PONE-D-23-41815Spindle checkpoint activation by fungal orthologs of the S. cerevisiae Mps1 kinasePLOS ONE

Dear Dr. Fabritius,

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As you can see, the reviewers (one submitted the critique directly to me, not through the system; I append it below) took different views on the overall evaluation. However, they both found the work technically sound, and I believe the comments are easy to address with some editing, without new experiments. The work will be helpful in the field, and I look forward to receiving a revised version accommodating their suggestions. Because the edits are straightforward, I think a re-review will not be necessary.

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Additional Editor Comments:

As you can see, the reviewers (one submitted the critique directly to me, not through the system, I append it below) took different views on the overall evaluation. However, they both found the work technically sound, and the comments are easy to address with some editing, without new experiments. The work will be helpful in the field, and I look forward to receiving a revised version accommodating their suggestions.

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(Additional review sent directly to the Academic Editor by another reviewer)

1. Recommendation: Major revision

2. Is the manuscript technically sound, and do the data support the conclusions?

- Yes, for the most part.

3. Has the statistical analysis been performed appropriately and rigorously?

- NA. Some scale bars need to be added to images.

4. Does the manuscript adhere to the PLOS Data Policy? Additional details can be found at http://journals.plos.org/plosone/s/materials-and-software-sharing.

- Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

- Yes

6. Review Comments to the Author (minimum 200 characters)

- The manuscript is well written and logically presented. Much of the data is fine. However, there are several inconsistencies that need to be addressed and some of the supplementary data improved before publication.

Figure 1: Is part B necessary here? I am not convinced it is.

Figure 2

A: just a few colonies are growing in the strain expressing C.albicans. Is this reproducible? I wonder if these rare isolates are really expressing Mps1 still? They may account for most of the liquid growth in panel B (after 12 hours).

C. How does this VF dataset relate to S3 Fig.B, where apparently no large-budded arrests are seen in wild-type cells? The latter seems odd – wouldn’t mitotic delays be expected here too?

D: could the position of 1C and 2C control peaks (for the VF strain) simply be labelled here, on each plot? ‘DNA content index’ isn’t so helpful.

Figure 3. Scale bars should be added for these IF images.

Fig. S2

A: These blots are not ideal and in too many pieces. These should be improved or deleted.

The position of relevant molecular weight markers should be labelled.

B: The GFP quantitation, in panel B, more closely matches the western results from wild-type cells.

Was expression of the 5 other Mps1-GFP fusion proteins detected (by western), in the VF strains that did not show significant growth defects (Cg, Ag, Rg, Cp, Bd)?

Fig. S3

A: why does the wild-type strain over-expressing ScMps1 grow so well, when the Nud1/Tub1 equivalent does not?

B: why no large budded delay/arrest in GAL, in these WT strains? Cf Fig.2C and Nud1/Tub1 panel.

C: the gossypii-expressing strain delays/arrests as large budded (in the tagged Nud1/Tub1 strain) and colonies grow slowly. This doesn’t seem to happen in wild-type or VF. Could it be a synthetic phenotype with the tagged cytoskeletal markers (not fully functional)? Why is there no large-budded delay in the Nud1/Tub1 strain expressing C.auris?

B.d. overexpression looks unhappy, but only in the VF. Any large budded arrest there?

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-Yes.

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-Kevin G. Hardwick

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript by Fabritius et al. describes the phenotype of overproduction of Mps1 fungal orthologs in budding yeast. The authors have found that heterologous expression of the MPS1 orthologs from C. albicans and C. auris with the GAL promoter leads to budding yeast cell lethality, the phenotype of which is comparable to that of overproduction of the endogenous Mps1. By observing the mitotic spindle morphology, the authors demonstrate that the cell growth defect caused by Mps1 and Mps1 ortholog overproduction is likely due to the activation of the mitotic spindle checkpoint. Surprisingly, overproduction of Mps1 orthologs from other related yeast species tested in this work show no detectable cell growth defect. Thus, the main conclusion of this work is limited, i.e. Mps1 from C. albicans and C. auris, which are closely related to budding yeast, function wise is comparable to budding yeast Mps1, whereas the other Mps1 orthologs tested by the authors are not. The experiments were conducted in a seemingly rigorous fashion, however, there is no experimental evidence presented in this work to support their main claim that heterologous expression of Mps1 orthologs in budding yeast can serve as a platform for drug screening.

The figure legends are very sketchy, they should be self-evident and detailed enough for the reader to understand the experiments.

Fig 2A should include a YEP panel as shown in Fig S3A.

Fig 3 needs quantification of spindle length and the number of cells observed.

Fig S2. Panel B is not the quantification of panel A, please explain the logic.

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Reviewer #1: No

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Spindle checkpoint activation by fungal orthologs of the S. cerevisiae Mps1 kinase

PONE-D-23-41815R1

Dear Dr. Fabritius,

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Kind regards,

Michael Polymenis, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: