Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number
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Abstract
Centrosomes act as the main microtubule organizing center (MTOC) in metazoans. Centrosome number is tightly regulated by limiting centriole duplication to a single round per cell cycle. This control is achieved by multiple mechanisms, including the regulation of the protein kinase PLK4, the most upstream facilitator of centriole duplication. Altered centrosome numbers in mouse and human cells cause p53-dependent growth arrest through poorly defined mechanisms. Recent work has shown that the E3 ligase TRIM37 is required for cell cycle arrest in acentrosomal cells. To gain additional insights into this process, we undertook a series of genome-wide CRISPR/Cas9 screens to identify factors important for growth arrest triggered by treatment with centrinone B, a selective PLK4 inhibitor. We found that TRIM37 is a key mediator of growth arrest after partial or full PLK4 inhibition. Interestingly, PLK4 cellular mobility decreased in a dose-dependent manner after centrinone B treatment. In contrast to recent work, we found that growth arrest after PLK4 inhibition correlated better with PLK4 activity than with mitotic length or centrosome number. These data provide insights into the global response to changes in centrosome number and PLK4 activity and extend the role for TRIM37 in regulating the abundance, localization, and function of centrosome proteins.
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Reply to the reviewers
1. General Statements [optional]
We thank the reviewers for their critical review of our manuscript. We are excited to see that the reviewers agree that we have presented high-quality data that advances the centrosome field and is worthy of publication following revision. The authors also agree with the reviewers that the data presentation requires improvement, that some experiments require additional replicates with robust statistical analyses and that a model or summary would help clarify the differences between previously published results and ours. We will address all these concerns in the revised version of our manuscript. The reviewer comments in …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
1. General Statements [optional]
We thank the reviewers for their critical review of our manuscript. We are excited to see that the reviewers agree that we have presented high-quality data that advances the centrosome field and is worthy of publication following revision. The authors also agree with the reviewers that the data presentation requires improvement, that some experiments require additional replicates with robust statistical analyses and that a model or summary would help clarify the differences between previously published results and ours. We will address all these concerns in the revised version of our manuscript. The reviewer comments in their entirety can be found below in italic followed by our response in bold.
__Considering that the manuscript was very well received we believe it makes a strong candidate for publication in eLife. In terms of editors at eLife, we believe that Anna Akhmanova and Jeremy Reiter would be very well suited to handle this manuscript. __
We hope that you will concur with us that the revision plan detailed below adequately addresses the reviewers’ comments.
2. Description of the planned revisions
Reviewer 1, Major points
- Previous data suggested that an important role of TRIM37 was to limit accumulation of CEP192 levels, yet here CEP192 levels appeared unchanged in TRIM37 knockout cells that stably express wild-type or RING domain mutant TRIM37. However, in agreement with previous work, transient expression of TRIM37 reduced CEP192 levels along with those of other PCM and centriole components in an E3-dependent manner. These data are rather confusing in light of the literature, and the current report does not really deal with these discrepancies but to me they suggest that high levels of TRIM37 can target multiple centrosome components for degradation, but this may be an experimental artefact.* We agree that acutely overexpressed TRIM37 results in decreased CEP192 levels and is consistent with published results. We also provide evidence that CEP192 levels are not correspondingly increased in the absence of TRIM37, nor are they decreased in a cell line that stably overexpresses FLAG-BirA TRIM37. This suggests that the decreased CEP192 (and PCNT and CEP120) after acute overexpression of TRIM37 might be short-lived or a consequence of overexpression. We will discuss this possibility more clearly in the revised mansucript. In addition, we will perform Western blots for TRIM37 in wild type cells, cells stably expressing FLAG-BirA TRIM37 and cells induced to express TRIM37-3xFLAG to more directly compare the amount of TRIM37 present in these cell lines.
- The choice of cells for particular experiments is not always stated or explained. For instance, in Figure 3A: Trim37 KO pool used while in Figure 3B TRIM37 single KO. These are then combined with both transient and stable expression of TRIM37 mutants.*
We apologize for this and will clarify the choice of cell lines in the results section. Importantly, because some of our results challenge previously published reports, we performed critical experiments using multiple cell lines. For example, we show that centrinone B-induced growth arrest is independent of TRIM37 E3 ligase activity using a single RPE-1 TRIM37-/- clone, an RPE-1 TRIM37-/- pool and an A375 TRIM37-/- pool. We feel this is a highlight of our work and this new data will be included in the revised version of the manuscript and will be emphasized.
- Two different concentrations (200 nM and 500 nM) of centrinone were used to compare responses of too many or no centrosomes in RPE1 and A375 . While these concentrations result in centrosome amplification (200 nM) and loss (500 nM) in RPE1 cells, the phenotypes seem much less clear-cut in A375 cells. At 200nM 70% of cells have 0 or 1 centrioles (~35% each category) and only about 15% have centrosome amplification, whereas centrosome amplification occurs in 30% of RPE1 with 0-1 centrioles seen in fewer than 10% (Figure 4 - figure supplement 1H). Hence the different outcomes of centrinone treatment makes conclusions about cell-type specific responses difficult. This difference may be due to differences in drug uptake/efflux, PLK4 activity or in expression of other components of these pathways. In fact, 167nM centrinone B in A375 cells would have been a much closer match to the 200nM treatment of RPE-1. These points should be discussed as they impact the conclusions.*
The reviewer rightly points out that the response to centrinone appears to differ between cell types, as shown previously (Meitinger et al., 2020 and Yeow et al., 2020), and that this difference may impact our conclusions. Although we don’t think that the major conclusions drawn will change, we will discuss these caveats within the results and discussion of the manuscript.
