Author response:
The following is the authors’ response to the original reviews.
We sincerely thank the reviewers and the Reviewing Editor for their careful evaluation of our manuscript and for their constructive and insightful comments. Their suggestions have helped us to improve the clarity, rigor, and presentation of our work. In response to these comments, we have substantially revised the manuscript and performed several additional analyses and experiments, as summarized below.
Major additions and modifications made during revision
In response to the reviewers' comments, we have substantially revised the manuscript and performed several additional analyses and experiments:
New analyses
- Quantification of MyoF recovery following auxin washout using MyoF-mAID-HA immunofluorescence (Figure 8B, Figure S10D).
- Quantification of maternal MIC2 fluorescence intensity following 24 h MyoF depletion and subsequent redistribution after auxin washout (150 micronemes per condition; Figure S10A,B).
- Pearson correlation analysis of ANKER1-Halo and HDEL-GFP localization (Pearson's R = 0.92 ± 0.04; n = 30 parasites).
- Additional probability-based analysis supporting regulated microneme inheritance.
- Expanded analysis of microneme redistribution across larger replication stages (Figure S5D).
New figures
- Figure S6: Dual-labelling analysis of additional Group 2 organelles (ER, apicoplast, glideosome).
- Figure S10A, B: MIC2 fluorescence intensity analysis following RB retention and redistribution.
- Figure S10D: Correlation between MyoF recovery and phenotype rescue.
- Figure S11: Schematic overview of quantification and analysis workflow.
Additional experimental efforts
- Generation of a MIC2-Halo / IMC1-mKATE / Cb-Emerald parasite line to improve visualization of RB-associated trafficking.
- Multiple attempts to perform higher-temporal-resolution live-cell imaging. However, prolonged acquisition resulted in severe phototoxicity, replication arrest, and parasite death, preventing reliable long-term recordings.
Textual and methodological revisions
- Expanded Materials and Methods section with detailed descriptions of fluorescence quantification, colocalization analyses, and statistical procedures.
- Re-evaluation of statistical analyses using two-tailed tests throughout.
- Revision of manuscript text to clarify the evidence supporting RB-associated trafficking and to better acknowledge current limitations.
Public Reviews:
Reviewer #1 (Public review):
Summary:
This work asks the question of how different organelles and structures in the apicomplexan parasite Toxoplasma gondii are recycled and/or segregated to the daughter cells during cell replication. In particular, they consider an unusual cell structure called the residual body that links replicating cells during the intracellular infection stage of this parasite. The residual body has historically been considered a 'dumping ground' for unnecessary relics of the mother cell during division, but this notion is increasingly being revised. Indeed, cell replication in Toxoplasma is often misinterpreted as cell division (cytokinesis), but in fact, the cell replicates its organelles and structures to multiple 10s of copies in seemingly distinctly formed daughter cells, but cytokinesis is delayed for many such cycles and typically only occurs simultaneously with parasite egress from its host cell. The residual body is, in fact, the connection between these pre-cytokinetic replicated daughters, and effectively, this is still a single cell at this stage. The authors have previously shown that an actin network extends through the residual body between these daughter cells, and ER and mitochondria common to all cells are also linked through this structure. This study examining the fates of organelles during cell replication is timely for continuing our understanding of how this fascinating component of the cell participates in these processes. The authors use Halo-tags as their principal tool to track discrete populations of proteins, labelling their organelle locations, and this provides beautiful insight into these processes.
Strengths:
Using dyes conjugated to Halo tags, this work elegantly tracks the fates of proteins synthesised by an original 'mother' cell over several replication cycles of pre-cytokinetic 'daughters'. Using this tool, they show that some organelles are made intact just once and that some of these can be subsequently sorted to the daughters (micronemes and rhoptries) while others are dismantled (IMC) and the daughters must make their own. A third set of organelles (largely synthesis, sorting, and metabolic compartments) is divided and inherited, and new daughter-synthesised proteins are added to the preexisting maternal proteins in these structures. A role for actin and myosin is clearly demonstrated for micronemes and rhoptries, and this correlates with their relatively late inheritance into the developing daughters. Overall, this work gives clarity to the behaviours of several cell structures during replication and paves the way to a better understanding of the mechanisms that drive the differences between structures and the universality of these processes in other apicomplexan parasites.
Weaknesses:
In addressing the question of residual body participation in sorting of organelles, it would be useful to clearly define this structure and when and where it is delineated from the posterior of a mother cell during the formation of daughter structures. This might seem like a moot point, but it would give clarity to notions of recycling and 'reservoirs'. Mother cells retain their active invasion apparatus until very late in daughter formation, and the need for micronemes and rhoptries to be released from this service late in the process might explain why they are only then trafficked to the cell posterior and then into the daughters. So, is this a distinct 'residual body' body function/reservoir or just a spatial constraint of this sequence of daughter formation? In subsequent cell replications (4, 8, 16... stages), is there a separation between the residual body that links them all and the posterior of each new 'mother cell', and if so, when is this distinction lost? This is important because without a definition, we might be confusing different processes.
We thank the reviewer for this excellent and thoughtful question. The residual body (RB) emerges at the end of the first replication cycle, where it is delineated by the basal complex and persists as an IVN-associated compartment connecting all daughter parasites through both plasma membrane and cytoplasm. Previous EM and live-cell studies, including ours, have shown that the RB is not a passive remnant but a dynamic structure dependent on F-actin and unconventional myosins, supporting recycling, inter-parasite connectivity, and synchronous growth (Delbac et al., 2001; Muñiz-Hernández et al., 2011; Frénal et al., 2017; Periz et al., 2017).
In the present study, the MyoF reversibility experiment provides strong support for a model in which RB functions as an active recycling hub. Upon MyoF depletion, maternal microneme and rhoptry proteins accumulate within the RB. Following restoration of MyoF expression, this material is redistributed to daughter organelles. We interpret this reversible phenotype as evidence that the RB represents a distinct and regulated trafficking intermediate rather than simply a by-product of late daughter cell formation.
