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Reply to the reviewers

We would like to thank our reviewers for their constructive criticism and for their appreciation and enthusiasm for our study. Some reviewers expressed opposing views, particularly when it came to the function and identity of the Cdt1-related protein in Toxoplasma gondii. To avoid redundancy in our response, we would like to make a brief statement. Toxoplasma gondii and other apicomplexan parasites utilize unique and highly unusual modes of cell division; numerous studies suggest that multiple phases can run concurrently in apicomplexan cell cycles. The best-known examples include the asynchronous S/M cycles in schizogony and concurrent mitosis and budding in Toxoplasma endodyogeny. These overlapping phases are not a feature exclusive to apicomplexans, since in budding yeast, cytokinesis initiates in G1 phase by marking the location of budding on the surface of the mother. Based on years of previous research and from our experience, we adjusted our approach by focusing on the processes that are associated with each cell cycle phase rather than on their temporal order. While the model of a conventional cell cycle guides our studies, we “follow the breadcrumbs” that we discover and the published studies to create a more accurate model of apicomplexan cell cycle instead of relying on the traditional cell cycle map employed by distantly related eukaryotes. Below are point-to-point responses to reviewers’ comments.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: Hawkins et al. employ a reverse genetic approach to analyze the molecular function of the Toxoplasma gondii kinase Crk4 and the Toxoplasma gondii cyclin 4. The authors combine inducible depletion with imaging, (phospho-)proteomics, molecular modeling, and protein-protein interaction studies.

Major comments: - The major conclusion of the manuscript is that TgCrk4/TgCyc4 regulate entry into mitosis and that the primary role of TgCrk4 is to suppress DNA re-replication and chromosome re-duplication (lines 105-106). The authors also provide evidence that TgCrk4 interacts with TgCdt1, a DNA licensing factor ("TgCdt1" is missing in line 107). (had been corrected) By sequence homology, the authors found homologues of TgCrk4 only in apicomplexan parasites with binary division and concluded that the dominant division mode, presumably schizogony, is repressed in these organisms in favor of binary division. Indeed, internal budding and daughter cell formation is defective in the inducible depletion mutants of TgCrk4 and most experiments focus on this developmental stage. However, the analysis of preceding events, such as DNA replication is rather brief. If G2 is indeed regulated by TgCrk4/TgCyc4, one would assume that the parasites are post-S phase and the nucleus contains two copies of the genome, as indicated in Fig. 2C. The data shown in Fig. 3H and 7A, however, show that the TgCrk4 and TgCTD1 depletion induces a developmental arrest pre-S phase. This contradicts the main conclusions of the manuscript.

*We agree that the G2 location is odd for a conventional cell cycle model. Given the high possibility that cell cycle phases can overlap in apicomplexans, we determined the relative position of G2 phase in Toxoplasma endodyogeny by instead focusing solely on the processes that are attributed to a specific cell cycle phase (such as DNA replication for S phase, DNA re-replication for G2 phase, DNA segregation for mitosis). Our approach shows that Toxoplasma G2/M checkpoint operates upstream of SAC, which led to enrichment of parasites with replicated DNA (Fig. 3H and Fig. 7A), which places G2 at the end of S-phase. Our focus in the present study is on the G2 functions, the control of centrosome and chromosome reduplication, but we appreciate the suggestion to examine DNA replication in Toxoplasma, which could be investigated in future studies. *

Indeed, many data of this manuscript could support an alternative conclusion, i.e., that TgCrk4 regulates entry into S-phase (similar to Plasmodium falciparum Crk4: PMID: 28211852). This alternative conclusion is supported by the data showing that TgCyc4 is in the nucleus during S-phase (Fig. 1H) and that TgCrk4 interacts with TgCdt1, which has a well-known role in origin of replication licensing and loading of the MCM complex. MCM subunits were less phosphorylated in absence of TgCrk4, which could also suggest a role for TgCrk4 in S phase. Together, it seems more parsimonious to interpret the data as a DNA replication phenotype rather than a phenotype in G2.

*We understand some confusion from prior data, but PfCrk4 is not orthologous to TgCrk4 (Alvarez & Suvorova, 2017); The true TgCrk4 ortholog had not been found in Plasmodium genomes. Our understanding is that nuclear accumulation of TgCyc4 in S-phase activates TgCrk4, which leads to repression of the DNA reduplication. One of the possible mechanisms involves interfering with loading of the MCM complex on chromatin mediated by hyper-phosphorylated TgiRD1 (former TgCdt1), which has been reported in other eukaryotes. We also believe that increased MCM phosphorylation indicates entry into or active S-phase, while the reduced phosphorylation that was detected in Crk4-depleted cells supports a block at the end of S-phase (G2). *

• *

The currently provided data on the DNA content are, however, clearly insufficient to draw firm conclusions. The gating strategy (dotted lines in Figs. 3H, 7A) is unclear. Why are populations, e.g., not separated at the lowest part of the depression in the histogram, but shifted towards lower DNA content? This seems to overestimate the percentage of cells that have a higher DNA content and the statement in lines 269-271, i.e., that TgCrk4 deficient parasites break the "once and only once" rule, is not supported by data.

*We corrected the gating of the FACScan plots to separate G1, S, G2+M, and parasites with over-duplicated DNA. Please note that, in general, the cell cycle gating of FACScan data is relative and somewhat subjective when it comes to the gaussian curve. Independent of the chosen gates, our data show that removal of either TgCrk4 or TgiRD1 led to substantial decrease of the G1 population (reduction of 1N peak) accompanied by increase of parasites in the process of replication, completed replication (increase of 1.8 N peak), as well as undergoing DNA re-replication, which supports our claim in lines 269-271. In the case of TgiRD1, the number of parasites with re-duplicated DNA nearly doubled upon 8h of factor deficiency. *

• *

It is also unclear how may biological replicates are represented by these data (Figs. 3H, 7A), a critical wild type control at t = 4 h is missing, as well as a statistical analysis. Alternatively, the authors could use microscopy to quantify the DNA content of individual nuclei, which would yield a direct read out on whether a nucleus is in pre-S phase, S-phase or post-S phase. Defining the onset of S-phase indirectly by the number of centrosomes per cell seems imprecise, given the small size of the structure and the resolution of the microscope. Without solving these issues, the major conclusions and several minor statements throughout the manuscript are in question.

*Thank you for your point, we performed a minimum of three independent experiments to evaluate the DNA content of TgCrk4- or TgiRD1- (former TgCdt1) depleted tachyzoites and have now indicated this in the figure legends. The 0h time point is a “wild type” control, since the parasites that expressed factors were incubated without auxin (mock treated) for 4h. The DNA content of Toxoplasma has been thoroughly studied and we are thus confident our 0h data is a good representation of asynchronous healthy populations. Although the parental strain had been examined, due to the data density mentioned in the reviews, we included only relative results (control and two experimental points) for clarity. Our concern with using microscopy to analyze DNA content is that it can be highly subjective, hinging on the quality of staining and imaging, while flow cytometry produces more unbiased datasets. We have considered the concern that the start of centrosome duplication can be difficult to identify, but the centrin-positive centrosomes move apart by the middle of S-phase. The independent structures are then distinct and easy to resolve, providing a popular means of marking G1/S transition in Toxoplasma. *

• Lines 187-189: The mentioned checkpoint is unclear and so is the "specific cell cycle population". Fig. 2B analyses budding, but as the final step in the cell cycle, the knock down parasites may have arrested at various other stages of the cell cycle. In addition, it is unclear on which primary data Fig. 2B is based. It appears these may be at least partially shown in Fig. 3. If so, please reorganize as this is highly misleading.

*“A checkpoint” in the indicated lines refers to G2/M and SAC, which are regulated by TgCrk4 and TgCrk6, respectively. We refer to “specific cell cycle population” since each transgenic parasite that is subject to G2/M or SAC arrest can allow us to isolate very different cell cycle stages. TgCrk6-dependent arrest had been confirmed by the presence of unresolved centrocone (not shown but was previously reported in Hawkins et al., 2022), while we thoroughly examined the novel TgCrk4-dependent block by focusing on many parameters, such as joint centrosomes, single-bud assembly, or unresolved apicoplast. Fig. 2 and Fig. S2 summarize our rigorous quantifications of these phenotypes. For convenience, we used budding efficiency as a readout to compare arrest and release of G2/M and SAC, which was incorporated in Fig. 2B. Table S4 contains the primary data used in all figures in the manuscript, including Fig. 2B. *

• Line 246-254: It is unclear how many biological replicates were performed and how many cells were analyzed to conclude that TgCrk4 deficient parasites cannot form a bipolar spindle (Fig. 2H, S3B). This, together with the possibility that the developmental arrest occurs pre-S phase (Fig. 3H), does not support the statement, that the G2/M transition is regulated by the novel TgCrk4-TgCyc4 complex.

