Decision letter | Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

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Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

Decision letter

Affiliation details

University of Glasgow, United Kingdom; Cancer Research United Kingdom Beatson Institute, United Kingdom; University of Vermont, United States
Anna Akhmanova, Reviewing editor, Utrecht University, Netherlands

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Anna Akhmanova as the Senior and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Isabelle Tardieux (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This manuscript addresses the organization of actin filaments in the protozoan Toxoplasma gondii. The authors use an actin probe based on camelid antibodies (chromobody, Cb) to trace endogenous actin during intracellular life cycle. This approach has already proved successful in monitoring actin dynamics in various systems ranging from mammalian cell lines to the whole zebrafish. This approach is highly relevant in the context of Toxoplasma and related parasites, since the understanding of actin dynamics (i.e. assembly/disassembly/2D and 3D organization) remains quite elusive in this organism and the processes of formation and turnover of polymerized actin pool are subject of controversy. Use of a chromobody helps to avoid the drawbacks of direct tagging or overexpression. This study clearly brings novelty to the field but also challenges the existing data in particular concerning the amount of F-actin, localization and network architecture in Toxoplasma. While the authors do not yet provide mechanistic explanations on how a particular F-actin bundle-like network that spreads in the vacuolar space, could nucleate, maintain the polarity of the progeny within the vacuole and possibly assist daughter cell individualisation, the first identification of this actin-based population provides a new angle for future dissection of the features of actin dynamics during Toxoplasma replication and possibly for uncovering additional F-actin sub-populations of distinct dynamics.

Essential revisions:

The strength of the tool/approach and the high informative value of some experiments (some live and static assays, especially the Figure 3A, which is gorgeous) are somewhat weakened by inaccuracies in the text and also by limited sampling (quantitative data and statistics are not presented). To make this contribution stronger, we thus recommend to improve the clarity in the introductive part, to reorganize data presentation (the authors might consider showing the static study first, followed by the live study), to correct some editing errors throughout the text and to provide additional information as requested below. Please also check that for each figure presenting graphs, the definition of the error bars, the n numbers, the number of experiments and the statistical test used are included in the corresponding figure legends. Please also confirm that the statistical test used was appropriate (e.g., a t-test can be applied when the test statistic would follow a normal distribution).

1) The Abstract needs to be rewritten to help the reader on the rationale of the experimental approach (the actin chromobody approach is not mentioned) and the main results.

2) Abstract and Introduction:

Toxoplasma parasite needs to be introduced.

Egress needs to be introduced.

It is difficult to understand why the possible role(s) of actin described is/are unexpected? In addition, the basic reference to CC needs an explanation and for instance it should take into account the polar nature of the actin filament and therefore the intrinsic actin self-assembly and disassembly and the additional layer of extrinsic level of regulation by ABPs.

Introduction: Define briefly isodesmic actin model by comparison to conventional actin dynamics.

3) Results:

General remarks:

It is unclear whether Cb binds to F-actin, G-actin or both. It appears in some places to be argued one way and at times the other way. A normal anti-actin antibody staining would be expected to give background whereas Cb doesn't.

Furthermore, the authors may be correct that Cb may not adversely affect actin functions too much but they should be careful in interpreting this as evidence that it has no effect on actin polymerisation etc. in the cell. One might be affected without the other, and they have no direct measurement of actin in the cell.

Figure 1:

Please keep the same scale between WT and KO panels. It seems that the KO tachyzoites are longer despite they have a flat end. This is also visible in the Figure 2 (FRAP). If this is real, please comment this point.

Subsection “Depletion of actin results in the loss of the residual body”: If there is a loss of progeny synchronisation, it needs to be shown more convincingly, i.e. qualitatively and quantitatively (number tachyzoite/ vacuoles for n vacuoles and zoom staining). In Figure 1B, the IMC labelling of the Act1cKO does not seem to support asynchrony but rather shows a problem of IMC biogenesis. In fact, the SEM (Figure 1C) shows tachyzoites that have lost connection through the RB and are dispersed in the vacuolar matrix. The red arrows in SEM panels point to the end of division but it is not clear what the authors want to say. Do they mean the closure as individual cells (a step very poorly documented)?

Figure 2:

It should be explained why the bleached cell does not recover. It is proposed that GFP transport from surrounding cells is affected – but why is there no recovery from the newly synthesised GFP made in that cell?

FRAP assays: while only one pre-bleach panel is sufficient, the visualization for the FRAP effect should be improved. Delineating the FRAP area will also help. Why is there a range of exposure time (100-200 ms) for FRAP recording? Is it different from experiment to experiment? FRAP data should represent a certain number of experiments that should be mentioned. Could the authors clarify the following statement which as it is now, seems to lack real ground “Of note, digital tracking of vesicles suggested movement along a tubular or filamentous structure”.

Figure 3:

Panel B, on the right: the scale bar shows 10 micron unlike all the other figures.

Figure 5:

This figure is confusing as it does not bring strong support of actin in the RB with this imaging technique (it is even the opposite as the signal is everywhere despite the fact that the antibody is presented as kind of F-actin-specific). The chromobody characterized as a good marker of F-actin in the Toxoplasma vacuole (previous data) is much more convincing.

Figure 6:

This is a very important figure which would deserve to be better documented and clarified. Quantitative data on the network detection should be given since even in the jasplakinolide-treated cells, one vacuole containing 2 parasites seems not to display F-actin labelling (Figure 6A)? Does that mean that the network is not always present at the same stage of parasite development in the vacuole? This is also puzzling for the loxActin (Figure 6B) where few filaments (bundles as nicely seen later Figure 8) are observed among the tachyzoites in the vacuole. The description of the network that here looks as lying between tachyzoites within the vacuole is a little different from Figure 3, which shows a network in the connecting posterior structure throughout the replication cycle. This is to be partly answered with the Figure 8.

Figure 7 and the accompanying text:

Subsection “Cb specifically binds to parasite actin and does not alter the total amount of F-actin”. 2% of actin is in the F actin form: the authors might do a calculation (even a ballpark one) of the amount of actin they estimate as a percentage of the total protein, then the number of molecules and then the amount of F-actin that would be likely. Then look at images and see if reasonable. One might do it the other way as well. Does it really seem likely that the actin is as rare a protein as previous studies have suggested if 2% can be assembled into such extensive networks? It is hugely unlikely that MS proteomics detected no other proteins in the pull down. The statement “could not be identified" is ambiguous, please describe the results more clearly.

Figure 8:

What is the reason for qualifying the network as tubules (subsection “Inter-parasite actin tubules are dynamic during parasite replication and egress”)?

In the first paragraph of the aforementioned subsection. The filamentous actin is within tubules. If the authors are really sure that this is true – i.e. the correlative EM sections shown actually do fit in 3D with the LM images, this raises a really fundamental piece of biology that they must address in their discussion. How does actin (normally a cytoplasmic protein) get inside a membrane-lined tubule? We know a lot about how proteins cross membranes. However, here we have a claim that a protein that normally does not, does so in this system – and with all of its cohort proteins that facilitate filament formation and no doubt dynamics. This is not easy to imagine, so a discussion of how this might occur would be useful.

To improve the logic of presentation, the figure that shows the data related to the network dynamics during replication and the high-resolution description of the network by CLEM might be presented after Figure 3.

DOI: http://dx.doi.org/10.7554/eLife.24119.028