PONE-D-24-25002The evolutionarily conserved PhLP3 is essential for sperm development in Drosophila melanogasterPLOS ONE

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This work was made possible by NSF MRI #1828164, which provided for the purchase of the Zeiss LSM880 confocal microscope. This work was supported by internal research awards to C. P., E. K., S. W., M. M., B. S., C. C., J. C. J., and S. M. K. from Loyola University Chicago.

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Thank you to the Bloomington Drosophila Stock Center and the Vienna Drosophila Resource Center for fly stocks. The rat anti-Vasa monoclonal antibody, developed by A. C. Spradling and D. Williams, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Thanks to E. O’Flaherty and Danielle Talbot for helpful discussions and comments on this project. This work was made possible by NSF MRI #1828164, which provided for the purchase of the Zeiss LSM880 confocal microscope. This work was supported by internal research awards to C. P., E. K., S. W., M. M., B. S., C. C., J. C. J., and S. M. K. from Loyola University Chicago.

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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: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript presents a strong initial characterization of Drosophila melanogaster PhLP3. The authors use structural modeling and alignment to demonstrate the protein’s conserved structure with PhLP3 homologs from other species, run a biochemical assay to show its redox activity, and then use two complementary genetic approaches (a P-element insertion allele and RNA interference) to establish that ablation of PhLP3 results in male sterility. This is accompanied by cytological studies that identify a lack of individualization complexes in spermatids, an interesting observation given the potential for PhLP3 to interact with cytoskeleton components. This is a very solid manuscript overall. It makes good use of a wide variety of techniques and approaches and will make a helpful contribution to the literature on phosducin-like proteins and on fly spermatogenesis. I have only minor comments, which I expect can be addressed without any additional experiments. (Some of the comments are essentially copy-editing, which the instructions ask reviewers to do, since the journal does not provide this service.)

The excision of the P-element to rescue the wild-type phenotype is a strength and helps validate the P-insertion allele as the cause of the fertility defect. The use of RNAi to confirm the requirement of PhLP3 for fertility is another important strength, making it less likely that the P-element mutant effect was due to some other mutation in that background.'

Minor points:

Figure 1:

-Give units for the distance measure included at the top of the phylogenetic tree in Fig. 1B.

-Specify in the caption whether the structure modeled in Fig. 1C is the Drosophila PhLP3, or another ortholog.

-If you haven’t already, consider comparing the SWISS-MODEL structure to the AlphaFold prediction (https://alphafold.ebi.ac.uk/entry/Q9VGV8) to determine which is the better one to include in the figure. To my non-expert eye, they appear fairly similar, though the AlphaFold prediction shows a longer second alpha helix at the N-terminus (in line with the discussion section description, line 549). If you feel SWISS-MODEL is better, then no need to change. If you think the AlphaFold prediction is more reliable, then you could do the same for the structures in Fig. S1.

-If any amino acids are missing from the structure, you could clarify this in the caption (e.g., it looks like positions 1-18 and 185-216 are not represented?).

-If possible, remove Microsoft’s red-squiggly line under Trx.

Line 114: The data in Fig. S2 do not seem to distinguish between “germline” and somatic cells of the testis, so consider re-wording this sentence (or, clarify that germline-specific expression is based on scRNA-seq data and/or your later data in Fig. 6, rather than the graphs in the supplemental figure?).

Figure 2:

-Add to the caption that “dc” indicates the dense complex in Fig. 2B.

Line 151: no italics for “mutants”

Figure 3:

-If possible, remove Microsoft’s red-squiggly line under Trx.

Figure 4:

-Fig. 1 also presents a PhLP3 structure and an alignment, so aspects of these figures are redundant with each other. Since there doesn’t appear to be anything anomalous in the alignments or structures that is noteworthy for the manuscript (i.e., the structures align well, the cysteine is conserved, etc.), can these figures be condensed into one, with any other information being moved to supplemental material?

-In the caption describing Fig. 4A, clarify which structure is shown in part with the green curves.

-Line 190: unnecessary underlining of ref. [16].

Line 200: it may be helpful to give a brief (one line) description of “the thioredoxin system” here, since this system might be unfamiliar to some readers, or simply add “(describe in the next paragraph)”, where it’s clarified.

Line 202: Do you mean Cys95 in D. melanogaster, rather than Cys93? (Also, be consistent with how you write the amino acids; cysteine is abbreviated as C in line 195, Cys here.)

Figure 5:

-It may be helpful to show the chemical reaction in Fig. 5B with the same protein names as you use in the text. For example, in line 215, you write about D. melanogaster “Trx1”, but it’s just “Trx” in Fig. 5B. Overall, though, these look like great data!

Figure 6:

-This is a nice demonstration that the P-element insertion abolishes PhLP3 expression.