We have indicated our replicates in the M&M. As addressed for Fig. 3H above, these IFA experiments were performed in at least three independent experiments.

* * Minor comments: - Throughout the manuscript, please reorganize and present the figures in order of appearance in the text. Also, Fig. 1G summarizes data that are only presented in Fig. 1H. Please reorder. Similarly, Fig. 2C appears to summarize data that are only presented later.

*Thank you for the suggestion, however we must abide by the standards of the publishers. The order of the figures must be maintained, but there is a substantial degree of freedom in organizing panels within figures. Fig. 1G summarizes data shown in Fig. 1F, H, while Fig. 2C summarizes many panels including preceding Fig. 2B and Fig. S2. Most of our schematics are placed at the top of figures to provide guidance for the relevant experiments. *

• Why was only the "G1" timepoint quantified in Fig. 1H? Do the other images shown in F and H represent the majority of cells analyzed?

*You are correct, we indicated the percentage of factor-positive parasites only when the factor emerges during a specific cell cycle phase. For example, the TgCyc4-positive parasites with 1 centrin dot were quantified to show that TgCyc4 emerges in the middle of G1 phase. The lack of a number indicates that the image represents all the parasites progressing through this phase; we have added this explanation to the figure legends. *

• Several micrographs lack scale bars (Fig. 1B, D; 2E, F, H, I; 6D; 7F, H and S2G, S3A, B; S5A, B, D).

*Thank you, we have added the scale bars to indicated images.

*

• Lines 83-85 and 93-95: Recently several publications investigated the cell cycle of the apicomplexan parasite Plasmodium and data are accumulating, showing that there may be a gap between the last S phase and segmentation (e.g., PMID: 35731838; PMID: 35353560), which may be interpreted as a G2 phase. Thus, these statements could be revised to reflect the current literature.

*The studies mentioned provide very valuable insights into S-phase dynamics; the gap that was detected between S-phase and segmentation includes mitotic events such as prophase, metaphase, and anaphase prior to telophase (karyokinesis to segmentation). However, studies using means like stage-specific markers could help resolve the composition and order of events in the apicomplexan cell cycle. We used processes specific to G2 (repression of DNA and centrosome reduplication) and identified TgCrk4/TgCyc4 as the first G2 markers in apicomplexans. *

• Fig. 4 shows the effect on protein abundance and phosphorylation upon TgCrk4 depletion. Fig. 4B seems somewhat redundant as a more detailed analysis with two timepoints is shown in the rest of the figure.

*Fig. 4B is provided in contrast to the plot in Fig. 4A. It demonstrates that TgCrk4 depletion results in a far more pronounced effect on global phosphorylation rather than on proteolysis. While Fig. 4B highlights the checkpoint arrest, panels C and D are dedicated to the search for TgCrk4 substrates: the phospho-sites that immediately lost intensity of phosphorylation and remained low during the 4h block. *

*

*

• Lines 146-148: This statement is confusing in light of the expression data in Fig.1 F and H. If they stabilize each other, how is TgCrk4 stabilized in G1, when TgCyc4 is absent?

We believe that multiple mechanisms contribute to the stability and function of TgCrk4. We tested one and found that depleting the cyclin partner led to reduced expression of TgCrk4, and were able to conclude that the complex is stable when both subunits are expressed. Please note that we probed the mixed cell cycle populations by WB, and our proteomics data show that TgCrk4 interacts with many partners (Fig. 1E). Thus, it is likely that G1 stability may have been mediated by other partners, or by a higher transcription/translation rate, which could be evaluated in further experiments that focus on the regulation of TgCrk4/TgCyc4 complex.

• *

• Fig. 2D, and G: Please provide representative images of what has been quantified, as E/F and H/I are apparently UxEM images.

The corresponding images are included in Fig. S2.

• Line 236-243: This statement seems to be based on a single IFA shown in Fig. 2K. If so, the manuscript would benefit from clearly stating that this is a singular observation.

*Thank you, we have provided clarification as described in previous points. *

*

*

• Lines 301-304: In the cited publication, the TgOTUD3A knockout could not be complemented, which raises the possibility that other factors are involved. Thus, this statement would benefit from revision.

*The lack of TgOTUD3A KO complementation is an example of the unappreciated complexity of apicomplexan cell cycle regulation by controlled proteolysis. We highlighted the similarity of TgCrk4 and TgOTUD3A deficiencies, which indirectly confirms their partnerships in the G2 network. Fig. 8A shows that, in addition to TgOTUD3A, the G2 network contains numerous factors. *

*

*

• Lines 421-422: PfCdt1 was annotated in PlasmoDB some time ago and this statement needs to be revised.

*Please see our response to comments made by Reviewer 2. Briefly, we agree with Reviewer 2 comment that TgCdt1 does not function as conventional DNA replication licensing factor CDT1. Therefore, we named TGME49_247040 TgiRD1 – inhibitor of DNA and centrosome ReDuplication 1. *

• *

• Lines 448-450 and Fig. 6F: Are these data from a single biological replicate and how many cells were analyzed for the different time points? Given the insufficient data on the DNA content, the paper would benefit form more conservative conclusions on the role of TgCdt1. The numbers of biological replicates were added throughout the text, also please refer to our response to Reviewer 2 and the comment above.

Reviewer #1 (Significance (Required)):

• This manuscript investigates the role of TgCrk3, TgCyc4 and TgCdt1s and provides a large amount of data.
• These data will contribute to our understanding of the unusual division modes of Apicomplexa, a field of research that recently gained momentum.
• These data will be interesting to the community of cell and molecular biologist, which work on the fundamental biology of eukaryotic microorganisms.
• My field of expertise is the cell biology of Apicomplexa.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary: In this Manuscript, Hawkins et. al. describe advances in the apicomplexan parasite cell cycle, which is reminiscent but distinct from mammalian cell cycle regulation. These differences include a presumed lack of G2 phase and the ability to replicate in either a multinuclear (schizogony) or binary (endodyogeny) manner. Using Toxoplasma gondii (TG) as a model, the authors seek to expand the current understanding of how these highly variable parasitic cell cycles are regulated by describing a previously unreported G2 phase. Building on the authors earlier work, this manuscript defines the function of TgCrk4 and identifies a novel binding partner, TgCyc4. Crk4 and Cyc4 control a G2/M checkpoint by regulating centrosome duplication and separation.

The authors also identify 247040, a protein with previously no known function, as a binding partner and substrate of TgCrk4/TgCyc4 and several replication fork proteins such as MCM and PCNA. Results indicate that the protein negatively regulates replication and centrosome duplication. The authors propose to rename this protein TgCDT1 despite "low sequence similarity" and having a completely opposite function to eukaryotic CDT1. Using Swiss-Prot modeling the authors claim 247040 bears a "partial resemblance" to mammalian CDT1. Indeed, both of these proteins show high intrinsic disorder and have 2 folded domains. While 247040, like hCDT1, does contain cyclin interacting motifs (Cy), a collection degrons (not all shared with other CDT1 orthologs), and an NLS, the list of nuclear cell cycle proteins that also contain Cy and degron motifs would be very long. Further, 247040 is regulated in an opposite manner to all other CDT1 orthologs because it is absent in TG G1 and present in TG S phase; eukaryotic CDT1 is either degraded or relocalized to the cytoplasm in S phase, and evidence for degradation via APC/C is minimal. Crucially, loss of 247040 resulted in inappropriate replication ("re-replication"), whereas all other eukaryotic CDT1 orthologs are essential for replication. Re-replication in eukaryotic cells can be caused by excess or hyper-active CDT1, not by loss of CDT1 activity as shown here for 247040. Clearly 247040 is a negative regulator of DNA replication, and as such, is not a candidate for the TgCDT1 ortholog. If anything, it is functionally analogous to metazoan geminin, the negative regulator of metazoan CDT1; of note, geminin also has centrosome-related phenotypes. We cannot support naming 247040 TgCDT1 because it will cause confusion in the field.

Aside from this major issue, the study is well-executed, rigorous, quantitative, and thorough; it has many strengths from the unbiased interaction screens. The authors' sequence analysis also suggests broader possibilities for cyclin structures than had previously been appreciated. We appreciate the legend in Figure 2 to the organism-specific terminology.