- I find the different outcomes of stable versus acute expression of TRIM37 ligase mutant confusing. Here, stable expression of TRIM37 ligase mutant increases mitotic length compared to that of TRIM37 wild-type, which contradicts a recent report by (Meitinger et al. 2021). What could be the potential reason for these differences? *
It is unclear why we obtain results that differ from Meitinger et al. We are using similar cell lines (RPE-1 hTert vs. RPE-1 hTert Cas9) with similar TRIM37 constructs (TRIM37-3xFLAG) that are induced in similar ways (both are doxycycline inducible but using different systems). For our experiment, we used a single TRIM37 KO clone. As an independent validation, we will repeat this experiment using our TRIM37 KO pools in both RPE-1 and A375 cells and discuss these results and implications.
*What could be the mechanism for TRIM37 action in regulating spindle assembly/mitotic duration and cell proliferation upon centrosome loss? How do those acentrosomal MTOCs form that decrease mitotic duration and promote proliferation? *
These are insightful questions that we feel lie at the heart of TRIM37 function. Current models posit that in the absence of TRIM37, PLK4 condensates form and are required to nucleate ectoptic accumulations of PCM components (ex. CEP192) that facilitate mitosis (Meitinger et al. 2020). A number of our findings are not consistent with this model. First, PLK4 is detected in the Cenpas/condensates only using a single antibody (Wong et al., 2015) (two other antibodies have been reported to be used (Sillibourne *et *al., 2010, Moyer et al., 2015) and we have used another (Millipore MABC544 clone 6H5) - none of these three detect PLK4 at the condensates). Additionally, the PLK4 signal observed is not sensitive to PLK4 siRNA (Balestraet al. 2021, Figure 4 – figure supplement 1I). In our manuscript we also provide evidence that overexpressed PLK4-3xFLAG cannot be detected (using PLK4 or FLAG antibodies) at these strucures. Moreover, our experiments using TRIM37 mutants show that Cenpas formation and ectopic PCM assembly are mechanistically distinct; Cenpas are not resolved after expression of TRIM37 C18R, yet ectopic PCM structures are suppressed (Figure 5E and G). Our data do, however, suggest that the ability to form ectopic PCM structures is inversely correlated to growth arrest activity (i.e. cells that form ectopic PCM fail to arrest). How these structures form and how they affect growth arrest are still critical, open questions. We will discuss these possibilities further in the revised manuscript.
*Do the authors find a difference in the % of cells expressing TRIM37 mutants upon stable or acute expression? This part needs a better summary, and again a table would help. I also wonder about protein expression levels; wild-type FB-TRIM37 seems to be expressed at much lower levels than the mutants in Figure 5B. *
The differences in overall abundance are not due to heterogenous expression within the population. The TRIM37 mutants are expressed in all cells after stable and acute expression. We will provide quantification of immunofluorescence images and statistics to show this. TRIM37 mediates its own degradation in an E3-dependent manner (Meitinger et al. 2021, Figure 3f). Our results are consistent with this as the TRIM37 C18R and TRIM37 ____D____RING mutants have a higher overall abundance compared to TRIM37 or TRIM37 ____D____505-709. These experiments are ongoing and we will discuss this further in the revised manuscript and provide a summary table.
- Other means of centrosome depletion (Cenpj, SAS6 etc) would have been useful to include in the manuscript in support of E3 ligase dependent and independent roles of TRIM37. It is not essential to perform these experiment but if data are available, including these would improve the paper. *
We will generate new data using a double TRIM37 KO, SASS6 KO line to address TRIM37 ligase-dependent and -independent functions.
- The authors show that TRIM37 regulates PLK4 phosphorylation and that this modification could only be observed in HEK293T and not in RPE1. Why would there be a difference between HEK293 and RPE1?*
We will address this by surveying a panel of cell lines to determine if there any cell type dependent differences in TRIM37 modification. Any potential differences will be addressed in the discussion.
- Statistical analysis for graphs should be included. Figure 5 is ok but graphs in Figures 3, 4, 6, 7 would benefit.*
This point is well taken. In the revised manuscript, we will ensure that all experiments are performed in biological triplicate and that proper statistical analyses are included to support our conclusions.
- The authors characterise TRIM37 localisation. They detect it at centrosomes (as shown by Yeow et al 2021) and more specifically at the PCM, but apparently the signal is not present in all cells. They should also provide a quantification of the % of cells with centrosomal TRIM37 signal and compare this to cells expressing Flag-tagged Trim37. The specificity of the antibody signal using TRIM37-/- should be confirmed. *
We will perform immunofluorescence experiments using wild type and TRIM37-/- cells to demonstrate the specificity of the antibody signal. We will also provide a more detailed analysis regarding TRIM37 localization noting 1) the number of cells with centrosomal TRIM37 2) cell cycle correlation with centrosomal TRIM37 and 3) a comparison with FLAG-BirA tagged TRIM37.
Reviewer 1, Minor points
1.Page 3: "A recent screen for mediators of supernumerary centrosome-induced arrest identified PIDDosome/p53 and placed the distal appendage protein ANKRD26 within this pathway [31]". It appears that the reference for Burigotto et al. is missing.
This reference will be inserted.
2.Page 6: The authors state that: TP53BP1, USP28 and CDKN1A are also suppressors in the Nutlin-3a screen and suggest that they act in a general p53 pathway. However Meitinger et al (2016) showed that depletion of TP53BP1 or USP28 did not affect the upregulation of p53 and p21 upon Mdm2 inhibition.
Our data is consistent with previous reports that TP53BP1 and USP28 are required for cell arrest after Nutlin-3a treatment (Cuella-Martin R et al. 2016). We will discuss possible explanations for the results observed by Meitinger et al.
3.Page 9: "First, we performed live cell imaging to measure mitotic length in cells grown in centrinone". For consistency the authors should say centrinone B here as wellI
We will change the text to indicate using centrinone B.
4.Page 9: "Cells lacking TRIM37 suppressed the growth arrest from 150 to 500 nM centrinone B in RPE-1 and 167 to 500 nM in A375 cells". The growth data for the A375 cells seem to be missing from the figures.
We refer to Figure 4D and Figure 4 – figure supplement 1G that contain the RPE-1 and A375 growth data, respectively. We will modify the text to more clearly refer to the data.