We agree with the reviewer that mother cells retain a functional invasion apparatus until very late during daughter formation, and that the delayed release of micronemes and rhoptries likely contributes to their late trafficking toward the cell posterior. However, our data indicate that once released, these organelles transit through a defined RB compartment that actively participates in their recycling rather than merely reflecting positional constraints. This has been previously well illustrated for micronemes, which are trafficked along F-actin filaments within the residual body (Periz et al., 2019).
At later rounds of replication (4, 8, 16 parasites), previous studies have demonstrated the presence of multiple residual body centres within the same vacuole. However, the precise temporal and structural distinction between the RB linking parasites within the vacuole and the posterior of newly formed mother cells remains insufficiently resolved and is beyond the scope of the present study. Importantly, available ultrastructural and live-cell imaging supports the persistence of shared RB compartments connecting parasites within a vacuole, arguing against a simple conflation of posterior membranes and residual body material.
While the primary aim of the current work was to investigate the RB's role in organelle recycling, we fully agree that a more precise definition of when and how the RB is formed, remodelled, and ultimately resolved during successive replication cycles will be essential to distinguish recycling from spatial constraints. We have revised the Discussion to better acknowledge this limitation and to avoid overinterpreting the role of the RB in organelle inheritance.
Are rhoptries/micronemes that originate in one 'mother' able to be sorted to the 'daughters' from a distinct mother in this syncytium? If so, this would make it a sorting centre, but otherwise we could be just capturing the activities at the posterior of any given cell during replication. The authors' further thoughts on this would be very interesting.
We agree with the reviewer that our current data do not definitively demonstrate whether rhoptries or micronemes originating from one “mother” parasite can be redistributed to daughters derived from another mother within the same syncytial vacuole. Nevertheless, our MyoF chase experiments are consistent with a model in which the RB/IVN functions as an active recycling and sorting hub rather than simply representing posterior trafficking events associated with individual parasites.
Upon MyoF depletion, maternal micronemes accumulated within the RB. Following restoration of MyoF expression, these accumulated micronemes were subsequently redistributed to daughter parasites. This reversible redistribution is more consistent with an active recycling process than with passive accumulation alone.
To further support this interpretation, we expanded the analysis presented in Figure S5 by including additional vacuoles and larger replication stages (new panel D). These analyses show that maternal micronemes are redistributed broadly and relatively evenly among daughter parasites. We additionally performed a probability-based analysis demonstrating that the recurrent and homogeneous redistribution patterns observed are highly unlikely to arise from stochastic capture events occurring independently at the posterior end of each parasite during replication. Together, these analyses support the interpretation that microneme redistribution is a regulated process.
Direct demonstration of recycling between all parasites within a vacuole would require a system allowing simultaneous differential labeling of (i) daughter parasites derived from a specific mother cell and (ii) the maternal organelles originating from that same mother during a subsequent replication cycle. To our knowledge, such an approach is not currently technically feasible. Nevertheless, our live-cell imaging experiments provide additional support for communal redistribution, as microneme material accumulated within the RB was subsequently observed redistributing, albeit unevenly, across multiple tachyzoites within the same vacuole.
The Group 2 structures are described as those that are divided between daughters and receive newly synthesised proteins that add to the maternal protein of these compartments. While this is a logical conclusion for several that are mentioned, where the maternal protein signal is seen to be depleted with replication (including for the apicoplast, ER, glideosome, and Golgi). Data for the addition of new proteins to these existing structures is actually only presented in direct support of this for the Golgi.
We thank the reviewer for this important clarification. We initially selected the Golgi as a representative example because its morphology and restricted localization provide the clearest visualization of the dual-labeling dynamics. However, the same experimental approach was applied to all Group 2 organelles analyzed in this study. To address the reviewer's concern more directly, we have now included a new supplementary figure (Figure S6) showing that the same pattern is also observed for the apicoplast, ER, and glideosome.
We would also like to clarify that the maternal protein signal is not lost during replication but instead becomes progressively diluted as these organelles expand, are partitioned into daughter parasites, and incorporate newly synthesized proteins. The Golgi was originally highlighted because these dynamics are most readily visualized in this compartment; however, the same principle applies to all Group 2 organelles analyzed in this study, as now illustrated in Figure S6.
Reviewer #2 (Public review):
Summary:
Toxoplasma gondii is an obligate intracellular parasite and the causative agent of Toxoplasmosis. Parasite invasion into host cells, intracellular replication, and then egress, which results in the destruction of the infected cell, is central to pathogenicity. This manuscript focuses on understanding how maternal resources (in this case, cellular organelles) are shared between daughter parasites during cell division. Many organelles are single copy, meaning that division and inheritance by the daughters is crucial for successful replication. The major strength of this study was the use of a Halobased pulse chase assay to characterize patterns of organelle inheritance. The results show that both microneme and rhoptries (secretory vesicles) previously thought to be synthesized de novo are inherited by daughter parasites. Thus, this paper adds new insight to our understanding of cell division in this important parasite.
Strengths:
This study demonstrated that pulse labeling of proteins can be used to monitor protein synthesis, turnover, and movement. This approach will be of great interest to the field. Using this method, the authors demonstrate three main modes of organelle inheritance.
(1) Organelles, where there are multiple copies (such as secretory vesicles, micronemes, and rhoptries), are divided between the daughter parasites, with additional contribution of newly formed vesicles. New and old material remain as separate entities in the cell.
(2) Single-copy organelles, which are expanded to include newly synthesized material prior to division, such as the Golgi and apicoplast.
(3) Cytoskeletal structures that are synthesized anew during each round of division. These studies provide more refined insight into patterns or organelle inheritance and demonstrate that secretory organelles are not made de novo during each round of division as previously thought. The paper has a logical flow, and overall, the data is presented in a clear and organized fashion.
Weaknesses:
(1) Descriptions of methodology and statistical analysis were incomplete.
We agree with the reviewer that the description of the methodology and statistical analyses required further clarification. To address this, we have added a new supplementary figure (Figure S11) illustrating the experimental workflow, quantification strategy, and analysis pipeline. We have also expanded the Materials and Methods section to provide detailed descriptions of the experimental design, fluorescence quantification procedures, statistical analyses, and the number of biological replicates. These revisions provide a clearer and more comprehensive description of the methodology and data analysis.