Major comments: The spatiotemporal dynamics of 247040, its role in repressing TG DNA replication, lack of PIP motif and winged helix domain indicate that some other nomenclature, other than TgCdt1 will be a better name for this protein of previous unknown function.

We would like to thank Reviewer 2 for this highly insightful comment. We agree that TGME49_247040 functions as a CDT1 inhibitor rather than as CDT1 itself, so conserving the name would produce confusion in the cell cycle field. Based on TGME49_247040 protein function we decided to name this factor TgiRD1 – inhibitor of DNA and centrosome ReDuplication 1. We revisited our data, looked deeper into the protein structure, and adjusted our conclusions. Our new Figure S5 shows differences in the predicted folding of HsCDT1 and TgiRD1. We could not ignore the fact that TgiRD1 is phylogenetically related to CDT1 in ancestral branches and metazoans (Fig. 6B), but we identified substantial differences that may indicate a selective loss (or inheritance) of protein features. For example, TgiRD1 does not interact with ORCs that are critical for the licensing step, but TgiRD1 retained an MCM binding domain (winged helix-turn-helix) that plays a role in licensing and firing. Rather than CRL4Cdt2 degrons, TgiRD1 contains APC/C degrons that would be activated late in mitosis (similar to regulation of Geminin). Together with the lack of DNA licensing control in G1 and its opposing expression profile, we concluded that TgiRD1 represents a Cdt1-related protein that controls DNA and centrosome reduplication in S and G2 phases.

Minor comments:

1. For clarity, please include the number of replicates in the figure legends where appropriate. We added the requested information.

For microscopy/imaging, how were representative cells/images chosen? The representative images constituted the most common phenotype of the feature we aimed to highlight, and most are accompanied by quantifications.

In addition to the ELM analysis, the authors could also employ fold recognition software (such as Promal) to analyze 247040 structural models to show similarity to known protein structures.

We use a variety of folding prediction software, including AlphaFold2, PyMol, and template-based SWISS-PRO module to examine protein structures in our study, indicated in the text and figure legends. Our new TgiRD1 (former TgCdt1) analysis is based on an AlphaFold2 prediction (Fig. S5). All the software we used is listed in the M&M section.

Line 107: missing words "TgCdt1"

*We corrected the sentence.

*

Line 141: the interpretation that the C terminus is "unstable" is misleading if it is simply that the protein cannot tolerate a fusion to the C-terminus.

*We successfully incorporated a tag at the C-terminus (confirmed by sequencing across the recombinant gene) but could not detect protein expression. If our protein could not tolerate a recombinant tag, the transgenic parasites would not survive because TgCyc4 is essential protein. Therefore, since the parasites survived, we concluded that the lack of TgCyc4-AID-HA expression was due to native truncation at the C-tail (instability). *

Line 221: word choice "reminisced" We have changed the wording.

Line 348 refers to Orc4 expression in Figure 4A, but the data point is not labelled. Fig. 4A references GO group (DNA replication/licensing factors), and the raw data is included in Table S6, which is now indicated in the text.

Lines 407-8 and 510-11: Reference Fig 1E We added the reference.

Line 408: please define what is meant by "dominant interactor" We meant that TgiRD1 is the most prominent interactor of TgCrk4 and TgCyc4. To clarify the confusion, we changed the wording to “primary interactor”.

Reviewer #2 (Significance (Required)):

This manuscript makes great strides in defining apicomplexan cell cycle control and genome replication. These strides include defining a previously unrecognized G2/M checkpoint controlled by TgCrk4 and the novel TgCyc4. Further, the authors identify a binding partner and substrate of the novel Crk4/Cyc4 kinase complex, 247040 that acts as a repressor of replication.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary The present study Hawkins et al have described the important role of Cyclin-CDK complex in an apicomplexan parasite Toxoplasma(Tg) which exhibit binary mode of cell division like many other eukaryotes. In the apicomplexan field it is generally shown that G2 phase of cel cycle is either absent or has very little role. The authors here demonstrate that the combination of Tg CRK4 and Tg Cyclin4 works during the G2 phase of cell cycle such as chromosome rereplication and centrosome reduplication. In order to show the function of Cyclin-CRK function they used Auxin degradation system to down regulate or deplete the protein and study parasite growth during cell cycle as well as they used tagged parasite to identify the protein complex with these two molecules. In the study they showed that these two molecules Cyc4 and cRK4 formed the complex in protein pulldown method and show identical function in the cell cycle. In addition to thiese two proteins they also found another interacting partner Cdt1 that was further analysed to be involved in controlling Chromosome rereplication and centrosome. So overall the study is nicely performed and three molecules of Cyclin4-CRk4-Cdt1 and their role is illustrated in the binary mode of cell division in Toxoplasma.

Comments 1.Though no new experiments need to be performed but it will be good if some details are given as to which stage of tachyzoite cycle the protein complex were performed and if there is difference in the various phases of cell cycle especially the s phase and the M phase. Are these period changed. Since G2 is suppose to be absent in many apicomplexan do the authors suggest that G2 phase is only coupled to binary mode of cell division. Please discuss how it is then linked to the other part of cell cycle.

*You are correct, we propose that the presence of G2 phase is linked to binary division in apicomplexans and our hypothesis is supported by the overall evolution of the cell cycle (see Discussion section). We also entertained the hypothesis that G2 operates in multinuclear division since all apicomplexans encode TgiRD1 orthologs (please, see the Discussion section). For the first time, we identified the major functions of G2 functions (repression of the DNA and centrosome reduplication) in the apicomplexan cell cycle. However, given the unresolved organization of the Toxoplasma (or any apicomplexan) cell cycle, it is currently impossible to define the boundaries of G2. According to our study, TgCrk4 and TgCyc4 control G2/M transition or the end of G2 phase, and we still lack markers of G2 entry. In our comparative synchronization study (Fig 2), we uncovered the temporal link between G2/M and SAC regulatory points, which is discussed in the results section. *

Ganter et al have studied CRK4 in Plasmodium previously and they do find in their phosphoproteome study the similar association with the DNA replication machinery with CRK4 but no cyclin was identified in their study. In the cyclin study by Roques et al it has been shown that no cell cycle cyclins are found in Apicomplexan so can the author discuss more how these complex can be different in two apicomplexan species. They describe that Crk4 is novel cell cycle kinase though this has been studied earlier. Authors have almost not discussed these previous finding with respect to their in this study.

*We would like to clarify this confusion. We have not discussed Ganter et al. studies because PfCRK4 is not orthologous to TgCrk4, but rather it is related to TgCrk6. Unfortunately, the Plasmodium and Toxoplasma Crk nomenclature was published almost concurrently. Our previous (Alvarez & Suvorova, 2017) and current study show that Plasmodium and other apicomplexans that divide by multinuclear division do not encode TgCrk4 orthologs (and/or TgCyc4). Additionally, the mentioned studies by Roques and Ganter were released prior to newer genome annotations that include additional cyclin-domain proteins, including 10 Toxoplasma cyclins (5 new) that we categorized in our recent publication (Hawkins et al., 2022). Although the newly annotated cyclins are not related to conventional cell cycle cyclins, we had proven empirically that TgCyc1 together with TgCrk6 controls SAC, and now, the specific interaction of TgCyc4 with TgCrk4 controls G2 processes. Lastly, we call TgCrk4 “a novel” kinase only in the meaning that it is a novel cyclin-dependent kinase that is not related to known CDKs in other eukaryotes. The identification of TgCrk4 in our previous study (Alvarez & Suvorova, 2017) is described in the Introduction section and at the opening of the Results. *

The manuscript is too dense, in terms of both figures and text. At times loses the focus and hence can be organised with most important finding in the figure and text. Especially Fig2, Fig4 and Fig7. Fig5 does not give too much in terms of the real finding an in fact take away from the focus. Some parts of these figures can be simplified or moved to supplementary. Some of the figures in Fig2 and 7 are missing the scale bars.

We respectfully disagree with some conclusions made by the Reviewer. Our study contains ample material that is intended to guide the reader through the complexity of the Toxoplasma cell cycle and the intricate structures contained in the parasite. We have also introduced a few novel approaches that require additional schematics and dedicated discussions.

• Fig 2*. The G2/M block, as well as the G2 phase, had never been detected in apicomplexans. We created a new approach to determine the timing of the G2/M checkpoint, which involves comparison to a known cell cycle block. Panels A, B, and C provide visuals and summarize our findings. The main events are highlighted with arrows (Panel C), while graphs (panel B) show differences in responses. The rest of the figure is devoted to quantification of the primary events caused by TgCrk4 deficiency, since the G2 block had never been examined. While the U-ExM images of the entire vacuole (2-4 parasites) may seem overwhelming, they represent that the deficiency is consistent. *
• Fig 7* is devoted to the major Crk4/Cyc4 interactor TgiRD1 (former TgCdt1). This is one of the first mechanistic studies of central cell cycle regulators in Toxoplasma. This Cdt1-related protein was examined at the molecular level to support the main claims of its control of G2 Nevertheless, we moved two panels from Fig. 7 into the supplement. *
• 4* is organized as follows. Top row: panels A, B visualize the G2/M checkpoint block at the protein level. Middle row: panels C, D, and E represent the workflow to find TgCrk4 substrates. Bottom row: panels F, G highlight TgCrk4 substrates of interest that are discussed in the paper. *
• 5* is an in-depth analysis of the central cell cycle regulators across Apicomplexa phylum, a key figure of the study. Its comparative nature supports our main message: binary division is regulated by TgCrk4/TgCyc4, which are only expressed in a subgroup of apicomplexans that divide in a binary mode. *

May be bit more discussion of ORC in relation to their Cyclin-CRK complex as they did find upregulation of the ORC in their genome profiling. So may be instead of CDT1 these are more important in the licencing of DNA replication.

*Our choice to focus on Cdt1-related protein was driven by the fact this protein is a major component of the TgCrk4/TgCyc4 complex, while the ORCs act downstream (as TgCrk4 substrates). Shifting focus to ORCs opens an entire new project, which will be explored in the future. *

5 The model in Fig8B does not take Cyc4 into consideration and I feel is bit oversimplified as there are many factors that may be responsible for centrosome non separation. The S and G2 are no separated in the Cell cycle as given in this Fig.

Referring to comment 3, we focused on empirically supported, central findings and created the first model of centrosome cycle regulation in T. gondii. We intentionally drew focus to TgCrk4, which was extensively studied, while TgCyc4 received less attention due to difficulties in modulating its expression. We have used transcriptional downregulation to evaluate TgCyc4 (tet-OFF model), which is unfavorable for cell cycle studies because it exceeds the duration of the cell cycle. The unclear cell cycle borders are addressed in the introduction to this response. Briefly, the organization of apicomplexan cell cycle is currently unclear, thus most of the schematics are approximate.

It is not clear from the data with CDt1 if this linking the inner and outer centrocone or its down regulation breaks the bipartite centrosome. May be some reflection it will be useful.

*Our model suggests that both TgCrk4 and TgiRD1 (former TgCdt1) affect only the inner core of the centrosome, which we propose is comprised of two types of linkers. The arrows in Fig. 8 point specifically to the linkers whose stability depends on the expression of TgCrk4 or TgiRD1. *

Minor comments

I what is SAINT analysis as it is not described in methods.

*We added the description of our SAINT analysis to M&M.

*

How was budding quantified

*We supplemented the figure legend with the required information. *

Western blot can have predicted size

*Due to density of the figures, we did not supply the predicted MW of the proteins when they display the proper PAGE motility. *

what does red star mean in Blot 1C

*We added the description to the figure legend.

*

What does the number in Fig1H means please explain in the legends and same for Fig6F. In fig 1, removing the inhibition for 5 hours led to very less budding, but in fig 3, removing inhibition showed increased budding (50% in 2 hours). Please explain

*Please see our response to the reviewer 1 minor comment regarding Fig. 1H and 6F. *

*We presume that there is some confusion regarding figure numbers. Perhaps the Reviewer refers to Fig. 2B. Indeed, the 4h block at G2/M led to reduced budding (Fig. 2B), while release from the block for 2 hours (Fig. 3C, post-recovery) allows parasites to continue cell cycle progression and reach the next stage –budding. The numbers over the Fig. 3A, B, and C panels are from the plots in Fig. 2B to help give a comprehensive representation of the analyzed timepoint. *

Fig2 has no scale bars -please add- this figure is too dense. May be fig2A, B,C can be in supplementary, legend in the figure can be in the figure legend.

Please see our response to comment 3. We have included scale bars.

Also this figure2 H and I in not quoted in line 231. Also this figure2 has no panel J but goes directly from I to K

*The alphabetical order was corrected, and the reference added. *

Fig3 the FigG can be more relevant in the Figure 8 while describing about the Crk4 and Cyc4 and CDt1 in binary mode of cell division. Also please define what stars mean either in legend or methods section in terms of significance.

*Thank you for the suggestion. The Fig. 3G schematics summarize the overall findings of the Figure and acts as an intermediate conclusion in this study. We added the meaning of the stars in the M&M section. *

Line 107 the sentence is incomplete

We have corrected the sentence.

Line 217 may be the figure could be referred as then it is not cleat about the description.

Due to the density of the figures and well-established dynamics of the centrocone and basal rings, we included the reference to a publication rather than as a figure panel.

**Referees cross-commenting**

The study is quite rigrous and with analyses of CRK4-CYC4 and CDT. However it will be better if authors please revisit their conclusions on G2 phase of cell cycle in Toxoplasma based on their findings. The study will have important bearing on the community studying apicomplexan parasites and DNA replication as well as who work on eukaryotic cell cycle.

Reviewer #3 (Significance (Required)):

Significance In the manuscript by Hawkins etal have illustrated that in the apicomplexan parasite that have binary mode of cell division present a Cyclin-Crk complex with detailed analysis of Tg Crk4-Cyc4 that are novel in these group pf parasite infect humans and animal alike like malaria parasite and ones affecting cattle and chicken. So these finding are novel as very little is known about this interaction. The significant finding is to show how the G2 phase of cell cycle may be regulated in these parasites and how DNA licencing factor Cdt1 is highly divergent but part of this CRK-Cyclin complex.

So though it discusses more on the Toxoplasma but it may be of interest to the scientist working on eukaryotes with divergent mode of cell cycle.

General Assessment - The findings are novel but the manuscript is too dense and at time loses the focus. May be both text and Figures could be made less dense so that important finding are revealed in better way.

Advance - It does give important insight into the cell cycle in apicomplexan parasite and how even though there are no cell cycle cyclin in Apicomplexa. The findings here suggest how different complexes can substitute for the function. It does extend the knowledge in the field of Cell division in divergent parasites both in terms of mechanistic, functional and technical way.

Author Response

The following is the authors’ response to the original reviews.

First of all, we'd like to thank the three reviewers for their meticulous work that enable us to present now an improved manuscript and substantial changes were made to the article following reviewers' and editors' recommendations. We read all their comments and suggestions very carefully. Apart from a few misunderstandings, all comments were very pertinent. We responded positively to almost all the comments and suggestions, and as a result, we have made extensive changes to the document and the figures. This manuscript now contains 16 principal figures and 15 figure supplements.

The number of principal figures is now 16 (1 new figure), and additional panels have been added to certain figures. On the other hand, we have added 7 additional figures (supplement figures) to answer the reviewers' questions and/or comments.

Main figures

▪ Figures 1, 4, 5, 10, 11, 12, 13, 14: unchanged ▪ Figure 7 and 8 were switched.

▪ Figure 2: we added panel F in response to reviewer 3's and request for sperm defect statistics

▪ Figure 3: the contrast in panel B has been taken over to homogenize colors

▪ Figure 6: This figure was recomposed. The WB on testicular extract was suppressed and we present a new WB allowing to compare the presence of CCDC146 in the flagella fraction. Using an anti-HA Ab, we demonstrate that the protein is localized in the flagella in epididymal sperm. Request of the 3 reviewers.

▪ Figure 7 (old 8): to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum. Moreover, the WB was removed and is now presented in figure 6 (improved as requested).

▪ Figure 8. Was old figure 7

▪ Figure 9: figure 9 was recomposed and improved for increased clarity as suggested by reviewer 2 and 3.