5.Page 10: "Our results confirmed that PLK4 and TRIM37 form a complex in RPE-1 cells (Figure 3G)" It appears the authors referred to the wrong figure, it should be Figure 4B.
Our apologies. The correct figure reference will be used.
*6.Figure 1C: The nuclear p53 signal is not apparent with 500 nM centrinone B in the exemplary cells. Did the authors use thresholding to quantify p53/p21 positive cells? *
The p53 staining in centrinone-treated cells is somewhat variable. To quantify the data, we used automated image analysis and set a cut off based on p53 intensity in DMSO-treated cells to indicate p53-positive cells. To improve the figure we will repeat the experiment and use a lower magnification image to show a more representative field of cells stained for p53. The quantification pipeline will be better explained in the methods section.
7.Figure 4D and Figure 4 - Figure supplement 1G: The graph is misleading and should not be presented as a continuous line.
We are sorry that the reviewer finds the graph misleading. We will change the way this data is presented to make it easier to understand and to facilitate indicating statistical differences. Instead of a scatter plot of all the data, we will present the data as individual boxplots at each centrinone B concentration with statistical differences indicated. We hope this will address any confusion regarding these data.
8.Figure 5A and C: A direct and statistical comparison mitotic timing upon expression different Trim37 mutants to wildtype and trim37-/- cells is missing
In Figure 5A we compare RPE-1 WT to TRIM37-/- at each centrinone B concentration and within each line we compare each centrinone B concentration to DMSO. Perhaps we do not understand the reviewer’s concern here, but we do not think any comparisons are missing from this panel. In Figure 5C, we compare the mitotic lengths between cell lines expressing TRIM37 WT or TRIM37 C18R since we focus on the requirement for the E3 ligase activity of TRIM37. For this experiment we did not include a wild-type control, but we will perform statistical analyses between control cells expressing FLAG-BirA and those expressing FB-TRIM37 WT or FB-TRIM37 C18R. We hope this addresses this concern.
9.Figure 6B: A loading control/Ponceau staining is missing as well as the quantification of protein levels
This experiment will be repeated for proper quantification and we will include a loading control for our representative results.
*10.Figure 6D: It is unclear if the centrosomal signal intensity was quantified in interphase or mitotic cells *
The centrosomal signal was quantified in mitotic cells only. This results and figure legend will be updated to more clearly indicate this.
11.Figure 7C: A loading control/Ponceau staining is missing
The experiment will be repeated and a sample will be taken prior to immunoprecipitation to indicate the input amounts for each sample.
12.Figure 2 - figure supplement 2F and G: It would help if the authors could highlight the cell line, e.g. RPE-1 (F) or A375 (G) in the venn diagrams.
In Figure 2 – figure supplement 2G we highlight the genes found in RPE-1 and A375 screens only in the overlap of the Venn diagram using font colour. We will colour code the hits from each cell line in panels (F) and (G). We thank the reviewer for this suggestion.
13.Figure 4 - figure supplement 1E: it appears that the BirA antibody gives only an unspecific signal. It would be useful to show if the different TRIM37 variants are able to localise to the centrosomes. Furthermore it appears that centrosomes are missing in the C18R and 505-709 variants. It would be useful if the authors quantify centrosome numbers upon expression of different Trim37 variants as shown in Figure 4 - figure supplement 1. To make the identification of the cell easier it would help to include a DNA signal or indicate the outline of the cell.
__The anti-BirA antibody does give a slightly diffuse signal, although we disagree that it is unspecific considering that the BirA signal is only observed in cells expressing FLAG-BirA alone or BirA fusion proteins. __
We agree with this reviewer that we did not make any statements about the centrosomal localization of the TRIM37 mutants. We will re-analyze our images to quantify relative centrosomal localization of these proteins. The images as displayed in this Figure panel appear to be somewhat confusing to the reviewer. In terms of scale, only a small portion of the cell surrounding the centrosome is shown, therefore a nuclear or cell outline cannot be displayed on these images. In each image a centrosome is present, even in the C18R and 505-709 samples. We will show images of entire cells with insets to highlight the region surrounding the centrosome.
*14.The generation of stable and dox-inducible cell lines is missing in the material and methods *
We apologize for this omission. This information will be added.
Reviewer 2, Major points
- The centrosomal localization of endogenous TRIM37 should be validated by comparing control and knockout/knockdown cells.* We will perform these experiments as outlined in response to Reviewer 1, Major point 8.
- Some of the quantifications are derived from only two experiments and in many cases no statistical testing was done. The authors should test the observed effects and add extra replicates to make the data more robust, where required. *
We will ensure experiments are performed in biological triplicate and that appropriate statistical analyses are performed (see comment to Reviewer 1, Major point 7)
- Fig. 5 supplements: panels showing effects on marker proteins in cells by IF lack quantification of the claimed effects. Without providing some type of quantifications for key findings, it is unclear how strong or penetrant the effects are.*
Quantification and statistical testing will be performed for these experiments.
Reviewer 2, Minor points
I would suggest a final, summarizing schematic that illustrates the main findings in a cartoon/flow chart manner.
We will improve the discussion of our main findings as well as provide a model/table of comparisons to improve the clarity of our manuscript.
- Please revise incorrect abstract sentence: "We identify TRIM37 as a key mediator of growth arrest when PLK4 activity is partially or fully inhibited but is not required for growth arrest triggered by supernumerary centrosomes." * In our screens, we find that TRIM37 is required for growth arrest after treating cells with 200 and 500 nM centrinone B. Treatment of cells with 200 nM centrinone B causes centriole overduplication and our initial hypothesis was that centriole overduplication alone is inducing growth arrest. To test this in a parallel manner, we also overexpressed PLK4 to induce centriole overduplication. Surprisingly, but consistent with recently published results (Evans et al., 2020), TRIM37 was not required for growth arrest after PLK4 overexpression. Thus, TRIM37 is required for growth arrest after 200 nM centrinone treatment, but not PLK4 overexpression, yet both of these conditions induce centriole overduplication. This concept will be highlighted, discussed and clarified in the text. We will change the abstract sentence to ‘We identify TRIM37 as a key mediator of growth arrest when PLK4 activity is partially or fully inhibited, but it is not required for growth arrest after PLK4 overexpression’.