(2) There are inconsistencies between the data in Figures 1 and 5. In Figure 1, a small amount of maternal IMC is visible in stage 2 parasites. Although this is a ~90% reduction, these parasites should be quantified as parasites with material IMC. However, the graph in Figure 5C indicates that no material parasites have GAPM1a, given that graph 5C is a binary measure (present vs. absent), one would expect a non-zero percent of parasites to have maternal material.
We agree with Reviewer 2 that, based on the raw fluorescence signal, one might expect a non-zero percentage of parasites to retain maternal IMC material after the first replication. The apparent discrepancy between Figures 1 and 5 reflects our thresholding strategy rather than inconsistent data.
Figure 5C presents a binary analysis (presence versus absence) using a threshold calibrated from stage 1 parasites and applied uniformly across all markers. Under this criterion, the residual GAPM1a signal after the first replication falls below the detection threshold, resulting in 0% positive vacuoles. Although normalization to stage 2 parasites would detect this weak residual signal, such a protein-specific threshold would compromise direct comparison across the dataset.
To clarify this point, we have updated the Figure 5C legend to explain the analytical approach and the asterisk associated with GAPM1a. The residual maternal IMC signal visible in Figure 1 represents a rare example selected to illustrate the remaining ~10% signal and is consistent with the absence of detectable maternal IMC1 after replication in Figures 2C and 5E.
(3) The conclusion from Figure 6 was not justified based on the data. I agree with the author's conclusion that the accumulation of micronemes and rhoptries in the residual body was timedependent. In Figure 6A, the signal observed in the residual body at times 6:30, 13, and 14 hours is not observed in subsequent time points. However, the fate of these micronemes and rhoptries is unclear. It cannot be concluded that these vesicles are recycled back to the mother. They could also have been degraded. In fact, the graphs of microneme inheritance in Figure 2B show a decrease in maternal signal from 100% to 80% between stages 1 and 2, indicating that some microneme degradation is taking place.
We agree with the reviewer that Figure 6 alone does not definitively establish the fate of micronemes and rhoptries accumulating within the residual body (RB), and that both recycling and degradation remain possible interpretations. Our conclusion that maternal micronemes are predominantly recycled is therefore based on the integration of Figure 6 with our MyoF depletion and recovery experiments, additional quantitative analyses, and previous work demonstrating F-actin-dependent microneme trafficking through the RB (Periz et al., 2019).
Consistent with this model, MyoF depletion results in the accumulation of maternal micronemes within the RB, whereas restoration of MyoF expression following auxin washout leads to their redistribution across multiple tachyzoites within the same vacuole (Figure 8). Furthermore, maternal microneme signal remains detectable even after prolonged MyoF depletion (up to 48 h) and multiple rounds of replication (Figures 7 and 8), arguing against extensive degradation.
To further address this possibility, we quantified the fluorescence intensity of individual maternal MIC2-positive micronemes retained within the RB after 24 h of MyoF depletion and following redistribution after auxin washout (150 micronemes per condition). No significant difference in fluorescence intensity was observed compared with control maternal micronemes (Figure S10A,B), indicating that maternal microneme signal is preserved during RB retention and redistribution.
We therefore interpret the decrease in maternal microneme signal observed between stages 1 and 2 in Figure 2B primarily as a consequence of redistribution and dilution rather than degradation, consistent with the stable fluorescence intensity of individual micronemes (Figure 3). Regarding rhoptries, we note that the majority (~90%) are incorporated into daughter parasites before budding is complete, limiting their accumulation within the RB and suggesting that RB-associated trafficking primarily reflects redistribution rather than bulk degradation.
(4) To convincingly demonstrate that the redistribution of micronemes and rhoptries was due to recovery of MyoF protein levels after auxin washout, a Western blot should be performed to show MyoF protein levels over time. In addition, the decrease in mMIC2 protein levels in the residual body in Figure 8F should be measured and normalized for photobleaching. Both apical and basal signals appear to be reduced over the time course of imaging.
We agree with the reviewer that demonstrating MyoF recovery following auxin washout is important. Rather than performing a Western blot, we monitored MyoF recovery by immunofluorescence using the HA tag in the MyoF-mAID-HA strain, allowing direct correlation between MyoF reappearance and microneme redistribution at the single-vacuole level. These data are now included in Figure 8B, with the corresponding MyoF presence–phenotype association analysis presented in Figure S10D.
Regarding photobleaching, we agree that fluorescence loss during time-lapse imaging is an important consideration. However, in this experiment, changes in fluorescence intensity reflect not only photobleaching but also biological redistribution of micronemes and movement of parasites in and out of the imaging plane. In the absence of a stable internal reference fluorophore, applying a standard photobleaching correction could therefore introduce additional inaccuracies. For this reason, we did not quantify fluorescence intensity during the redistribution phase.
Instead, to assess whether maternal microneme signal is lost during RB retention and redistribution, we quantified the fluorescence intensity of individual maternal MIC2-positive micronemes following 24 h of MyoF depletion and subsequent auxin washout (Figure S10A,B). No significant difference was observed compared with control maternal micronemes, supporting the conclusion that redistribution occurs without substantial loss of the maternal microneme pool.
Reviewer #3 (Public review):
Summary:
Knoerzer-Suckow et al. explore the mechanisms of organelle inheritance during endodyogeny in Toxoplasma gondii using an innovative dual-labeling approach to track the distribution of maternal organelles into daughter parasites. They can clearly distinguish between maternal and daughterderived organelles using their dual-labeling Halo Tag approach. They reveal that different organelles are trafficked to daughter parasites in three broad patterns, which they have binned into groups. Their findings reveal a role for MyoF in the inheritance of micronemes and rhoptries, and notably, they observe that the inner membrane complex (IMC) is not recycled. Instead, the IMC undergoes a pronounced relocalization to the posterior of the maternal cell, where it is likely targeted for degradation.
Strengths:
The data surrounding their MyoF knockdown experiments, IMC degradation, and trafficking of MIC2 after auxin washout are compelling. These data add to the knowledge of how organelle inheritance occurs in T. gondii, increasing the field's understanding of endodyogeny.