▪ Figure 16 was before appendix 11

Figure supplements and supplementary files

▪ Figure 1-Figure supplement 1 New. Sperm parameters of the 2 patients. requested by editor (remark #1) by the reviewer 1 (Note #3)

▪ Figure 2-Figure supplement 1 new. Sperm parameters of the line 2 (KO animals) requested by the reviewer 1 (Note #5)

▪ Figure 4-Figure supplement 1 New. Experiment to evaluate the specificity of the human CCDC146 antibody. Minimal revision request and reviewer 1 note #8

▪ Figure 6-Figure supplement 1 New. Figure recomposed; Asked by reviewer 2 note #4 and reviewer 3

▪ Figure 8-Figure supplement 1 New. We now provide new images to show the non-specific staining of the midpiece of human sperm by secondary Abs in ExM experiments; Asked by reviewer 2

▪ Figure 10-Figure supplement 1 New. We added new images to show the non-specific staining of the midpiece of mouse sperm by secondary Abs in IF (panel B). Rewiever 1 note #9 and reviewer 2 note #5

▪ Figure 12-Figure supplement 1 New. Control requested by reviewer 3 Note #23

▪ Figure 13-Figure supplement 1 New. We provide a graph and a statistical analysis demonstrating the increase of the length of the manchette in the Ccdc146 KO. Requested by editor and reviewer 3 Note 24

▪ Figure 15-Figure supplement 1 New. Control requested by reviewer 2. Minor comments

▪ Figure supplementary 1 New. Answer to question requested by reviewer 2 note #1

All the reviewers' and editors’ comments have been answered (see our point to point response) and we resubmit what we believe to be a significantly improved manuscript. We strongly hope that we meet all your expectations and that our manuscript will be suitable for publication in "eLife". We look forward to your feedback,

Point by point answer

Please note that there has been active discussion of the manuscript and the summarize points below is the minimal revision request that the reviewers think the authors should address even under this new review model system. It was the reviewers' consensus that the manuscript is prepared with a lot of oversights - please see all the minor points to improve your manuscript.

All minimal revision requests have been addressed

Minimal revision request

1) Clinical report/evaluation of the two patients should be given as it was not described even in their previous study as well as full description of CCDC146.

We provide now a new Figure 1-figure supplement 1 describing the patients sperm parameters

2) Antibody specificity should be provided, especially given two of the reviewers were not convinced that the mid piece signal is non-specific as the authors claim. As both KO and KI model in their hands, this should be straightforward.

To validate the specificity of the Antibody, we transfected HEK cells with a human DDK-tagged CCDC146 plasmid and performed a double immunostaining with a DDK antibody and the CCDC146 antibody. We show that both staining are superimposable, strongly suggesting that the CCDC146 Ab specifically target CCDC146. This experiment is now presented in Figure 4-Figure supplement 1. Next, to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum.

3) The authors should improve statistical analysis to support their experimental results for the reader can make fair assessment. Combined with clear demonstration of ab specificity, this lack of statistical analysis with very few sample number is a major driver of dampening enthusiasm towards the current study.

Several statistical analyses were carried out and are now included:

1) distribution of the HA signal in mouse sperm cells (see point 2 Figure 7 panel B)

2) quantification and statistical analyses of the defect observed in Ccdc146 KO sperm (figure 2 panel E)

3) Quantification and statistical analyses of the length of the manchette in spermatids 13-15 steps (Figure 13-Figure supplement 1 new)

4) The authors need to clarify (peri-centriolar vs. centriole)

In figure 4A, we have clearly shown that the protein colocalizes with centrin, a centriolar core protein in somatic cells. This colocalization strongly suggests that CCDC146 is therefore a centriolar protein, and this is now clearly indicated lines 211-212. However, its localization is not restricted to the centrioles and a clear staining was also observed in the pericentriolar material (PCM). The presence of a protein in PCM and centriole was already described, and the best example is maybe gamma-tubulin (PMID: 8749391).

or tone down (CCDC146 to be a MIP) of their claim/description.

Concerning its localization in sperm, we agree with the reviewer that our demonstration that CCDC146 is MIP would deserve more results. Because of that, we have toned down the MIP hypothesis throughout the manuscript. See lines 491495

Testis-specific expression of CCDC146 as it is not consistent with their data.

We have also modified our claim concerning the testis-expression of CCDC146. Line 176

Reviewer #1 (Recommendations For The Authors):

Major comments

1) As described in general comments, this study limits how the CCDC146 deficiency impairs abnormal centriole and manchette formation. The authors should explain their relationship in developing germ cells.

In fact, there are limited information about the relationship between the manchette and the centriole. However, few articles have highlighted that both organelles share molecular components. For instance, WDR62 is required for centriole duplication in spermatogenesis and manchette removal in spermiogenesis (Commun Biol. 2021; 4: 645. doi: 10.1038/s42003-021-02171-5). Another study demonstrates that CCDC42 localizes to the manchette, the connecting piece and the tail (Front. Cell Dev. Biol. 2019 https://doi.org/10.3389/fcell.2019.00151). These articles underline that centrosomal proteins are involved in manchette formation and removal during spermiogenesis and support our results showing the impact of CCDC146 lack on centriole and manchette biogenesis. This information is now discussed. See lines 596-603

2) The authors generated knock-in mouse model. If then, are the transgene can rescue the MMAF phenotype in CCDC146-null mice? This reviewer strongly suggest to test this part to clearly support the pathogenicity by CCDC146.

We indeed wrote that we created a “transgenic mice”, which was misleading. We actually created a CCDC16 knock-in expressing a tagged-protein. The strain was actually made by CRISPR-Cas9 and a sequence coding for the HA-tag was inserted just before the first amino acid in exon 2, leading to the translation of an endogenous HA-tagged CCDC146 protein. We have removed the word transgenic from the text and made changes accordingly (see lines 250-253). We can therefore not use this strain to rescue the MMAF phenotype as suggested by the reviewer.

3) Although the authors cite the previous study (Coutton et al., 2019), the study does not describe any information for CCDC146 and clinical information for the patients. The authors must show the results for clinical analysis to clarify the attended patients are MMAF patients without other phenotypic defects.

We have now inserted a table, indicating all sperm parameters for the patients harboring a mutation in the CCDC146 gene (Figure 1-Figure supplement 1) and is now indicated lines 159-160

4) The authors describe CCDC146 expression is dominant in testes, However, the level in testis is only moderate in human (Supp Figure 1). Thus, this description is not suitable.

In Figure 1-figure supplement 2 (old FigS1), the median of expression in testis is around 12 in human, a value considered as high expression by the analysis software from Genevestigator. However, for mouse, it is true that the level of expression is medium. We assumed that reviewer’s comment concerned testis expression in mouse. To take into account this remark, we changed the text accordingly. See line 176.

5) Although the authors mentioned that two mice lines are generated, only one line information is provided. Authors must include information for another line and provide basic characterization results to support the shared phenotype within the lines.

We now provide a revised Figure 2-figure supplement 1CD, presenting the second line and the corresponding text in the main text is found lines 178-183.

6) In somatic cells, the CCDC146 localizes at both peri-centriole and microtubule but its intracellular localization in sperm is distinguished. The authors should explain this discrepancy.

The multi-localization of a centriolar protein is already discussed in detail in discussion lines 520-526. We have written:

“Despite its broad cellular distribution, the association of CCDC146 with tubulin-dependent structures is remarkable. However, centrosomal and axonemal localizations in somatic and germ cells, respectively, have also been reported for CFAP58 [37, 55], thus the re-use of centrosomal proteins in the sperm flagellar axoneme is not unheard of. In addition, 80% of all proteins identified as centrosomal are found in multiple localizations (https://www.proteinatlas.org/humanproteome/subcellular/centrosome). The ability of a protein to home to several locations depending on its cellular environment has been widely described, in particular for MAP. The different localizations are linked to the presence of distinct binding sites on the protein…. “

7) Authors mention CCDC146 is a centriolar protein in the title and results subtitle. However, the description in results part depicts CCDC146 is a peri-centriolar protein, which makes confusion. Do the authors claim CCDC146 is centrosomal protein?

In figure 4A, we have clearly shown that the protein colocalizes with centrin, a centriolar core protein. This colocalization strongly suggests that CCDC146 is therefore a centriolar protein in somatic cells, and is now clearly indicated lines 211-212. However, its localization is not restricted to the centrioles and a clear staining was also observed in the pericentriolar material (PCM). The presence of a protein in PCM and centriole was already described and the best example is maybe gamma-tubulin (PMID: 8749391).

8) Verification of the antibody against CCDC146 must be performed and shown to support the observed signal are correct. 2nd antibody only signal is not proper negative control.

It is a very important remark. The commercial antibody raised against human CCDC146 was validated in HEK293-cells expressing a DDK-tagged CCDC146 protein. Cells were co-marked with anti-DDK and anti-CCDC146 antibodies. We have a perfect colocalization of the staining. This experiment is now presented in Figure 4-figure supplement 1 and presented in the text (lines 206-208).