-The first line of the figure caption (line 243) states that panels “(A-B)” show in situ data, but it looks like only panels A and C show this, while panel B shows qPCR data. Please clarify.

Figure 8:

-These data are convincing that PhLP3-/- males do not produce mature sperm and do not undergo individualization.

-To support the text on lines 322-323, is it possible to show images with only the phalloidin channel here, to illustrate the presence of ICs for controls and the absence of them for the mutants? (If not, that is okay; this is a small point.)

Figure 9:

-Is it possible to add to the caption whether these images represent zoomed in portions of whole-mount (intact) testes (which seems to be what the Methods section implies on line 450), or whether they come from shreds to release spermatid bundles?

It is a bit surprising that the P-element excision line (with 33% of WT levels of PhLP3 transcripts) is fertile, while the RNAi line (with 23% of WT levels of PhLP3 transcripts) is near-sterile. Either the authors have serendipitously narrowed down the exact threshold level of PhLP3 transcripts required for functional spermatogenesis, or there are other biological or technical explanations. (Example biological: there could be post-transcriptional regulation that acts differently between the delta P line and the RNAi progeny or some strain background differences with the RNAi line. Example technical: there could be variation in the RT-PCR measurements. The bam-gal4 control line measurement in Fig. 10 has large error bars, for instance, while error bars appear to be missing for the +/+ genotype in Fig. 6.) This doesn’t require extensive comment, but if the authors had thoughts about it, they could briefly include them.

Figure 10:

-Optional: if possible, it would be useful to show the scattered actin cones in the RNAi flies at higher magnification, but in my view, it is not necessary to re-do this experiment if you don’t already have the data available.

Line 419: “or control” doesn’t need to be in italics

Line 474-475: it’s not clear why Trx1 primers were used, since the manuscript doesn’t report on Trx1 purification. If the authors needed to produce and purify Trx1 for the biochemical assay, this can be briefly added to the manuscript, or they can simply state that Trx1 was produced as previously described in ref. 4.

Given the high expression of CG4511 in ovaries, did the authors observe whether homozygous P-element insertion females were sterile?

Lines 568-569: Related to the point above: This part of the discussion seems to raise the possibility that PhLP3 could be a redox target of Dhd in the ovaries. There doesn’t yet appear to be any evidence that PhLP3 is part of mature sperm (for example, while not definitive evidence for absence, CG4511 was not detected amongst the 3,000+ sperm proteome proteins; Garlovsky et al. 2022, PMID: 35985624), so it isn’t clear that testis-expressed PhLP3 would have the opportunity to interact with Dhd. Alternatively, since the data in Fig. S2 show high PhLP3 expression in ovaries as well as testes, it could be possible that ovary-expressed PhLP3 is a target of Dhd. Thus, consider re-phrasing here.

Line 571-572: Clarify this sentence a bit. What aspect of the kinetics data presented here (and, compared to what other data) suggests that PhLP3 is unlikely to be an effective antioxidant scavenger?

Line 638: remove the extra ) after the parentheses

Line 641: “…described by Fabrizio have … [68].”

Reviewer #2: The paper entitled The evolutionarily conserved PhLP3 is essential for sperm development in Drosophila melanogaster provides an analysis of the role and function of CG4511, a conserved member of the phosducin-like family of proteins. In brief, the authors examine the structural similarity of the protein to other known members of the PhLP family, and perform several experiments examining sperm development, noting an arrest of spermatogenesis in PhLP3¬-/- flies. A significant impact on fertility in PhLP3 null animals is noted both P-element excision and RNAi knockdown are used to further interrogate the results.

In accordance with the PLOS ONE publishing criteria, please find my assessment below:

1) The study presents the results of primary scientific research.

a. Yes

2) Results reported have not been published elsewhere.

a. To the best of my knowledge, these results are novel. The entry in flybase would also indicate this CG4511 is understudied.

3) Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail.

a. Yes.

4) Conclusions are presented in an appropriate fashion and are supported by the data.

a. The format of the article may require some adjustment for clarity, but is sufficient to replicate the experiments.

b. I have significant concern in two areas, given what is reported in the article:

i. In line 349-351, the authors denote that the p-element excision used in the article only restores expression to the level of 33%, but restores fertility in the animals. However, knockdown by RNAi (by 77%, as per line 356-357) is sufficient to render the animals infertile. Based on the p-element data, I would expect the knockdown to not render the animals sterile based on expression. I believe that further explanation is required beyond the expression levels to be confident in the validity of this data.

ii. Only one RNAi line is used in the paper. I would much prefer to see at least two different lines, especially given the potential issue raised prior.

5) The article is presented in an intelligible fashion and is written in standard English.

a. Yes.

6) The research meets all applicable standards for the ethics of experimentation and research integrity.

a. Yes.

7) The article adheres to appropriate reporting guidelines and community standards for data availability.

a. Yes.

