Tracking maternal proteins uncovers a central role for the residual body in organelle recycling during Toxoplasma gondii replication
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eLife Assessment
This study presents valuable new insights into the patterns of organelle inheritance in the protozoan parasite Toxoplasma gondii. An innovative dual-labeling approach used in this study to track maternal-derived and de novo synthesized organelles provides a technical advance with potential to be more broadly applied. Solid evidence is provided that different organelles show distinct inheritance fates during cell replication; however, the data describing the residual body component in this process is incomplete.
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Abstract
Toxoplasma gondii replicates through endodyogeny, an unconventional form of internal budding in which two daughter cells are assembled within a single mother cell. During this process, daughter cells must acquire a full complement of organelles, which may be inherited from the mother, formed de novo, or assembled through a combination of both mechanisms. To date the fate of maternal components during replication remains poorly understood. We previously showed that F-actin–driven dynamics generate the intravacuolar network, which defines the residual body (RB) and facilitates recycling of microneme proteins. However, the inheritance and recycling of other organelles have not been systematically analysed.
To address this, we employed a dual HaloTag-based pulse-chase fluorescence labelling strategy to distinguish between de novo–synthesized and recycled proteins in replicating tachyzoites. This approach reveals three distinct organelle inheritance patterns: (1) direct transmission of intact maternal organelles (e.g., rhoptries, micronemes), (2) expansion and division of pre-existing maternal organelles with incorporation of newly synthesized components (e.g., Golgi apparatus, apicoplast), and (3) degradation of maternal structures without recycling (e.g., inner membrane complex). Furthermore, we identify Myosin F (MyoF) as the key motor protein that mediates the selective recycling of maternal organelles via the RB. These findings redefine the RB as an active trafficking hub and reveal a selective, regulated system of organelle inheritance and recycling that is critical for intracellular organization and parasite development.
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eLife Assessment
This study presents valuable new insights into the patterns of organelle inheritance in the protozoan parasite Toxoplasma gondii. An innovative dual-labeling approach used in this study to track maternal-derived and de novo synthesized organelles provides a technical advance with potential to be more broadly applied. Solid evidence is provided that different organelles show distinct inheritance fates during cell replication; however, the data describing the residual body component in this process is incomplete.
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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 …
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. 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.
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.
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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 Halo-based 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 …
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 Halo-based 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.
(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.
(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 time-dependent. 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.
(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.
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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 daughter-derived 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 …
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 daughter-derived 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.
(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.
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