Please also see similar comment to Reviewer 3, Major point 1.
- In various figures and supplements showing centrosome and condensates/Cenpas, these are very difficult to distinguish due to their small size. I suggest to magnify regions of interest and/or add arrowheads in different colors marking the specific structures.*
This comment is similar to Reviewer 1, Minor point 13. We will use coloured arrowheads to indicate different structures. Where possible, we will use magnified regions to improve clarity.
- Fig. 2A: What is the purpose of the schematics on the right of panel A? The labels in the graph are unreadable and the network diagram without any labels is also not very useful. This could be removed. *
The schematics on the right indicate a ‘generic analysis’ using the NGS sequencing data. We agree it is not essential and it will be removed.
- Fig. 2B: The network presentation is not very easy to read. What are the functional groups/pathways here? The clusters should be labeled accordingly. What is the meaning of the different sizes of the circles? Maybe key interactions (e.g. TRIM37) could be indicated in a different color shade to highlight these? *
In our figure we tried to highlight 1) the connectivity among screening conditions and 2) complexes that were identified by the screens. In our figure, each node (other than the six hub nodes that denote a screen condition) represents a hit from the screens. Thus, the nodes are connected by edges only to the screening conditions, not to each other. In this scenario, highlighting TRIM37 ‘interactions’ would only highlight the screening conditions for which TRIM37 was a hit (200 nM RPE-1, 500 nM RPE-1, 200 nM A375, 500 nM A375). We could try to overlay functional enrichment data on the graph, but this data is presented separately in Figure 2 – figure supplement A-D. The large circles represent hits found in previous screens and is indicated in the legend. Given the challenges of this figure we will modify it to improve its clarity.
Reviewer 3, Major points
- The presentation throughout the manuscript sometimes made it difficult to follow exactly what the authors meant when they referred to the various doses of Centrinone used in their experiments-often using the terms "low" or "high" without specifying exactly what they mean. In Figure 1A, for example, they present a growth inhibition curve using a log10 scale of Centrinone concentration, and they conclude that growth was inhibited "at concentrations above 150nM, with full inhibition observed at concentrations greater than 200nM". I presume this is just sloppy language, as it appears that growth is significantly inhibited at 150nM and full growth inhibition is achieved at 200nM. However, in Figure 4D, the authors show another growth inhibition curve (this time presented on a linear scale) where significant growth inhibition is seen well below 100nM and full inhibition appears to be achieved at ~125nM. The discrepancy between these experiments is not noted, nor any reason for it explained.* We agree with the reviewers and apologize for using ‘low’ and ‘high’ as they are ambiguous. We will ensure that we refer specifically to each concentration of centrinone B used (ex. 50 nM, 150 nM etc.). The comparison between Figure 1A and Figure 4D is not straightforward. The experiments presented were performed approximately 6 years apart and in slightly different ways. As reviewer 3 indicates, Figure 1A is presented in a log scale; this makes it difficult for the reader to determine the exact concentrations of centrinone B used. For this panel, we used, 0 (DMSO), 10, 30, 75, 165, 200 and 500 nM centrinone B. For Figure 4D, we used 0, 50, 125, 150, 167, 200 and 500 nM. The only point that might be anomalous is 75 nM in Figure 1A. We do see approximately 25% inhibition using 50 nM centrinone B in Figure 4D, but no inhibition using 75 nM in Figure 1A. We can offer two explanations for this discrepancy. First, we noticed small deviations in the potency of centrinone B batches. Second, for Figure 1A, cells were assayed using a passaging assay where they are continuously plated, counted and re-seeded. Cells in Figure 4D were assayed using a clonogenic assay where cells are plated at low density and allowed to grow over the course of approximately two weeks. It is possible that a combination of these factors led to the highlighted discrepancy. We feel that the discrepancy is a minor one and we propose the following as a solution. We will present the growth data in Figure 1A as a scatter / box plot using only 200 and 500 nM centrinone B since these are the drug concentrations we use for the screen conditions and the key conclusions are derived only from these concentrations (i.e. both concentrations result in p53-dependent growth arrest where centrioles are overduplicated after 200 nM centrinone B, while centrioles are lost after treatment with 500 nM). We hope that this explanation and changes satisfy the reviewers. *
While discrepancies such as this may seem trivial, they make it hard to interpret some of the authors conclusions. For example, in their initial screen, the "low" dose of Centrinone (200nM) leads to centriole amplification and genes that block centriole duplication or PIDDosome function (which normally signals the presence of extra centrioles) are required for the growth arrest triggered by this concentration of the drug (Figure 1B). To me, this suggests that centriole amplification is required for this growth arrest at 200nM. However, when the authors test a more graded series of concentrations they conclude "excess centrioles might not be the trigger for this arrest at low Centrinone B concentrations". I assume they are using "low" here to indicate concentrations at or below 150nM (even though they use low to mean 200nM in their initial screen)? In the Discussion, they state that TRIM37 is "required for the growth arrest in response to partially or fully inhibited PLK4, but this activity was independent of the presence of excess centrioles". Again, it is not clear to which experiments they are referring when they talk about "partially" or "fully" inhibited PLK4, but, if this is correct, then why are genes required for centriole duplication and PIDDosome function identified in their initial screen as being required for the growth arrest at 200nM but not 500nM? Do they consider 200nM to be fully inhibiting PLK4? *
We observed that cells arrested after treatment with either 200 or 500 nM centrinone B. Additionally, we observed centriole over-duplication after 200 nM but centriole loss at 500 nM. Our initial hypothesis was therefore that either centriole overduplication or loss resulted in growth arrest. Our subsequent results with TRIM37 caused us to question this simple interpretation. To determine if centriole overduplication caused by 200 nM centrinone B triggers growth arrest in this case, we induced centriole overduplication by overexpressing PLK4 and, surprisingly, TRIM37 was not required for growth arrest in these conditions, similar to that observed by ____Evans et al., 2020. Thus, we have two conditions where centriole overduplication is observed where the growth arrest in only one condition is dependent on TRIM37. This is an important difference that we will better highlight in our revised manuscript. We will also present a better model and/or table outlining our most salient results. Briefly, it is thought that partially inhibited PLK4 blocks its own auto-phosphorylation and therefore blocks its degradation. The overall abundance of PLK4 therefore increases under these conditions and overduplication occurs. In our hands, we consider PLK4 to be partially inhibited in RPE-1 or A375 cells at any concentrations of centrinone B at 200 nM or lower.
Please also see similar comment to Reviewer 2, Minor point 1.
Presumably it will only require textual changes to address this point, but it is hard to assess the broader significance of the paper until these points are clarified: is the main point of this paper that the cells response to Centrinone treatment is complicated and the role of TRIM37 equally so; or, is there a narrative that leads to a clear hypothesis that can explain these surprising findings? *
We don’t currently have a model that explains all the results we observe with TRIM37. We have data that is consistent with some previously published results and data that challenges some of these recent reports. The current model suggests that TRIM37 E3-dependent remodeling of CEP192 underlies its growth arrest activity after centriole loss. Importantly, we find that TRIM37 supports growth arrest in an E3-ligase-independent manner. We will discuss this further in our revised manuscript, as well as providing additional hypotheses based on our other observations of TRIM37 function.
- It seems a striking omission that the authors show that p53 and p21 are induced by 200nM and 500nM Centrinone (Figure 1D), but they don't assay these proteins at any concentration lower than this. Perhaps they are saving this data for a subsequent manuscript, but the authors certainly seem to draw conclusions from several experiments they perform at concentrations below 200nM, so they should at least explain why they don't assay p53 and p21 status in these experiments. *
We apologize for not including this data in the original version of the manuscript. It will be included in the revised version.
Reviewer 3, Minor points
- In the abstract the authors claim that the way in which altered centrosome numbers cause a p53-dependent growth arrest is evolutionarily conserved. This is misleading, as it implies that the loss and gain of centrosomes trigger the same arrest (which is probably not correct), and most of the data to date suggests that flies and worms (two popular models for centrosome research) do not have such a growth-arrest pathway.* This is a good point. We will modify this statement to indicate that p53-dependent arrest is confined to mammalian cells: “Altered centrosome numbers cause a p53-dependent growth arrest in both mouse and human cells through mechanisms that are still poorly defined”.
Reviewer 3, comment in ‘significance’
I could not discern, however, whether one could draw any broader conclusions than this, in part due to the presentation problems described above. Moreover, in the abstract the authors propose that altering PLK4 activity alone is sufficient to signal growth arrest. This would be an important conclusion, and I presume this refers to the very low dosage Centrinone experiments that trigger growth arrest without altering centrosome numbers and which does not require TRIM37? If so, this arrest is poorly characterised here and will be the subject of a future investigation, so it seems to strange to have this as a major conclusion in the abstract.
We agree. As reviewer 3 points out, based on our findings we hypothesize that altered PLK4 activity could itself signal growth arrest. As this is not supported experimentally, we will remove it from the abstract and discuss this tantalizing possibility within the discussion.
3. Description of the revisions that have already been incorporated in the transferred manuscript
Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.
Most of the experiments are currently ongoing and the preliminary results we have obtained discussed in the previous section. The revised manuscript will be modified to address each and every concern of the three reviewers as detailed above.
4. Description of analyses that authors prefer not to carry out
Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.
We will carry out all the experiments requested by the reviewers as detailed above.
References
Balestra FR et al., TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin. Elife. 2021 Jan 25
Cuella-Martin R *et *al., 53BP1 Integrates DNA Repair and p53-Dependent Cell Fate Decisions via Distinct Mechanisms. Mol Cell. 2016 Oct 6;64(1):51-64
Evans LT et al., ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification. EMBO J. 2021 Feb 15;40(4)
Meitinger F *et *al., TRIM37 controls cancer-specific vulnerability to PLK4 inhibition. Nature. 2020 Sep;585(7825):440-446
Moyer TC et al., Binding of STIL to Plk4 activates kinase activity to promote centriole assembly. J Cell Biol. 2015 Jun 22;209(6):863-78
Sillibourne JE et al.,Autophosphorylation of polo-like kinase 4 and its role in centriole duplication. Mol Biol Cell. 2010 Feb 15;21(4):547-61
Wong YL et al., Cell biology. Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science. 2015 Jun 5;348(6239):1155-60
Yeow ZY et al., Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer. Nature. 2020 Sep;585(7825):447-452
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Referee #3
Evidence, reproducibility and clarity
Summary:
In this manuscript, Tkach et al. analyse the molecular pathways that lead to the growth arrest of either RPE-1 or A375 cells in response to varying doses of the PLK4 inhibitor Centrinone B (hereafter Centrinone). They show that both 200nM and 500nM Centrinone cause a strong growth arrest, but the lower concentration actually leads to centrosome amplification, while the higher concentration leads to centrosome loss. They identify the Ubiquitin E3 ligase TRIM37 as a key mediator of the growth arrest at both drug concentrations, although they confirm previous findings that TRIM37 is not required for the growth arrest induced …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary:
In this manuscript, Tkach et al. analyse the molecular pathways that lead to the growth arrest of either RPE-1 or A375 cells in response to varying doses of the PLK4 inhibitor Centrinone B (hereafter Centrinone). They show that both 200nM and 500nM Centrinone cause a strong growth arrest, but the lower concentration actually leads to centrosome amplification, while the higher concentration leads to centrosome loss. They identify the Ubiquitin E3 ligase TRIM37 as a key mediator of the growth arrest at both drug concentrations, although they confirm previous findings that TRIM37 is not required for the growth arrest induced by the supernumary centrosomes that are formed when PLK4 is overexpressed. Perhaps most importantly, the authors test the ability of various mutated forms of TRIM37 to function in the growth arrest induced by Centrinone treatment, and they conclude that, surprisingly, the E3 ligase activity of TRIM37 is not required for this growth arrest.