Weaknesses:
(1) The evidence provided to support the claim that microneme and rhoptry inheritance specifically traffics through the residual body does not sufficiently substantiate the claim. The temporal resolution of the imaging is inadequate to precisely trace the path of microneme and rhoptry inheritance. From the data shown in the manuscript, it can be concluded that at least some of the micronemes and rhoptries might be recycled through the residual body, but it is unclear whether many or most of these organelles do so.
We thank the reviewer for this important comment and refer also to our response to Reviewer 1 above.
Previous work has demonstrated F-actin-dependent trafficking of micronemes within the residual body (RB) (Periz et al., 2019). Consistent with these findings, our data support a model in which RB-mediated trafficking contributes to maternal microneme recycling. We acknowledge, however, that the temporal resolution of our imaging does not allow continuous tracking of every individual organelle throughout the entire replication process.
In contrast, our observations indicate that the majority of maternal rhoptry material is incorporated into daughter cells before replication is complete and therefore does not necessarily transit through the RB under normal conditions (Figure 6). Nevertheless, rhoptry inheritance remains dependent on the actin–MyoF trafficking machinery, as MyoF depletion results in the accumulation of rhoptry material within the RB (Figures 6 and 7).
Taken together, our data support a model in which the RB serves as an important recycling hub for maternal micronemes and can, under conditions of impaired trafficking, also transiently accommodate rhoptry material. However, our imaging resolution does not allow us to conclude that all microneme or rhoptry inheritance obligatorily transits through the RB, and we have revised the manuscript to reflect this limitation more explicitly.
(2) The absence of specific markers for the residual body brings into question whether microneme inheritance occurs through a discrete residual body or simply via the basal end of the maternal parasite. The authors need a robust way to visualize and define the residual body to claim that micronemes and rhoptries are specifically transported through this structure.
We agree with the reviewer that the absence of a dedicated residual body (RB) marker remains a limitation and that such a tool would improve the precision of our analyses. To date, no specific RB marker has been identified (see also our response to Reviewer 1). The most reliable proxy currently available is the F-actin chromobody, which labels the dense F-actin network associated with the RB. Using this approach, previous work demonstrated F-actin-dependent trafficking of micronemes within the RB (Periz et al., 2019).
Building on these findings, our data support a model in which RB-associated trafficking contributes to maternal microneme recycling, whereas rhoptries are more frequently incorporated directly into daughter cells without obvious RB transit. In addition, functional perturbation of the actin–MyoF transport machinery, through MyoF depletion and subsequent recovery, supports the interpretation that the RB represents a discrete actin-associated compartment involved in organelle redistribution. Nevertheless, we acknowledge that our current imaging resolution does not allow us to determine the extent to which all microneme or rhoptry inheritance occurs through the RB, and we have revised the manuscript accordingly.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
Comments for revision where either the clarity or accuracy could be improved:
(1) The methods could do with some further detail with respect to the fluorescence intensity measurement. For example, where the Z-series was taken, and for measurements, were maximum projects taken or single Z-planes? Were all measurements made on unprocessed images, or was any deconvolution, etc, undertaken?
We updated the Materials and Methods for better clarity and now read as: “Parasites were labeled as described above and allowed to replicate for 24 h on HFF-coated Ibidi live-cell dishes. Approximately 15 fields of view were imaged using Z-stacks spanning 3 μm centred on the vacuoles. For each replication stage (1, 2, 4, and 8 parasites per vacuole), individual tachyzoites were sampled across multiple vacuoles.
Maximum-intensity projections were generated from non-deconvolved images. Fluorescence intensity (FI) was quantified as the maximum grey value measured within regions of interest (ROIs) drawn on individual tachyzoites from each vacuole stage. ROIs excluded overlapping parasites, neighbouring vacuoles, and regions with atypical signal intensity. For each biological replicate, up to 25 tachyzoites per replication stage were analyzed, and the mean FI value was calculated for each stage. The highest mean FI observed among the stages within a replicate was defined as 100%, and FI values for the other stages were expressed relative to this maximum. Relative FI values were then averaged across three independent biological replicates. Data are presented as mean ± SD.”
(2) Line 93: Is more JF549 added at each of stages 2, 4, and 8? I assume so to get the progressive increase, but it would help to clarify this here.
As illustrated in Figure 1a, the second dye is added only once, after 24 h of replication, and is then washed off before imaging. It is not maintained throughout the replication steps. The observed increase in signal is related to the presence of newly synthesized (de novo) proteins generated during replication. The Halo tags of these new proteins are initially free of ligand, as no ligand is present during replication. During the second labelling step, all free Halo tags can bind the dye. The signal intensity at each replication step therefore reflects the amount of de novo material present, which is higher at step 4 than at step 2, because the proteins generated de novo during stage 2 are also present in stage 4. The signal will be determined by both the number of newly produced molecules and their concentration at the same localization.
(3) Line 103: Check that the Carruthers and Sibley, 1997, ref for Tic20 in the apicoplast is correct. I don't think this could be correct given the date.
The reference it will be corrected to G van Dooren et al. 2008.
(4) Figure legends: It would be useful to state what form of microscopy was used in each figure.
Following the reviewer's advice the legends has been updated.
(5) Figure 2, S2: How are single organelles tracked, such as rhoptries? I'd assume that a cell will gain new de novo organelles as well, and that this would reduce the signal per cell. Stage 2 has a rhoptry signal in both daughters, so I'd expect the signal to be roughly half for the whole cell, unless the authors can resolve individual rhoptries (which would surprise me with this microscopy). If individual rhoptries were resolved, how was this done, and what was the confidence in this (were there controls?)
We thank the reviewer for this important point. We do not resolve individual rhoptries with the imaging conditions used in this study. Instead, fluorescence intensity was measured as the maximum grey value within a representative region of interest (ROI) encompassing the apical rhoptry signal while excluding overlapping parasites and regions with atypical fluorescence intensity. The same ROI selection strategy was applied consistently across all replication stages, allowing direct comparison with stage 1 parasites.
The analyses presented in Figures 3 and S2 show an increasing proportion of tachyzoites lacking detectable maternal rhoptry signal as replication progresses, while the fluorescence intensity of the remaining maternal signal remains relatively stable. Together, these observations are consistent with the redistribution of intact maternal rhoptries rather than a progressive loss of rhoptry fluorescence.