9) In human sperm, conventional immunostaining reveals CCDC146 is detected from acrosome head and midpiece. However, in ExM, the signal at acrosome is not detected. How is this discrepancy explained? The major concern for the ExM could be physical (dimension) and biochemical (properties) distortion of the sample. Without clear positive and negative control, current conclusion is not clearly understood. Furthermore, it is unclear why the authors conclude the midpiece signal is non-specific. The authors must provide experimental evidence.

Staining on acrosome should always be taken with caution in sperm. Indeed, numerous glycosylated proteins are present at the surface of the plasma membrane regarding the outer acrosomal membrane for sperm attachment and are responsible for numerous nonspecific staining. Moreover, this acrosomal staining was not observed in mouse sperm, strongly suggesting that it is not specific.

Concerning the staining in the midpiece observed in both conventional and Expansion microscopy, it also seems to be nonspecific and associated with secondary Abs.

For IF, we now provide new images showing clearly the nonspecific staining of the midpiece when secondary Ab were used alone (see Figure 10-figure supplement 1B).

For ExM, we provide new images in Figure 8-figure supplement 1B (POC5 staining) showing a staining of the midpiece (likely mitochondria), although POC5 was never described to be present in the midpiece. Both experiments (CCDC146 and POC5 staining by ExM) shared the same secondary Ab and the midpiece signal was likely due to it.

Moreover, we now provide new images (figure 7C) in ExM on mouse sperm showing no staining in the midpiece and demonstrating that the punctuated signal is present all along the flagellum. Finally, we would like to underline that we now provide new IF results, using an anti-HA conjugated with alexafluor 488 and confirming the ExM results.

These points are now discussed lines 498-502 for acrosome and lines 503-511 for midpiece staining.

10) For intracellular localization of the CCDC146 in mouse sperm, the authors should provide clear negative control using WT sperm which do not carry the transgene.

This experiment was performed.

To avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum.

11) Current imaging data do not clearly support the intracellular localization of the CCDC146. Although western blot imaging reveal that CCDC146 is detected from sperm flagella, this is crude approach. Thus, this reviewer highly recommends the authors provide more clear experimental evidence, such as immuno EM.

We provide now a WB comparing the presence of the protein in the flagellum and in the head fractions; see new figure 6. We show that CCDC146 is only present in the flagellum fraction; The detection of the band appeared very quickly at visualization and became very strong after few minutes, demonstrating that the protein is abundant in the flagella. It is important to note that epididymal sperm do not have centrioles and therefore this signal is not a centriolar signal. We also now provide new statistical analyses showing that the immuno-staining observed in the principal piece is very specific (Figure 7B). Altogether, these results demonstrate unequivocally the intracellular localization of CCDC146 in the flagellum. This point is now discussed lines 480-489

12) Although sarkosyl is known to dissociate tubulin, it is not well understood and accepted that the enhanced detection of CCDC146 by the detergent indicates its microtubule inner space. Sperm axoneme to carry microtubule is also wrapped peri-axonemal components with structural proteins, which are even not well solubilized by high concentration of the ionic detergent like SDS.

We agree with the reviewer that the solubilization of the protein by sarkozyl is not a proof of the presence of the protein inside microtubule. Taking into account this point, the MIP hypothesis was toned down and we now discuss alternative hypothesis concerning these results; See discussion lines 490-497

13) SEM image is not suitable to explain internal structure (line 317-323).

We agree with the reviewers and changes were made accordingly. See lines 354-357

Minor comments

1) In main text, supplementary figures are cited "Supp Figure". And the corresponding legends are written in "Appendix - Figure". Please unify them.

Done Labelled now “Figure X-figure supplement Y”

2) Line 159, "exon 9/19" is not clear.

We have written now exons 9 and indicated earlier that the gene contains 19 exons

3) Line 188, "positive cells" are vague.

Positive was changed by “fluorescent”

4) Representative TUNEL assay image for knockout testes were not shown in Supp Figure 3B.

It was a mistake now Figure 2-figure supplement 2C

5) Please provide full description for "IF" and "AB" when described first.

Done

6) Line 262, It is unclear what is "main piece".

Changed to principal piece

7) Line 340, Although the "stage" information might be applicable, this is information for "seminiferous tubule" rather than "spermatid". This reviewer suggests to provide step information rather than stage information.

We agree with the reviewer that there was a confusion between “stage” and “step”. We change to step spermatids

8) Line 342, Step 1 is not correct in here.

OK corrected. now steps 13-15 spermatids

9) Line 803, "C." is duplicated.

Removed

10) Figure 3A, it will be good to mark the defective nuclei which are described in figure legends.

These cells are now indicated by white arrow heads

11) Figure 5, Please provide what MT stands for.

Now explained in the legend of figure 5

12) Figure 6. Author requires clear blot images for C. In addition, Panel B information is not correct. If the blot was performed using HA antibody, then how "WT" lane shows bands rather than "HA" bands?

The reviewer is correct. It was a mistake; The figure was recomposed and improved.

Reviewer #2 (Recommendations For The Authors):

Overall, editing oversights are present throughout the manuscript, which has made the review process quite difficult. Some repetitive figures can be removed to streamline to grasp the overall story easier. Some claims are not fully supported by evidence that need to tone down. Some figures not referenced in the main text need to be mentioned at least once.

All figures are now referenced in the text

Major comments:

1) 163-164 - Please clarify the claim that there is going to be an absence of the protein or nonfunctional protein, especially for the patient with a deletion that could generate a truncated protein at two third size of the full-length protein. Similarly, 35% of the protein level is present for the patient with a nonsense mutation. Some in silico structural analysis or analysis of conserved domains would be beneficial to support these claims.

Both mutations are predicted to produce a premature stop codons: p.Arg362Ter and p.Arg704serfsTer7, leading either to the complete absence of the protein in case of non-sense mediated mRNA decay or to the production of a truncated protein missing almost two third or one fourth of the protein respectively. CCDC146 is very well conserved throughout evolution (Figure supplementary 1), including the 3’ end of the protein which contains a large coil-coil domain (Figure 1B). In view of the very high degree of conservation, it is most likely that the 3’ end of the protein, absent in both subjects, is critical for the CCDC146 function and hence that both mutations are deleterious. This explanation is now added to the discussion. see lines 439-448

2) 173, 423 - Please clearly state a rationale of your mouse model design (i.e., why a mouse model that recapitulate human mutation is not generated) as the truncations identified in human patients are located further towards the C-terminus, and it is not clear whether truncated proteins are present, and if so, they could still be functional. Basically, the current mouse model supports the causality of the human mutations.

This is an important question, which goes beyond the scope of this article, and raises the question of how to confirm the pathogenicity of mutations identified by high-throughput sequencing. The production of KO or KI animals is an important tool to help confirm one’ suspicions but the first element to take into consideration is the nature of the genetic data.

Here we had two patients with homozygous truncating variants. In human, it is well established that the presence of premature stop codons usually induces non-sense mediated mRNA decay (NMD), inducing the complete absence of the protein or a strong reduction in protein production. In the unlikely absence of NMD in our two patients, the identified variants would induce the production of proteins missing 60% and 30% of their C terminal part. Often (and it is particularly true for structural proteins) the production of abnormal proteins is more deleterious than the complete absence of the protein (and it is most likely the purpose of NMD, to limit the production of abnormal “toxic” proteins). For these reasons, to try to recapitulate the most likely consequences of the human variants, without risking obtaining an even more severe effect, we decided to introduce a stop codon in the first exon in order to remove the totality of the protein in the KO mice.

The second element is to interpret the phenotype of the KO animals. Here, the human sperm phenotype is perfectly recapitulated in the KO mice.

Overall, we have strong genetic arguments in human and the reproduction of the phenotype in KO mice confirming the pathogenicity of the variants identified in men.

This point is now discussed see lines 433-438

3) Figure 6A - the labelling is misleading as it seems to suggest that the specific cells were isolated from the testes for RT-PCR.

We have modified the labelling to avoid any confusion.

Figure 6B -Signal of HA-tag is shown in WT, not in transgenic. Please check the order of the labels. Figure 6C - This blot is NOT a publication-quality figure. The bands are very difficult to observe, especially in lane D18. Because it is one of the important data of this study, replacing this figure is a must.

The figure has been completely remade, including new results. See new figure 6. Figure 6C was suppressed.

4) Supplementary fig 6 is also not a publication-level figure, and the top part seems largely unnecessary (already in the figure legend).

The figure has been completely remade as well (now Figure 6-Figure Supplement 1).

5) 261/267- The conclusion that mitochondrial staining in the flagellum (in both mice and humans) is non-specific is not convincing. Supplementary fig 8 shows that the signal from secondary only IF possibly extends beyond the midpiece - but it is hard to determine as no mitochondrial-specific staining is present. Either need to tone down the conclusion or provide supporting experimental evidence.

First, to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum. These experiments are now described lines 271-279

Second, we provide new images of the signal obtained with secondary Abs only that shows more clearly that the secondary Ab gave a non-specific staining (Figure 10-Figure supplement 1B). This point is discussed lines 503-511

6) Figure 9 A - Please relate the white line to Fig. 9B label in X-axis. The information from Fig 9A+D and 9E+F are redundant. The main text nor the figure legends indicate why these specific two sperm were chosen for quantification and demonstrating the outcomes. One of them could be moved to supplementary information or removed, or the two could be combined.

As suggested by the reviewer, we have combined the two sperm to demonstrate that CCDC146 staining is mostly located on microtubule doublets. Moreover, the figure was recomposed to make it clearer.

Minor comments:

All of the supplementary figures are referred to as Supp Fig X in the text, however, they are actually titled Appendix - Figure X. This needs to be consistent.

The figures are now referred as figure supplement x in both text and figures

Line 125 - edit spacing.

We think this issue (long internet link) will be curated later and more efficiently by the journal, during the step of formatting necessary for publication.

144 - With which to study  with which we studied?

We made the change as suggested.

151 - Supp Fig 1 - the text says that the gene is highly transcribed in human and mouse testes, but the information in the figure states that the level in mouse tissues is "medium"

We have corrected this mistake in the text; See line 176

165 - The two mutations are most likely deleterious. Please specifically mention what analyses done to predict the deleterious nature to support these claims.

Both variants, c.1084C>T and c.2112del, are extremely rare in the general population with a reported allele frequency of 6.5x10-5 and 6.5x10-06 respectively in gnomAD v3. Moreover, these variants are annotated with a high impact on the protein structure (MoBiDiC prioritization algorithm (MPA) score = 10, DOI: 10.1016/j.jmoldx.2018.03.009) and predicted to induce each a premature termination codon, p.(Arg362Ter) and p.(Arg704SerfsTer7) respectively, leading to the production of a truncated protein. This information is now given line 164-169

196-200/Figure 4 - As serum starved cells/basal body (B) are not mentioned in the main text, as is, Fig 4A would be sufficient/is relevant to the text. Please make the text reflect the contents of the whole figure, or re/move to supplement.

We agree with the reviewer that the full description of the figure should be in the text. We added two sentences to describe figure 4B see lines 217-218.

224 - spermatozoa (plural) fits better here, not spermatozoon

OK changed accordingly

236 - According to the figure legend, 6B is only showing data from the epididymal sperm, not postnatal time points; should be referencing 6C. Alignment of Marker label

As indicated above, the figure has been completely remade, including new results. See new figure 6. Figure 6C was suppressed. The corresponding text was changed accordingly see lines 249-266

255-256 - Referenced figure 7B3, however, 7B3 only shows tubulin staining, so no CCDC146 can be observed. Did authors mean to reference fig 7B as a whole?

Sorry for this mistake. We agree and the text is now figure 8B6 (figure 7 and 8 were switched)

305 - "of tubules" - I presume it is meant to be microtubules?

Yes; The text was changed as suggested

317-321 - a diagram of HTCA would be useful here

We have added a reference where HTCA diagram is available see line 363. Moreover, a TEM view of HTCA is presented figure 12A

322/Fig 11A - an arrow denoting the damage might be useful, as A1 and A3 look similar. The size of the marker bar is missing. Please update the information on figure legend.

Concerning, the comparison between A1 and A3, the take home message is that there is a great variability in the morphological damages. This point is now underlined in the corresponding text. We updated the size of the marker bar as suggested (200 nm). See line 365-367

323 - Please mark where capitulum is in the figure

Capitulum was changed for nucleus

Since Fig 11B2 is not referenced in the main text, it does not seem to add anything to the data, and could be removed/moved to supplement.

We added a sentence to describe figure 11B2 line 370

342-343 - manchette in step I is not seen clearly - the figure needs to be annotated better. However, DPY19L2 is absent in step I in the KO, but the main text does not reflect that - why is that?

We do not understand the remark of the reviewer “manchette in step I is not seen clearly”. The figure shows clearly the manchette (red signal) in both WT and KO (Figure 13 D1/D2).

For steps 13-15 WT spermatids, the size of the manchette decreases and become undetectable. In KO spermatids, the shrinkage of the manchette is hampered and in contrast continue to expand (Figure 13D2). We also provide a new Figure 13-figure supplement 1 for other illustrations of very long manchettes and a statistical analysis. In the meantime, the acrosome is strongly remodeled, as shown in figure 16-new, with detached acrosome (panel H). This morphological defect may induce a loss of the DPY19L2 staining (Figure 13 D2 stage I-III). This explanation is now inserted in the text line 396399

Figure 15B and 15C only show KO, corresponding images from the WT should be present for comparison.

WT images are now provided in Figure 1-figure supplement 1 new

Figure 12 - Figure 12 - JM?.

JM was removed. It does not mean anything

Figure 12C and Supplementary Fig 10 - structures need to be labelled, as it is unclear what is where

Done

338 - text mentions step III, but only sperm from step VII are shown in Figure 13

As suggested by reviewer 3, we changed stage by step. The text was modified to take into account this remark see lines 388-396

360 - This is likely supposed to say Supp Figure 11E-G, not 13??

Yes, it is a mistake. Corrected

388 Typo "in a in a".

Yes, it is a mistake. Corrected

820 - Fig 3 legend - in KO spermatid nuclei were elongated - could this be labelled by arrows? I am not convinced this phenotype is that different from the WT.

In fact, the nuclei of elongating KO spermatids are elongated and also very thin, a shape not observed in the WT; We have added arrow heads and modified the text to indicate this point line 200.

836 - Figure 5 legend says that in yellow is centrin, but that is not true for 5A, where the figure shows labelling for y-tubulin (presumably, according to the figure itself).

We have modified the text of the legend to take into account the remark

837- 5A supposedly corresponds to synchronized HEK293T cells, but the reasoning behind using synchronized cells is not mentioned at all in the main text; furthermore, how this synchronization is achieved is not explained in materials and methods (serum starvation? Thymidine block?).

Yes, figure 5A was obtained with synchronized cells. We have added one paragraph in the MM section. For cell synchronization experiments, cells underwent S-phase blockade with thymidine (5 mM, SigmaAldrich) for 17 h followed by incubation in a control culture medium for 5 h, then a second blockade at the G2-M transition with nocodazole (200 nM, Sigma-Aldrich) for 12 h. Cells were then fixed with cold methanol at different times for IF labelling. See line 224 for changes made in the result section and lines 700-704 for changes made in the MM section.

845- figure legend says that the RT-PCR was done on CCDC146-HA tagged mice, but the main text does not reflect that.

We made changes and the description of the KI is now presented before (line 240) the RT-PCR experiment (line 257).

949 - it is likely supposed to say A2, not B1 (B1 does not exist in Fig 15)

Yes, it is a mistake. Corrected

971 - Appendix Fig 3 legend - I believe that the description for B and C are swapped.

Yes, it is a mistake. Corrected

Furthermore, some questions to address in A would be: Which cross sections were from which animal/points? How many per animal? Were they always in the same location?

Yes, we have a protocol for arranging and orienting all testes in the same way during the paraffin embedding phase. The cross-sections are therefore not taken at random, and we can compare sections from the same part of the testis. The number of animals was already indicated in the figure legend (see line 1128)

Reviewer #3 (Recommendations For The Authors):

1) There are a number of grammatical and orthographical errors in the text. Careful proofreading should be performed.

We have sent the manuscript to a professional proofreader

2) The author should also check for redundancies between the introduction and the discussion.

The discussion has modified to take into account reviewers’ remarks. Nevertheless, we did our best to avoid redundancies between introduction and discussion.

3) Can the authors provide a rationale why they have chosen to tag their gene with an HA tag for localisation? One would rather think of fluorescent proteins or a Halo tag.

Because the functional domains of the protein are unknown, adding a fluorescent protein of 24 KDa may interfere with both the localization and the function of CCDC146. For this reason, we choose a small tag of only 1.1 KDa, to limit as such as possible the risk of interfering with the structure of the protein. This rational is now indicated in the manuscript lines 251-254. It is worth to note, that the tagged-strain shows no sperm defect, demonstrating that the HA-tag does not interfere with CCDC146 function.