The experiments presented here are generally of a high quality, although I found some aspects of the presentation a little confusing (as detailed below).
Major Comments:
- The presentation throughout the manuscript sometimes made it difficult to follow exactly what the authors meant when they referred to the various doses of Centrinone used in their experiments-often using the terms "low" or "high" without specifying exactly what they mean. In Figure 1A, for example, they present a growth inhibition curve using a log10 scale of Centrinone concentration, and they conclude that growth was inhibited "at concentrations above 150nM, with full inhibition observed at concentrations greater than 200nM". I presume this is just sloppy language, as it appears that growth is significantly inhibited at 150nM and full growth inhibition is achieved at 200nM. However, in Figure 4D, the authors show another growth inhibition curve (this time presented on a linear scale) where significant growth inhibition is seen well below 100nM and full inhibition appears to be achieved at ~125nM. The discrepancy between these experiments is not noted, nor any reason for it explained.
While discrepancies such as this may seem trivial, they make it hard to interpret some of the authors conclusions. For example, in their initial screen, the "low" dose of Centrinone (200nM) leads to centriole amplification and genes that block centriole duplication or PIDDosome function (which normally signals the presence of extra centrioles) are required for the growth arrest triggered by this concentration of the drug (Figure 1B). To me, this suggests that centriole amplification is required for this growth arrest at 200nM. However, when the authors test a more graded series of concentrations they conclude "excess centrioles might not be the trigger for this arrest at low Centrinone B concentrations". I assume they are using "low" here to indicate concentrations at or below 150nM (even though they use low to mean 200nM in their initial screen)? In the Discussion, they state that TRIM37 is "required for the growth arrest in response to partially or fully inhibited PLK4, but this activity was independent of the presence of excess centrioles". Again, it is not clear to which experiments they are referring when they talk about "partially" or "fully" inhibited PLK4, but, if this is correct, then why are genes required for centriole duplication and PIDDosome function identified in their initial screen as being required for the growth arrest at 200nM but not 500nM? Do they consider 200nM to be fully inhibiting PLK4?
Presumably it will only require textual changes to address this point, but it is hard to assess the broader significance of the paper until these points are clarified: is the main point of this paper that the cells response to Centrinone treatment is complicated and the role of TRIM37 equally so; or, is there a narrative that leads to a clear hypothesis that can explain these surprising findings?
- It seems a striking omission that the authors show that p53 and p21 are induced by 200nM and 500nM Centrinone (Figure 1D), but they don't assay these proteins at any concentration lower than this. Perhaps they are saving this data for a subsequent manuscript, but the authors certainly seem to draw conclusions from several experiments they perform at concentrations below 200nM, so they should at least explain why they don't assay p53 and p21 status in these experiments.
Minor comments:
In the abstract the authors claim that the way in which altered centrosome numbers cause a p53-dependent growth arrest is evolutionarily conserved. This is misleading, as it implies that the loss and gain of centrosomes trigger the same arrest (which is probably not correct), and most of the data to date suggests that flies and worms (two popular models for centrosome research) do not have such a growth-arrest pathway.
Significance
Significance and comparison to existing literature:
The question of how centrosome loss or amplification leads to senescence or apoptosis in many cell types is currently a hot topic, and TRIM37 has previously been identified as a potentially important player-most recently in two high-profile papers from the Oegema/Loncarek (Meitinger et al, Nature 2021) and Holland/Chapman (Yeow at al., Nature 2021) labs. In these papers, TRIM37 is shown to be overexpressed in certain cancer cells, where it appears to degrade PCM components (most notably Cep192) to prevent the formation of ectopic spindle poles that help to ensure mitotic fidelity in these abnormal cells. Moreover, mutations in TRIM37 cause Mulibrey nanism, which has recently been shown to be associated with the formation of ectopic Centrobin-dependent PCM condensates (Balestra et al., eLife 2021; Meitinger et al., JCB, 2021).
This manuscript makes an important contribution to this area, and it will be of considerable interest to researchers in several fields (most obviously the centrosome, but also ubiquitin ligase, cancer and Mulibrey fields). In its current form, this contribution is largely to illustrate that treating cells with Centrinone (which is widely used by many centrosome researchers) triggers a complex cellular response that varies with drug dosage, and that the role of TRIM37 in triggering this response also appears to be surprisingly complicated. These are significant points that are of sufficient importance to warrant publication.
I could not discern, however, whether one could draw any broader conclusions than this, in part due to the presentation problems described above. Moreover, in the abstract the authors propose that altering PLK4 activity alone is sufficient to signal growth arrest. This would be an important conclusion, and I presume this refers to the very low dosage Centrinone experiments that trigger growth arrest without altering centrosome numbers and which does not require TRIM37? If so, this arrest is poorly characterised here and will be the subject of a future investigation, so it seems to strange to have this as a major conclusion in the abstract.