(6) Line 150: it is unclear what is meant by 'regulated partitioning'. The more equal inheritance of micronemes versus rhoptries might not indicate a 'regulated partitioning' but just a more uniform distribution, given the larger number of micronemes versus rhoptries.
We agree with the reviewer that this statement required clarification, and we have revised the text accordingly. Our intention was to emphasize that microneme inheritance appears to be a regulated process, rather than to directly compare it with rhoptry inheritance. The observed differences between these organelles are likely influenced, at least in part, by their different abundances.
Across successive rounds of replication, daughter parasites consistently inherit comparable amounts of maternal micronemes, even at later replication stages (Figure S4). Given that a single mother parasite contains approximately 30–40 micronemes, whereas successive rounds of endodyogeny can generate up to 32 daughter parasites, a purely stochastic segregation would be unlikely to produce the relatively uniform distribution observed (~1–2 maternal micronemes per tachyzoite). To support this interpretation, we performed an additional probability-based analysis, which indicates that the observed redistribution patterns are unlikely to arise by chance alone. We therefore interpret these findings as supporting the existence of mechanisms that promote balanced microneme inheritance during parasite replication.
(7) Line 184: How is the maternal signal measured without detecting the internal daughter signal? Is this an average signal for the full parasite, or just for a cross-section of the IMC? And if the latter, how are the different profiles of mother and daughter accounted for? Also, the abbreviation in the brackets doesn't make sense here.
We thank the reviewer for this important point. We have revised the Materials and Methods section to provide a clearer description of the fluorescence intensity (FI) measurements and added a new supplementary figure (Figure S11) illustrating the analysis workflow.
Briefly, FI measurements were performed on maximum-intensity projections generated from Z-stack images without deconvolution. A representative region of interest (ROI) was selected, and the maximum grey value was used for quantification. This approach minimizes variability arising from differences in ROI size and provides a robust metric for comparison across replication stages.
Daughter cell fluorescence was measured using the same approach while excluding overlapping signals from neighboring daughter cells and the maternal IMC. Maternal and daughter signals were distinguished based on their spatial localization and fluorescence labeling. Finally, the abbreviation in brackets has been corrected for clarity.
(8) Line 191: Subheading a bit unclear. Distinct from other organelles, or are miconeme and rhoptry pathways distinct from each other?
We agree with the reviewer and have updated the subheading to “Whole-organelle inheritance of micronemes and rhoptries occurs via distinct recycling pathways”
(9) Line 193: The site of disassembly of the IMC (suggested RB here) might not be the same as the site of degradation. I suggest using 'disassembly' instead here.
We agree that “disassembly” is an appropriate term to describe the breakdown of the IMC at the residual body (RB). However, we also believe that the RB represents the primary site of IMC degradation, for two reasons. First, if IMC material were not degraded at this site, we would expect to detect Halo-positive signal elsewhere following IMC collapse, which we do not observe. Second, transport of IMC material to an alternative degradation site would be required, but no IMC-positive vesicles are observed, arguing against significant redistribution. Together, these observations support the conclusion that the RB is both the site of disassembly and degradation of maternal IMC.
(10) Line 215: The conclusion for a difference in timing of microneme and rhoptry segregation is not clearly supported by the data presented. Also, if there are more micronemes than rhoptries, then the frequency of observing a microneme being trafficked through the RB would need to be higher than for rhoptries if the mechanisms were the same. So, a difference in frequency here cannot be used to argue for a different mechanism.
We agree with the reviewer and the text have been edited to soften our conclusion.
(11) Line 234: 'segregation' might be a better term than 'recycling' here because it is actually the sorting into daughter cells that is the important process.
The text have been edited
(12) Line 235: I don't think this can be what the authors intend to say. If the maternally-inherited rhoptries are not trafficked through the RB (every time), then how do they get into the daughters? Perhaps this is a case where a clear definition of the RB is required.
Our observations indicate that maternally inherited rhoptries are frequently incorporated into daughter cells before collapse of the mother cell and establishment of the residual body (RB). Although F-actin is enriched within the RB, an actin network is also present throughout the parasite cytoplasm, where MyoF is likewise localized. We therefore propose that, unlike micronemes, rhoptries do not necessarily transit through the RB during every replication cycle but can be incorporated directly into developing daughter cells while still relying on the same actin–MyoF-dependent trafficking machinery.
(13) The MyoF Rescue, the experimental plan is not fully described in order to be clear. If the endomembrane architecture was disrupted by MyoF depletion, and this secondary effect caused the segregation phenotype, restoration of MyoF might also simply restore the endomembrane system. So a direct role for MyoF doesn't seem to have been tested in this case.
We appreciate the reviewer's concern that the segregation phenotype could, in principle, arise indirectly from disruption of endomembrane architecture following MyoF depletion. However, although Golgi morphology is altered in MyoF-depleted parasites, its core functions appear largely preserved. This is supported by the normal biogenesis of de novo micronemes, their correct targeting to the apical pole, their efficient secretion, and the previously reported preservation of parasite invasion. In addition, Golgi markers are not detected in the residual body, where maternally inherited micronemes accumulate, arguing against Golgi-mediated trafficking as the primary cause of the segregation phenotype.
Taken together, these observations support the interpretation that the segregation defects are more likely to reflect a direct role of MyoF in organelle trafficking and inheritance than a secondary consequence of generalized disruption of endomembrane organization.
(14) Line 279: Why call it a checkpoint? What is the evidence for its presence here being sensed before a further process is activated, which is what a checkpoint does?
We agree the reviewer that checkpoint is a misleading term and have been updated to trafficking hub.
(15) Line 287 confuses replication of the daughters from cytokinesis, which only happens when each cell loses cytoplasmic connectivity with the other.
We will clarify this point. In Toxoplasma gondii, cytokinesis represents the final step of daughter cell formation, during which the two fully assembled daughter parasites separate from the mother cell following collapse of the maternal cytoplasm. Historically, the residual body was proposed to arise simply as leftover material from this process. However, multiple studies have now shown that residual body formation is an active and regulated process, dependent on specific cytoskeletal and trafficking factors. Importantly, although cytokinesis marks the physical separation of daughter cells from the mother, parasites within a vacuole remain connected via the residual body and continue to share cytoplasmic and plasma membrane components until egress.