Reviewer #3: The authors find a phosducin-like (PhLP) protein family gene, CG4511, in Drosophila, which is named PhLP3. The gene is mainly expressed in testes and ovaries according to the published RNAseq data. An insertional mutation in the 5’ UTR caused a significant reduction of expression and the homozygous males are sterile because of a lack of mature sperm. Importantly, the defects are seen as early as the canoe stage, where the microtubule and actin-rich structure, dense body is elongated and leads to needle-shaped nuclear. stage. The authors also confirmed its redox activity by using recombinant protein and NADPH reduction assays.

Because PhLP proteins are known to play a role in regulation of the microtubule and actin cytoskeletons, it is convincing that the mutant of the Drosophila PhLP gene with testis-biased expression showed defective spermiogenesis and sterile. I think that this study finds a new role of PhLP protein in Drosophila spermiogenesis and the paper merits publication in PLOS ONE. However, I found several issues that the authors should address before accepting.

Line 114 “high expression in the testis, specifically in germline cells (S2 Fig)”

I do not see any evidence for expression in germline cells in the figure. Is there any more data?

Line 170 “ an N-terminal helix domain, the central Trx-domain, and the unstructured N-terminal tail”

“unstructured N-terminal tail” is right or unstructured C-terminal tail? This is because “mostly unstructured C-terminal tail” appears on line 543.

Line 239 “gene is expressed in germ cells”

It is difficult to see the exact location of expression in the figure. It may be recommended to add an enlarged view of A.

Figure 7

Nonparametric Welch’s t-test is recommended, particularly between the control and mutant as in Figure 10.

Fig. 8 and line 322

It is difficult to see phalloidin staining (actin cones) in A and also to find sperm in seminal vesicles. Is it possible to add more focused and enlarged view? In addition, it may also be better to combine Figures 8 and 9 in order to understand the phenotypes.

Figure 9

It takes time to understand which are open and solid arrows; alternative symbols are better.

Figure 10

“White arrows indicate clusters of elongating spermatid nuclei” This is phalloidin staining; the revision is needed. There are no “arrows” in C. I cannot see evidence for “there are few if any DAPI clusters” in D or cannot understand where I should see. It is also difficult to see exactly “deformed, scattered actin cones.” Because this is very important point of this paper, the authors should be strongly recommended to prepare double staining and enlarged views for this.

Line 460 “manufacturer’s instructions”

Which is the manufacturer?

Line 549 “Stirling et al.”

Reference number [6] should be just after the author name.

Line 613 “Others”

Reference or references should be provided here.

Reviewer #4: Successful completion of spermatogenesis is crucial for the perpetuation of the species. It is interesting for authors used Drosophila as model to investigate gene function in spermatogenesis. The authors uncovered an important role of PhLP3 in spermiogenesis. These results could deepen the basic theory of the regulatory mechanism of animal reproductive development. However, some revisions are required before this paper can be accepted.

Major comments

1. It needs to be clear the reason of male sterile is due to the early release of mature sperm in the experimental group leads to infertility or the absence of spermatid extension stage abnormalities leads to abnormal individualization.

2. PhPL3 is a chaperone protein involved in protein folding as well as a role as an enzyme. Whether the protein degradation pathways were activated in the absence of PhPL3, such as ubiquitination proteasomal degradation pathway, lysosomal pathway, caspase. It is preferable do detect some gene expression related to these pathway.

3. It is preferable do detect some gene expression related to spermatogenesis in the absence of PhPL3.

4. In the result session, authors should explain the reason of using bam-gal4.

Minor comments

5.It is perferable to mark “seminal vesicle” in figure 8 and 10, thus easy for readers.

6.“***” shouled be marked in figure 8A.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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The evolutionarily conserved PhLP3 is essential for sperm development in Drosophila melanogaster

PONE-D-24-25002R1

Dear Dr. Kanzok,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Abhinava Kumar Mishra, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. 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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciate the authors’ careful attention to my comments and those of the other reviewers. The approach of moving the structures to the new Fig. 4 looks to have worked effectively. I also appreciated the additional discussion of the threshold issue, but agree with the authors that it can be explored experimentally in future studies. Likewise, it’s cool that PhLP3 mutant females are sterile, but very reasonable to save that story for another paper. Overall, I think this revised submission looks good and will make an interesting contribution to the literature on fly spermatogenesis.

Reviewer #2: The authors have clarified the majority of my concerns, and the re-write of the paper is significantly improved. Based on the comments added, I would accept it for publication.

However, it may be possible to further clarify both the threshold level of PhLP3 transcripts required for

functional spermatogenesis (as reviewer #1 and I noted) and my concerns about the RNAi experiments by adjusting the temperature for the Gal4 driven RNAi experiments (as another line is not available). This would help to remove any concerns about a background effect or other potential off-target effects.

Reviewer #3: (No Response)

Reviewer #4: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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