off in the conventional cell cycle at the minute

   Some comment on the text in the manuscript:

   Line 137: describing the expression pattern: the following papers first described the expression pattern of PCNA1 and 2 can be cited in the result. https://doi.org/10.1016/j.molbiopara.2005.03.020

   Line 154: Provide schematic for AID HA cloning and confirmation.

   Line 157: Fig 2 after 4 h treatment FACS analysis shows more than 1 and less than 2n genomic content. Does this study have any -IAA treated control for 4h and 7h to compare as what should the standard genomic content to be there at this time point of development. At 4 h of development can the authors provide any statistical analysis with their 3 experiments to prove their point that the replication is actually stalled. Downregulation of TgPCNA1 as shown is western blot is still basal protein left to carry the genomic replication in 7 mins. Can authors also state that TgPCNA 2 which is although non-essential but has no redundant role in the replication machinery.

   Line 178 : typing error "that that

   Line 179: states the role of TgPCNA 1 in DNA1 replication, however line 159 and 160 states the TgPCNA1 deficient can fulfil DNA replication. Can author confirm this contrast in the results. Results trying to illustrate the same fact TgFUCCIs or TgPCNA1ng that TgPCNA1 first aggregates at centromeres and then distributed on many replication forks and disappears late during cytokinesis. The part of the result can be merged.

   Line 189: Typing error, should say "such as nucleus", currently as is missing

   Line 346-349: basically explaining the same thing twice.

   Line 347 - immunfluorescent should be immunofluorescence

   Line 395-399: does this study has any non-inhibited (-IAA control) at 4h and 7 h. for fig 7C & 7G. Can the authors provide any statistical analysis for the significance with their 3 experiments.

   Line 415: Why the authors have not used the TgFUUCI sc lines which expresses the TgPCNAng and IMCmch both. This could have helped to understand the real time dynamics of DNA replication and budding initiation (cytokenesis), rather then fixing and staining with TgIMC.

   Overall good technology development as FUCCI but the rest of the manuscript is extremely dense and the focus of the study is not clear after technology part. The complexity of the cell cycle is known and hence not much novelty here and extremely descriptive and hard read. Science can be simplified.

   Significance

   The development of FUCCI technology is significant part of the manuscript and to understand cellcycle may be they could have used CDT1 rather than PCNA as there is another PCNA 2 that also exist. The authors have given some convincing result for some aspect of cell cycle of which most are known and only it is quite incremental.at some part. The technology may contribute to the methodology development in Apicomplexa.

   Read the original source
5. 

   Version published to 10.1101/2024.09.02.610821 on bioRxiv

   Sep 3, 2024