(16) Line 298: I don't think there is direct evidence of degradation in the RB. There might be disassembly, but degradation implies proteolysis, which hasn't been tested for.
We agree with the reviewer that our data do not provide direct biochemical evidence of proteolysis within the residual body (RB) and primarily demonstrate disassembly of the maternal IMC at this site. However, several observations are consistent with local degradation. Following IMC collapse, we do not detect Halo-positive signal elsewhere in the parasite, nor do we observe IMC-positive vesicles or other structures that would suggest transport to a distinct degradation compartment.
In addition, previous work identified the E3 ubiquitin ligase CSAR1 as a mediator of protein turnover within the RB, supporting the idea that this compartment is associated with degradation-related processes (O'Shaughnessy et al., 2023). While we cannot formally demonstrate proteolysis, these observations support a model in which IMC disassembly is closely coupled to local degradation within the RB.
(17) The paragraph structure gets a bit confusing at times. See single sentence paragraph, Line 224. Does this sentence justify its own paragraph?
The text has been edited.
(18) Make sure Toxoplasma gondii is in italics throughout.
The text has been edited
(19) Line 279 cites Figure 10. But there is none.
The text has been edited
(20) I advocate introducing a few new acronyms, like DCs. I find that this ultimately reduces the ease with which readers read the work if they don't learn them all quickly.
We agree that excessive use of acronyms can negatively impact readability. In the present manuscript, all abbreviations used in the text are introduced at their first occurrence in the Introduction, including DCs (line 32), IMC (line 41), PV (line 35), ER (lines 38–39), RB (line 50), and IVN (line 49). We have carefully limited the use of abbreviations to commonly used terms in the field and to those that recur frequently throughout the manuscript, with the aim of balancing clarity and readability. Nevertheless, we are happy to reduce or remove specific abbreviations if the reviewer feels this would further improve clarity.
Reviewer #2 (Recommendations for the authors):
(1) Descriptions of methodology and statistical analysis were incomplete as follows:
(1a) It was unclear how the fluorescence intensity measurements (used to evaluate inheritance vs. new synthesis) were carried out. The y-axis on the graph is labeled average fluorescence intensity (% of max intensity). However, it does not state what was averaged (average fluorescence per vacuole?) and what was max intensity (max pixel intensity in each image or time point with the highest average intensity, relative to the other time points?)
We agree with the reviewer that the original description of the fluorescence intensity (FI) measurements lacked clarity. We have therefore revised the Materials and Methods section and added a new supplementary figure (Figure S11) illustrating the analysis workflow.
Briefly, vacuoles were imaged as Z-stacks, and maximum-intensity projections were used for analysis. FI was quantified as the maximum grey value measured within representative regions of interest (ROIs) drawn on individual tachyzoites, rather than as an integrated fluorescence signal across the vacuole. This approach minimizes variability arising from differences in ROI size and allows direct comparison between replication stages.
For each biological replicate, up to 25 tachyzoites per replication stage were analyzed. The mean FI for each stage was normalized to the highest mean value within that replicate, and data from three independent biological replicates were subsequently averaged.
(1b) Given the uncertainties with how these measurements were performed, it is difficult to interpret the data. For example, one would expect that the fluorescence intensity of newly synthesized IMC1 in 8-parasite vacuoles would be 4 times higher than that of a 2-parasite vacuole; however, based on the graph in Figure 1B, the measured increase was only 30%.
We agree that the original description of the fluorescence intensity (FI) measurements required further clarification and have revised the Materials and Methods accordingly. As the reviewer correctly notes, a fourfold increase in FI between 2- and 8-parasite vacuoles would be expected if total IMC fluorescence across the entire vacuole had been measured. However, this was not the parameter quantified.
Instead, FI was measured as the maximum grey value within representative regions of the daughter IMC, providing a per-cell rather than a whole-vacuole measurement. Using this approach, FI increases between the 2- and 4-parasite stages and then reaches a plateau.
This behavior is consistent with the biology of IMC biogenesis. Although the total amount of IMC per vacuole increases with parasite number, the amount of IMC protein incorporated into each daughter parasite remains relatively constant. Consequently, once daughter IMCs are fully assembled from de novo-synthesised material, additional rounds of replication increase the total IMC content per vacuole but not the fluorescence intensity measured for individual parasites.
(1c) T. gondii replicates in an asynchronous manner, so that at the 24-hour time point, a single dish can contain vacuoles containing 2, 4, and 8 parasites. This should be stated explicitly so readers unfamiliar with T. gondii's growth patterns can understand how the experiment was performed.
We agree with the reviewer and have updated the text line 93. “As Toxoplasma gondii replicates in an asynchronous manner, after 24 of replication, vacuoles containing 1, 2, 4, and 8 parasites can be observed in a single dish.”
(1d) Colocalization package in Fiji used for ANKER1-Halo with HDEL-GFP and MIC2/RON2 with CbEmeraldFP should be specified.
We thank the reviewer for this suggestion. Following this recommendation, we performed Pearson correlation analysis for the ANKER1–HDEL-GFP experiment using the Coloc 2 plugins of FiJi. ANKER1Halo and HDEL-GFP showed a strong spatial correlation (Pearson's R = 0.92 ± 0.04, n=30 parasites from three independent biological replicates), supporting localization of ANKER1 to the ER.
We note, however, that this analysis should be interpreted as evidence for co-distribution within the same organelle rather than direct molecular colocalization, as ANKER1 is a transmembrane protein whereas HDEL-GFP labels the ER lumen.
For all the rest of our analysis, no automated colocalization package or plugin in Fiji was used for the analyses involving ANKER1-Halo with HDEL-GFP or MIC2/RON2 with Cb-EmeraldFP. Colocalization was assessed manually across all experiments by inspecting both full Z-stacks and maximum-intensity projections to ensure robust spatial overlap.
For MIC2 and RON2, the presence of signal within the Cb-Emerald–positive filament was scored as either cytoplasmic, on the residual body or absence of colocalisation.