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Referee #2
Evidence, reproducibility and clarity
Summary:
The study by Tkach et al. investigates the molecular basis of the previously described, p53-dependent growth arrest that is triggered by manipulation of PLK4 kinase activity, a master regulator of centrosome biogenesis. To address this, they use CRISPR/Cas9 screening in human cell lines, gene-specific knockout and rescue experiments, and biochemical interaction assays. As in previously conducted similar screens they identify the E3 ligase TRIM37 as a key mediator of growth arrest after PLK4 inhibition, but not growth arrest induced by increased centrosome number. Importantly, contrary to suggestions in previous studies, …
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Referee #2
Evidence, reproducibility and clarity
Summary:
The study by Tkach et al. investigates the molecular basis of the previously described, p53-dependent growth arrest that is triggered by manipulation of PLK4 kinase activity, a master regulator of centrosome biogenesis. To address this, they use CRISPR/Cas9 screening in human cell lines, gene-specific knockout and rescue experiments, and biochemical interaction assays. As in previously conducted similar screens they identify the E3 ligase TRIM37 as a key mediator of growth arrest after PLK4 inhibition, but not growth arrest induced by increased centrosome number. Importantly, contrary to suggestions in previous studies, they find that TRIM37 function in growth arrest is independent of E3 ligase function, but may involve regulation of PLK4.
Major comments:
Overall, I found the key conclusions convincing, assuming the claimed effects are significant. In this regard, some data requires quantification and some of the quantifications may require additional replicates.
The centrosomal localization of endogenous TRIM37 should be validated by comparing control and knockout/knockdown cells.
Some of the quantifications are derived from only two experiments and in many cases no statistical testing was done. The authors should test the observed effects and add extra replicates to make the data more robust, where required.
Fig. 5 supplements: panels showing effects on marker proteins in cells by IF lack quantification of the claimed effects. Without providing some type of quantifications for key findings, it is unclear how strong or penetrant the effects are.
Minor comments:
Overall, I felt that the presentation of the data can be improved. After reading the abstract, it was not clear at all to me, what message the authors want to convey, also in comparison to previous work. In particular the final part of the abstract should be improved. The results part is well written, but may still be improved, by providing more summarizing statements that extract the key conclusion from particular experiments and by explaining better why particular experiments were done. The specific rationale may be clear to expert readers but less so to non-experts. Only after reading the discussion, the findings and how they relate to previous work became clearer. I would suggest a final, summarizing schematic that illustrates the main findings in a cartoon/flow chart manner.
Please revise incorrect abstract sentence: "We identify TRIM37 as a key mediator of growth arrest when PLK4 activity is partially or fully inhibited but is not required for growth arrest triggered by supernumerary centrosomes."
In various figures and supplements showing centrosome and condensates/Cenpas, these are very difficult to distinguish due to their small size. I suggest to magnify regions of interest and/or add arrowheads in different colors marking the specific structures.
Fig. 2A: What is the purpose of the schematics on the right of panel A? The labels in the graph are unreadable and the network diagram without any labels is also not very useful. This could be removed.
Fig. 2B: The network presentation is not very easy to read. What are the functional groups/pathways here? The clusters should be labeled accordingly. What is the meaning of the different sizes of the circles? Maybe key interactions (e.g. TRIM37) could be indicated in a different color shade to highlight these?
Significance
While the authors start out by essentially reproducing results from previously conducted screens, which may seem to be of limited novelty, the current work reaches conclusions that differ in important aspects from those in previous studies. Moreover, the current work nicely compares in different cell backgrounds PLK4 partial inhibition (extra centrosomes), full inhibition (less/no centrosomes), and p53 pathway inhibition, to obtain an integrated view of the mechanisms involved in growth arrest and tease apart molecular requirements. The results challenge some of the conclusions from previous studies, including high-profile papers where this pathway has been identified as a potential target for cancer treatment. For these reasons I consider this very important work.
My expertise is in centrosome biology and microtubule organization including mitotic spindle assembly.
Referee Cross-commenting
Hi everyone,
Overall it seems that we all agree that this is an important study. However, as noted by several comments, the presentation definitely needs to be improved and the new findings need to be highlighted better and contrasted with previous studies. I had relatively few major concerns, but, after reading the other reviews, I found the additional comments also important and useful.
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Referee #1
Evidence, reproducibility and clarity
Centrosome loss and gain both elicit a p53-dependent cell cycle arrest but the molecular pathways involved are still not fully understood. To address this question the Pelletier lab performed several genome wide CRISPR screens using two different concentrations of centrinone that cause centrosome amplification (low) or loss (high) in RPE1 and A375 cells. In order to distinguish between pathways that act by regulating p53 levels in cells vs those that mediate p53 response to abnormal centrosome numbers, they also performed a screen in cells where p53 levels were artificially elevated by Nutlin treatment. The top hits from the …
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Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Centrosome loss and gain both elicit a p53-dependent cell cycle arrest but the molecular pathways involved are still not fully understood. To address this question the Pelletier lab performed several genome wide CRISPR screens using two different concentrations of centrinone that cause centrosome amplification (low) or loss (high) in RPE1 and A375 cells. In order to distinguish between pathways that act by regulating p53 levels in cells vs those that mediate p53 response to abnormal centrosome numbers, they also performed a screen in cells where p53 levels were artificially elevated by Nutlin treatment. The top hits from the low/high centrinone screen confirmed previous results from other groups, highlighting the importance of the 53BP1/USP28/p53 complex and PIDDisome/ANKR26 complex in the cell cycle response. TRIM37 was shared between both centrinone conditions, while being absent from the Nutlin screen, and thus the authors focused their analysis on the function of TRIM37. Overall the data quality and presentation are both good and the manuscript reads well. The Crispr-Cas9 screens have been performed to a high standard and it is reassuring that the same candidates emerge as from previous screens focusing on centrosome loss and gain.
TRIM37 has been the subject of several high profile papers over the past year. This current manuscript has the potential to clarify some of the outstanding questions but in its present form the manuscript brings more confusion than clarity to his area of research. Although the authors conduct a careful analysis of TRIM37 function, unless someone is a die-hard specialist, it is difficult to follow what is already known, what the authors find and how or why their data fits/contradicts previous work. The key observations are that i) TRIM37 may not actually control CEP192 levels (unless overexpressed transiently), ii) its E3 ligase activity and its binding to PLK4 are independent of its ability to promote growth arrest upon centrinone treatment, iii) its influence on mitotic duration is independent of its E3 activity or its role in growth arrest upon centrinone treatment. The result that a TRIM37-dependent growth arrest may also exist without increased mitotic duration is another interesting finding, as is the link between TRIM37 and condensates of centrosomal proteins. Including a table that summarises which roles of TRIM37 require PLK4 binding, E3 ligase activity etc would be useful not only to non-specialists. Some of the data contradicts current models for TRIM37 function in growth suppression, so the authors should consider showing a revised model, too.