4) In the abstract, line 53, "provide evidence" is not the right term for something that is just suggestive. The term "suggests" would be more appropriate.

The text was modified to take into account this remark

5) Line 74: "genetic deficiency" sounds strange here, do the authors mean simply "mutation"?

Infertility may be due to several genetic deficiency such as chromosomal defects (XXY (Klinefelter syndrome)), microdeletion of the Y chromosome or mutations in a single gene. Therefore, mutation is too restrictive. Nevertheless, we modified the sentence which is now “…or a genetic disorder including chromosomal or single gene deficiencies”

6) Lines 163-164: the authors describe the mutations (premature stop mutations) and say that they could either lead to complete absence of the gene product, or the expression of a truncated protein. Did they test this, for example, with some immuno blot analyses?

As stated above, unfortunately, we were unable to verify the presence of RNA-decay in these patients for lack of biological material.

7) Line 184 and Fig 2E: the sperm head morphologies should be quantitatively assessed.

We provide now a full statistical analysis of the observed defects: see new panel in Figure 2 F

8) Fig 3: The annotation should be more precise - KO certainly means CDCC146-KO. The colours of the IH panels is different, which attracts attention but is clearly a colour-adjustment artefact. Colours should be adjusted for the panels to look comparable. It would be also helpful to add arrowheads into the figure to point at the phenotypes that are highlighted in the text.

We have added Ccdc146 KO in all figures. We have added arrow heads to point out the spermatids showing a thin and elongated nucleus. Concerning adjustment of colors, we attempted to make images of panel B comparable. See new figure 3.

9) Fig 6A: the authors use RT PCR to determine expression dynamics of their gene of interested, and use actin (apparently) as control. However, actin and CDCC146 expression levels follow the same trend. How is the interpreted?

The reviewer did not understand the figure. The orange bars do not correspond to actin expression and the grey bars to Ccdc146 expression but both bars represent the mRNA expression levels of Ccdc146 relative to Actb (orange) and Hprt (grey) expression in CCDC146-HA mouse pups’ testes. We tested two housekeeping genes as reference to be sure that our results were not distorted by an unstable expression of a housekeeping gene. We did not see significant difference between both house keeping genes. Actin was not used.

10) In line 235, the authors suggest posttranslational modifications of their protein as potential cause for a slightly different migration in SDS PAGE as predicted from the theoretical molecular weight. This is not necessarily the case, some proteins do migrate just differently as predicted.

We have changed the text accordingly and now provide alternative explanation for the slightly different migration. See lines 258-259

11) The annotation of Fig 6 panels is problematic. First, why do the authors write "Laemmli" as description of the gel? It would be more helpful to write what is loaded on the gel, such as "sperm". Second, in panels B and C it would be helpful to add the antibodies used. It is not clear why there is a signal in the WT lane of panel B, but not in the HA lane (supposing an anti-HA antibody is used: why has WT a specific HA band?). In panel C, it is not clear why the blot that has so beautifully shown a single band in panel B suddenly gives such a bad labelling. Can the authors explain this? Also, they cut off the blot, likely because to too much background, but this is bad practice as full blots should be shown. In the current state, the panel C does not allow any clear conclusion. To make it conclusive, it must be repeated.

Several mistakes were present in this figure. This figure was recomposed. The WB on testicular extract was suppressed and we now present a new WB allowing to compare the presence of CCDC146 in the flagella and head fractions from WT and HA-CCDC146 sperm. Using an anti-HA Ab, we demonstrate that in epididymal sperm the protein is localized in the flagella only. See new figure 6. The corresponding text was changed accordingly.

12) The authors have raised an HA-knockin mouse for CDCC146, which they explained by the unavailability of specific antibodies. However, in Fig 7, they use a CDCC146 antibody. Can they clarify?

The commercial Ab work for HUMAN CCDC146 but not for MOUSE CCDC146. We have added few words to make the situation clearer, we have added the following information “the commercial Ab works for human CCDC146 only”. See line 240

13) In Fig 7A (line 258), the authors hypothesise that they stain mitochondria - why not test this directly by co-staining with mitochondria markers?

We chose another solution to resolve this question:

To avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the whole flagellum.

14) It seems that in both, Fig 7 and 8, the authors use expansion microscopy to localise CDCC146 in sperm tails. However, the staining differs substantially between the two figures. How is this explained?

In figure 8 we used the commercial Ab in human sperm, whereas in figure 7 we used the anti-HA Abs in mouse sperm. Because the antibodies do not target the same part of the CCDC146 protein (the tag is placed at the N-terminus of the protein, and the HPA020082 Ab targets the last 130 amino acids of the Cter), their accessibility to the antigenic site could be different. However, it is important to note that both antibodies target the flagellum. This explanation is now inserted see lines 304-312

15) Fig 8D and line 274: the authors do a fractionation, but only show the flagella fraction. Why?

Showing all fractions of their experiment would have underpinned the specific enrichment of CDCC146 in the flagella fraction, which is what they aim to show. Actually, given the absence of control proteins, the fact that the band in the flagellar fraction appears to be weaker than in total sperm, one could even conclude that there is more CDCC146 in another (not analysed) fraction of this experiment. Thus, the experiment as it stands is incomplete and does not, as the authors claim, confirm the flagellar localisation of the protein.

We agree with the reviewer’s remark. We provide now new results showing both flagella and nuclei fractions in new figure 6A. This experiment is presented lines 253-256

16) Line 283, Fig 9D,F: The description of the microtubules in this experiment is not easy to understand. Do the authors mean to say that the labelling shows that the protein is associated with doublet microtubules, but not with the two central microtubules? They should try to find a clearer way to explain their result.

As suggested by reviewer 2, we have changed the figure to make it clearer. The text was changed accordingly. See new figure 9 and new corresponding legend lines 1006.

17) Fig 9G - how often could the authors observe this? Why is the axoneme frayed? Does this happen randomly, or did the authors apply a specific treatment?

Yes, it happens randomly during the fixation process.

18) Line 300 and Fig 10A - the authors talk about the 90-kDa band, but do say anything about what they think this band is representing.

We have now added the following sentence lines 340-342: “This band may correspond to proteolytic fragment of CCDC146, the solubilization of microtubules by sarkosyl may have made CCDC146 more accessible to endogenous proteases.”

19) Fig 11A, lines 321-322: the authors write that the connecting piece is severely damaged. This is not obvious for somebody who does not work in sperm. Perhaps the authors could add some arrow heads to point out the defects, and briefly describe them in the text.

We realized from your remark that our message was not clear. In fact, there is a great variability in the morphological damages of the HTCA. For instance, the HTCA of Ccdc146 KO sperm presented in figure 10A2 is quite normal, whereas that in figure 10A4 is completely distorted. This point is now underlined in the corresponding text. See lines 367-369

We also added the size of the marker bar (200 nm), which were missing in the figure’s legend.

20) Line 323: it will be important to name which tubulin antibody has been used to identify centrioles, as they are heavily posttranslationally modified.

The different types of anti-tubulin Abs are described in the corresponding figure’s legend

21) Fig 11B - phenotypes must be quantified to make these observations meaningful.

We agree that a quantification would improve the message. However, testicular sperm are obtained by enzymatic separation of spermatogenic cells and the number of testicular sperm are very low. Moreover, not all sperm are stained. Taking these two points into account, it seems to us that quantification could be difficult to analyze. For this reason, the quantification was not done; however, it is important to note that these defects were not observed in WT sperm, demonstrating that these defects are cased by the lack of CCDC146. We have added a sentence to underline this point; See lines 374-375

22) Line 329: Figure 12AB - is this a typo - should it read Figure 12B?

We have split the panel A in A1 and A2 and changed the text accordingly. See line 378

23) Why are there not wildtype controls in Fig 12B, C?

We provide now as Figure 12-figure supplement 1, a control image for fig 12B. For figure 12C, the emergence of the flagellum from the distal centriole in WT is already shown in Fig 12A1

24) Fig 13: the authors write that the manchette is "clearly longer and wider than in WT cells" (lines 342-343). How can they claim this without quantitative data?

We now provide a statistical analysis of the length of the manchette. See figure 13-figure supplement 1A. We also provide a new a new image illustrating the length of the manchette in Ccdc146 KO spermatids; See Figure 13-figure supplement 1B.

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