In total, more than 300 and 500 vacuoles were analyzed for MIC2 and RON2–Cb-Emerald colocalization respectively (stable expression of both markers), and more than 150 vacuoles were analyzed for ANKER1-Halo and HDEL-GFP colocalization (transient expression of HDEL-GFP).
This information has now been added to the Methods section.
(1e) Statistical methods should be described on an experiment-by-experiment basis. The authors should justify why a one-tailed t-test was conducted. A two-tailed t-test seems more appropriate.
We agree that statistical methods should be clearly justified on an experiment-by-experiment basis. All the statistical analysis have been performed using two tails and updated in the figures.
(1f) In Figures 5E and 5F, using boxes to indicate the exact areas of the cell that were used in the fluorescence intensity measurements, rather than arrows, would make this data easier to interpret.
The figure has been updated.
Reviewer #3 (Recommendations for the authors):
The current time-lapse images and videos do not clearly demonstrate microneme movement from the maternal parasite apical end to the residual body and back to the apical end of daughter parasites. As such, the route by which micronemes enter daughter parasites remains inconclusive. To strengthen their claims, the authors should employ higher temporal resolution imaging to definitively capture the movement of micronemes from the maternal apical region into the daughters. From the current data, it also seems plausible that the micronemes may be trafficked into the daughters through the conoid as well, as there is no evidence provided showing a movement of micronemes away from the apical end of the maternal parasite before being present in the daughter parasites.
We agree with the reviewer that higher temporal resolution imaging would provide a more definitive view of microneme trafficking. However, long-term live imaging of replicating Toxoplasma gondii requires a compromise between temporal resolution and parasite viability. In our experiments, images were acquired every 15–30 min over periods of up to 16 h, as more frequent acquisition consistently induced phototoxicity and prevented completion of parasite replication.
Despite this limitation, our imaging reliably tracked maternal micronemes over successive rounds of endodyogeny and consistently showed microneme signal associated with the residual body. These observations are in agreement with previous high-temporal-resolution studies, which demonstrated F-actin-dependent microneme trafficking within the residual body over shorter imaging periods (Periz et al., 2019).
We cannot formally exclude the possibility that some micronemes are transferred directly to daughter parasites through the apical end. However, together with previous studies showing enrichment of F-actin at the basal region of developing daughter cells rather than at the apical tip (Periz et al., 2017), our observations support a model in which RB-mediated trafficking contributes to maternal microneme inheritance.
The lack of a clear residual body marker needs to be addressed, as the distinction between the basal end of the maternal cell and a bona fide residual body must be explicitly defined to substantiate the major claim of the study. As it stands, it remains unclear whether micronemes and rhoptries as a whole travel through the residual body to be transported into the daughter parasites.
We agree with the reviewer that a marker specific to the residual body would strengthen this study. Unfortunately, no such marker has been identified to date. The F-actin chromobody is currently the best available proxy, as previous studies have shown that the F-actin network is enriched within the residual body (Periz et al., 2017; Kellermeier et al., 2024). Moreover, high-resolution live-cell imaging has previously demonstrated F-actin-dependent microneme trafficking within this compartment (Periz et al., 2019). We have revised the manuscript to more clearly acknowledge this limitation.
The conclusions drawn from the actin colocalization data in Figure 6C are based entirely on fixed samples, despite all experimental tools being compatible with live-cell imaging. Supplementing the fixed imaging with live cell data would increase its biological relevance. Published studies have shown that fixation of the actin chromobody results in the loss of resolution of an appreciable amount of the cytosolic F-actin network, and while the localizations analyzed here are primarily along the periphery, since the quantification and text make claims about the colocalization within the cytosol, this potential loss of cytosolic F-actin becomes an issue as there may be more actin available for analysis that is lost due to fixation within the cytosol of the parasites.
We thank the reviewer for raising this important point and agree that conventional fixation can compromise preservation of the F-actin network. However, we used the same fixation protocol described by Periz et al. (2019), which allows reliable visualization of the RB-associated F-actin network. We have also corrected the description of the fixation protocol in the Materials and Methods.
Fixation was necessary to image entire vacuoles with sufficient spatial resolution and signal-to-noise ratio for the volumetric analyses presented in Figure 6C. Although some loss of cytosolic F-actin cannot be excluded, this would be expected to reduce, rather than artificially increase, the detection of organelle–actin associations.
Importantly, previous live-cell imaging studies demonstrated F-actin-dependent microneme trafficking (Periz et al., 2019), and our observations are consistent with these findings. Moreover, the defects observed following MyoF depletion provide independent functional evidence that the trafficking events described here rely on the actin–MyoF transport machinery.
The statement of colocalization should be backed up by quantitative coefficients like Pearson's coefficient.
We thank the reviewer for this helpful suggestion. Following this recommendation, we performed a Pearson correlation analysis of ANKER1-Halo and HDEL-GFP using the Coloc 2 plugin in Fiji. ANKER1-Halo showed a strong spatial correlation with HDEL-GFP (Pearson's R = 0.92 ± 0.04, n = 30 parasites from three independent biological replicates), supporting localization of ANKER1 to the ER. As ANKER1 is a transmembrane protein and HDEL-GFP labels the ER lumen, this analysis should be interpreted as evidence of co-distribution within the same organelle rather than direct molecular colocalization.
In contrast, we do not consider Pearson's coefficient appropriate for evaluating the association of micronemes or rhoptries with F-actin. These organelles are predominantly concentrated at the apical pole and, when associated with F-actin, are typically positioned along rather than directly overlapping the filaments. Consequently, Pearson's coefficient would underestimate these biologically relevant associations. We therefore relied on morphological and spatial criteria, which we consider more appropriate for assessing organelle–cytoskeleton interactions.
In addition, from the methods and presented figure images, specifically in Figure 6C for RON2, how the cytosolic and residual body actin is separated is difficult to discern, as there is a clear residual body actin signal overlapping a parasite. The methods for how this was separated and analyzed should be clearer to remove doubts about how this area was measured, as the current description raises concerns about the counting of the residual body actin within the cytosol.
