Review Reports - Reduced RhoGDI2 Expression Disrupts Centrosome Functions and Promotes Mitotic Errors

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

I have reviewed the revised version. Although I still feel that only one positive result was shown for the interaction search, the authors have provided sufficient evidence to support it. This is the resubmitted version of the research manuscript. Since it addresses my original comments, I am now satisfied with it and have no further comments.

Author Response

Comment: I have reviewed the revised version. Although I still feel that only one positive result was shown for the interaction search, the authors have provided sufficient evidence to support it. This is the resubmitted version of the research manuscript. Since it addresses my original comments, I am now satisfied with it and have no further comments.

Response: We would like to thank the reviewer for his/her positive comment on the revisions we made to our manuscript

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript titled “Reduced RhoGDI2 expression disrupts centrosome functions 2 and promotes mitotic errors” reported that RhoGDI2 specifically regulates centrosome functions in human osteosarcoma cell lines as well as NK-92 immune cells while RhoGDI1 showed lesser to no effects. Key observations are listed below:

RhoGDI2 knockdown reduces U-2-OS and MG63 proliferation rates by evaluating newly synthesized DNA, number of mitotic cells after CDK1 inhibitor treatment, and BrdU assay results.
Co-precipitation and mass spectroscopy analysis of FLAG tagged RhoGDI2 suggested interactions with centrosome-related proteins. RhoGDI2 silencing induces centrosome supernumeracy. Reduced ciliogenesis and weakened immune cell response were observed in RhoGDI silenced cells, both of which suggested abnormal centrosome activities.
Knockdown of RhoGDI2 did not affect RhoA, Rac1, and Cdc42 expression, and only resulted in higher RhoA activity compared to the other two Rho GTPases. RhoA silencing partially restored the normal centrosome activities, suggesting that RhoA participated in the RhoGDI2 signaling pathway of centrosome regulation.

Generally, the authors performed well-rounded experiments to demonstrate characteristic RhoGDI2 functions. The information is valuable and deserves publication. My only criticism is that the analysis in Figure 3 is not enough support that RhoGDI2 silencing causes defects in chromosome congression. I am not convinced by the significant difference from the statistical analysis, and Figure 3B suggests a very weak effect if not the same compared to that from RhoGDI1 knockdown. Given that this experiment does not change the overall conclusion, modification or deletion of part 3.3 is recommended.

Author Response

Comment:

RhoGDI2 knockdown reduces U-2-OS and MG63 proliferation rates by evaluating newly synthesized DNA, number of mitotic cells after CDK1 inhibitor treatment, and BrdU assay results.
Co-precipitation and mass spectroscopy analysis of FLAG tagged RhoGDI2 suggested interactions with centrosome-related proteins. RhoGDI2 silencing induces centrosome supernumeracy. Reduced ciliogenesis and weakened immune cell response were observed in RhoGDI silenced cells, both of which suggested abnormal centrosome activities.
Knockdown of RhoGDI2 did not affect RhoA, Rac1, and Cdc42 expression, and only resulted in higher RhoA activity compared to the other two Rho GTPases. RhoA silencing partially restored the normal centrosome activities, suggesting that RhoA participated in the RhoGDI2 signaling pathway of centrosome regulation.

Response: We would like to thank the reviewer for his/her constructive comments on our manuscript. We agree with the reviewer that the data of figure 3 (in the previous version) show differences between siRhoGDI2 and siCtrl, but not between siRhoGDI2 and siRhoGDI1. As recommended by the reviewer, these data (fig 3 in the previous version, as well as the accompanying text throughout the manuscript) have been removed in this revised version.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This MS deals with reduced RhoGDI2 expression disrupts centrosome functions and promotes mitotic errors and also try to link the novel roles for RhoGDI2 in centrosome functions in human cancer and immune synapses in immune cells. The study provides compelling evidence that RhoGDI2 plays critical and multifaceted roles in both cancer cell biology and immune cell function. It appears excited to see how its context-dependent association with cancer prognosis, and positions it as a promising target for further clinical application!

However, two points could make more deeply:

The link between RhoGDI2 suppression and defects in centrosome integrity, chromosome congression, and ciliogenesis, all of which are critical for maintaining genomic stability and proper cell signaling, but no direct link to which signaling is really involved. Please specify them and make an investigation, such as in Supplementary table 3 listed, you identified Centrosomal protein of 192 kDa (Cep192), which is a protein crucial for centrosome function and cell cycle regulation. It plays a key role in recruiting pericentriolar material (PCM), promoting centrosome maturation, and facilitating centriole duplication. Cep192 also contributes to spindle assembly by activating Aurora A kinases within the centrosome (even more Centrosomal related proteins, PLKs, SAC……).
Lines 434-437 in Discussion section “creased activation of RhoA, but not Rac1 and Cdc42, upon inhibition of RhoGDI2 suggested that RhoA may be involved in the inhibition of primary cilia formation observed in our model.” vs to Result section: Figure 6 need make description more clearly for RhoA, Rac1 and Cdc42.