We agree with the reviewer that the distinction between cytosolic and residual body (RB)-associated F-actin required further clarification. All analyses were performed manually, as described in our response to Reviewer 2 (comment 1d). The RB was identified by the presence of thick, bundled F-actin filaments at the basal pole that formed a continuous structure connecting parasites within the vacuole, whereas cytosolic F-actin was defined as the thinner filamentous network within the parasite body.
No automated or threshold-based segmentation was used because the marked differences in filament morphology and fluorescence intensity make reliable thresholding difficult and prone to misclassification. Manual annotation based on spatial localization and filament morphology was therefore considered the most appropriate approach. We have clarified these criteria in the Materials and Methods section.
Line comments:
(1) 113: round to rounds - "did not obtain maternal organelles after successive rounds of replication...".
The text has been updated
(2) 146: grammatical, "As consequence a progressive" -> "As a consequence", or "Consequently".
The text has been updated
(3) 164-166: "Autonomous duplication" implies the separation and duplication of the Golgi occurs on its own, i.e., without any outside intervention, when we know from Carmeille et al. 2021 and Figure 7C here that the Golgi becomes fragmented over rounds of division in the absence of MyoF. I think this is primarily a word choice error with "autonomous".
We agree with the reviewer and the word autonomous has been removed
(4) 184: The wording suggests that DC's refers to daughter IMC's, when DC has already been given as an abbreviation for daughter cells previously.
The text has been updated to correct this error
(5) 189: de novo is not italicized.
The text has been updated
(6) 192: The data shown so far do not show that the RB plays a selective role in organelle recycling.
The text has been edited to fit better our results “Our data suggest that the organelles trafficking through the residual body (RB) have different fate”
(7) 208: State that it depends on F-actin, but never show that it is dependent on F-actin through actin disruption, such as cytochalasin D treatment or a specific conditional disruption of F-actin.
We agree with the reviewer that we did not repeat F-actin disruption experiments (e.g., cytochalasin D or jasplakinolide treatments) in this study. These experiments were performed in our previous work, where pharmacological disruption of F-actin was shown to impair microneme trafficking (Periz et al., 2019). We therefore chose not to repeat these assays.
Instead, the present study provides complementary evidence by demonstrating that depletion of Myosin F (MyoF), a motor that uses F-actin as a transport track (Kellermeier et al., 2024), disrupts the trafficking of both maternal micronemes and rhoptries. Together, our previous F-actin perturbation experiments and the MyoF depletion data presented here support the interpretation that these trafficking events depend on the actin–MyoF transport machinery.
(8) 209: This suggests that the chromobody was transiently expressed in the RON2-Halo line, but the methods suggest MIC2-Halo and RON2-Halo were integrated into a parasite line stably expressing Cb-Emerald.
The text has been edited.
(9) 229: The section is confusing with the mention of (now maternal). If I understand correctly, the point being made is that the de novo synthesized MIC2 at stage 2 is now the maternal MIC2 for stage 4, but coloring-wise within the figure, the now maternal MIC2 at stage 4 from stage 2 would still be green. The methods suggest these images were all taken simultaneously, and not at specific timepoints of the same vacuole, so the now maternal line remains confusing.
The text has been revised for clarity and now reads: “Because Toxoplasma gondii replicates asynchronously, vacuoles at different replication stages coexist within the same culture after 24 h. The second labeling step marks all proteins synthesized since the beginning of the experiment, allowing discrimination between proteins present in the original mother parasite and those synthesized during subsequent replication cycles. As daughter parasites form, they inherit material from their mother, such that proteins synthesized during one replication cycle become maternal proteins in the next. Under MyoF depletion, these newly synthesized protein pools accumulate within the residual body instead of being redistributed to daughter parasites during subsequent rounds of replication (Figure 7, stage 4).”
(10) 238: The sentences here indicate that Golgi inheritance occurs without issue: "golgi inheritance remained unaffected by MyoF depletion". But, it is evident from the images shown that the Golgi is extremely fragmented, with many more Golgi fragments by stage 8 than there are parasites. This could be solved by rewording and including a line along the lines of "in accordance with the results found in Carmeille et al. 2021".
The reference to this article is already stated later in the text now line 299-303 “This active role is further supported by the dependence of RB-mediated recycling on F-actin and the class XXII myosin MyoF, which we show to be essential for retrieval of maternal MIC2 and RON2 but dispensable for Golgi inheritance although we noticed a fragmentation of the Golgi, which has been described to depend on MyoF (Carmeille et al., 2021).”
(11) 319: missing a comma, "Many of these, particularly...".
The text has been edited.
(12) 437: Images -> imaged.
The text has been edited.
(13) 439: a fresh media -> and fresh media.
The text has been edited.
(14) 439: Replication -> replicate.
The text has been edited.
(15) 440: images -> imaged.
The text has been edited.
Other grammatical issues within the methods:
(1) Figure 5C: Within the y-axis label of Figure 5C, there is an asterisk with no asterisk explanation within the legend.
We thank the reviewer for pointing out this oversight. The figure legend has been updated to include an explanation of the asterisk, providing a clearer understanding of the results.
(2) Figure 5E: The addition of an IMC1 label to match the magenta color would be helpful to readers.
The figure has been updated
(3) Figure 6D: No statistics showing significance of colocalization.
We thank the reviewer for this comment. In Figure 6D, we report the frequency of observed colocalization between organelles (MIC2 and RON2) and F-actin filaments, based on manual analysis across >300 vacuoles for MIC2 and >500 vacuoles for RON2. Specifically, we observed cytoplasmic F-actin colocalization in 94% of vacuoles for MIC2 and 79% for RON2, and residual body F-actin colocalization in 85% of vacuoles for MIC2 and 11% for RON2. This analysis is descriptive and is not intended to compare MIC2 versus RON2 quantitatively; rather, it illustrates the general association of these organelles with F-actin filaments. Standard statistical measures such as Pearson correlation are not meaningful in this context because the organelles are punctate and primarily localized along filaments rather than overlapping continuously. Importantly, the percentages reported represent the fraction of vacuoles in which colocalization can be observed, not the percentage of colocalization between Cb-Emerald and MIC2/RON2 within individual vacuoles.
(4) Figure 7: The legend title of Figure 7 is at the end of the legend of Figure 6.
The text has been edited.