Reviewer 2 Report

Comments and Suggestions for Authors

Tripathi et al. investigated the role of RhoGDI2 in cancer cells by knocking down approaches. RhoGDIs bind to Rho GTPases and sequester them in the cytoplasm, keeping the GTPases in an inactive form. RhoGDI2 has been implicated in tumor progression and is considered to be a potential therapeutic target in cancer. In the manuscript, the authors knocked down RhoGDI2 and assessed the resulting physiological and biological changes in cancer and immune cells. They reported that RhoGDI2 knockdown led to reduced cell proliferation, impaired centrosomes and chromosome congression, and defective ciliogenesis, whereas RhoGDI1 KD did not produce similar effects. Their findings are interesting and may have clinical relevance in cancer therapy. However, the authors should significantly improve the quality of the data and provide more mechanistic insights to support their conclusions to meet the standards of Cells. Here are some major and minor points to help improve the manuscript.

Major points

Overall imaging quality is subpar and needs substantial improvement in both clarity and representation. Many images are blurry and are limited to only 2 channels (dapi and g- or a-Tub). To provide more comprehensive view of the observed phenotypes, the authors should include additional markers (kinetochore, microtubules, chromosomes, centrosome, etc). Also, they did not specify the imaging system they utilized in the methods. More detailed suggestions are listed below.
The authors need to present more clear mechanistic insights into how RhoGDI2 knockdown leads to defects in centrosome, chromosome alignment and ciliogenesis. I strongly recommend focusing on uncovering the underlying molecular mechanisms. Can the RhoA KD modify/rescue the other phenotypes (proliferation, supernumerary centrosome, etc)? Is the level of RhoA enough (should also show the dominant negative and constitutively active RhoA to assess rescue or enhancement of the observed defects). More importantly, what are the downstream mechanisms specifically regulated by RhoGDI2, but not by RhoGDI1? One potential model is that CDK2-cyclin E mediated phosphorylation of NPM, which regulates centrosome duplication through ROCK II (Rho associated kinase). Since centrosomal localization of ROCKII depends on active RhoA, the authors should explore whether and how RhoGDI2, but not GDI1, knockdown influences this pathway and also contributes to the centrosome and proliferation defects. This should be addressed experimentally rather than through speculative discussion.

Minor points

In Fig 1A and B, add statistical significance (*) although the knockdown effect seems clear.
For Fig 1C,D and Fig 2, the authors measured proliferation by DNA amount (Fig 1) and monitored mitotic event (Fig 2) – Fig1 and 2 can be combined. However, it is not clear which cells are mitotic from a-Tub staining due to low image quality. They should use additional markers (e.g. PH3) or utilize more definitive assays like BrdU assay.
The authors performed IP-MS using both N- and C-terminally tagged RhoGDI2, and listed the potential interactors in supplementary data. However, the number of unique peptides seems low. Nevertheless, the authors should include the raw data (unique and total peptide counts for each terminal tagged IP and negative control (empty vector)) as supplementary files, specify the n number of replicates in the methods, and also show a summary table in the main figures.
The authors listed some examples of supernumerary centrosomes in Fig 3a via g-Tub and dapi staining. Add scale bar, also include co-staining with a-Tub (or other MT markers) and a centrosome marker (e.g. Centrin) to confirm centrosome identity and polarity (does it show any aneuploidy?). Is this phenotype only observed in G1-S phase? If not, are the multiple centrosome phenotypes linked to other phenotypes described later? The authors should address these.
Fig 4, the authors claimed that RhoGDI2 KD results in chromosome congression defect. It is unclear how the ‘chromosome congression index’ (Fig 4B) was defined or measured. The authors should provide a detailed explanation, quantify chromosome alignment based on MT and kinetochore organization, and improve DNA staining quality. Generally, the chromosomal congress is assessed by the distance between aligned kinetochores.
Fig 7 RhoGDI2 in immune cells is irrelevant and can be removed.

Reviewer 3 Report

Comments and Suggestions for Authors

RhoGDI2 is a RhoGTPase regulator that has roles in cytoskeleton organization and cell survival, amongst others. The authors knocked down its expression in human cancer cell lines, and found that repression of RhoGDI2 expression, but not that of the closely related RhoGDI1, significantly reduces their proliferation rate. In parallel, RhoGDI2 suppression induces supernumerary centrosomes, defects of chromosome congression and inhibits ciliogenesis. The specific silencing of RhoA partially rescued the ciliary defects observed upon RhoGDI2 silencing. Altogether, these data suggest novel roles for RhoGDI2 in centrosome functions in human cancer and immune synapses in immune cells, which provides an explanation for its reported dual role in cancer.

Comments:

Lack of expression positioningof RhoGDI2 and RhoGDI1 in human cancer cell lines.
The interference efficiency needs to be verified by WB.
Lack of bar in Figure 3A.
The presentation of the figure needs to be improved.