Major Points:
- Previous data suggested that an important role of TRIM37 was to limit accumulation of CEP192 levels, yet here CEP192 levels appeared unchanged in TRIM37 knockout cells that stably express wild-type or RING domain mutant TRIM37. However, in agreement with previous work, transient expression of TRIM37 reduced CEP192 levels along with those of other PCM and centriole components in an E3-dependent manner. These data are rather confusing in light of the literature, and the current report does not really deal with these discrepancies but to me they suggest that high levels of TRIM37 can target multiple centrosome components for degradation, but this may be an experimental artefact.
- The choice of cells for particular experiments is not always stated or explained. For instance, in Figure 3A: Trim37 KO pool used while in Figure 3B TRIM37 single KO. These are then combined with both transient and stable expression of TRIM37 mutants.
- Two different concentrations (200 nM and 500 nM) of centrinone were used to compare responses of too many or no centrosomes in RPE1 and A375 . While these concentrations result in centrosome amplification (200 nM) and loss (500 nM) in RPE1 cells, the phenotypes seem much less clear-cut in A375 cells. At 200nM 70% of cells have 0 or 1 centrioles (~35% each category) and only about 15% have centrosome amplification, whereas centrosome amplification occurs in 30% of RPE1 with 0-1 centrioles seen in fewer than 10% (Figure 4 - figure supplement 1H). Hence the different outcomes of centrinone treatment makes conclusions about cell-type specific responses difficult. This difference may be due to differences in drug uptake/efflux, PLK4 activity or in expression of other components of these pathways. In fact, 167nM centrinone B in A375 cells would have been a much closer match to the 200nM treatment of RPE-1. These points should be discussed as they impact the conclusions.
- I find the different outcomes of stable versus acute expression of TRIM37 ligase mutant confusing. Here, stable expression of TRIM37 ligase mutant increases mitotic length compared to that of TRIM37 wild-type, which contradicts a recent report by (Meitinger et al. 2021). What could be the potential reason for these differences? What could be the mechanism for TRIM37 action in regulating spindle assembly/mitotic duration and cell proliferation upon centrosome loss? How do those acentrosomal MTOCs form that decrease mitotic duration and promote proliferation? Do the authors find a difference in the % of cells expressing TRIM37 mutants upon stable or acute expression? This part needs a better summary, and again a table would help. I also wonder about protein expression levels; wild-type FB-TRIM37 seems to be expressed at much lower levels than the mutants in Figure 5B.
- Other means of centrosome depletion (Cenpj, SAS6 etc) would have been useful to include in the manuscript in support of E3 ligase dependent and independent roles of TRIM37. It is not essential to perform these experiment but if data are available, including these would improve the paper.
- The authors show that TRIM37 regulates PLK4 phosphorylation and that this modification could only be observed in HEK293T and not in RPE1. Why would there be a difference between HEK293 and RPE1?
- Statistical analysis for graphs should be included. Figure 5 is ok but graphs in Figures 3, 4, 6, 7 would benefit.
- The authors characterise TRIM37 localisation. They detect it at centrosomes (as shown by Yeow et al 2021) and more specifically at the PCM, but apparently the signal is not present in all cells. They should also provide a quantification of the % of cells with centrosomal TRIM37 signal and compare this to cells expressing Flag-tagged Trim37. The specificity of the antibody signal using TRIM37-/- should be confirmed.
Minor Points
• Page 3: "A recent screen for mediators of supernumerary centrosome-induced arrest identified PIDDosome/p53 and placed the distal appendage protein ANKRD26 within this pathway [31]". It appears that the reference for Burigotto et al. is missing.
• Page 6: The authors state that: TP53BP1, USP28 and CDKN1A are also suppressors in the Nutlin-3a screen and suggest that they act in a general p53 pathway. However Meitinger et al (2016) showed that depletion of TP53BP1 or USP28 did not affect the upregulation of p53 and p21 upon Mdm2 inhibition.
• Page 9: "First, we performed live cell imaging to measure mitotic length in cells grown in centrinone". For consistency the authors should say centrinone B here as well
• Page 9: "Cells lacking TRIM37 suppressed the growth arrest from 150 to 500 nM centrinone B in RPE-1 and 167 to 500 nM in A375 cells". The growth data for the A375 cells seem to be missing from the figures.
• Page 10: "Our results confirmed that PLK4 and TRIM37 form a complex in RPE-1 cells (Figure 3G)" It appears the authors referred to the wrong figure, it should be Figure 4B.
• Figure 1C: The nuclear p53 signal is not apparent with 500 nM centrinone B in the exemplary cells. Did the authors use thresholding to quantify p53/p21 positive cells?
• Figure 4D and Figure 4 - Figure supplement 1G: The graph is misleading and should not be presented as a continuous line.
• Figure 5A and C: A direct and statistical comparison mitotic timing upon expression different Trim37 mutants to wildtype and trim37-/- cells is missing
• Figure 6B: A loading control/Ponceau staining is missing as well as the quantification of protein levels
• Figure 6D: It is unclear if the centrosomal signal intensity was quantified in interphase or mitotic cells
• Figure 7C: A loading control/Ponceau staining is missing
• Figure 2 - figure supplement 2F and G: It would help if the authors could highlight the cell line, e.g. RPE-1 (F) or A375 (G) in the venn diagrams.
• Figure 4 - figure supplement 1E: it appears that the BirA antibody gives only an unspecific signal. It would be useful to show if the different TRIM37 variants are able to localise to the centrosomes. Furthermore it appears that centrosomes are missing in the C18R and 505-709 variants. It would be useful if the authors quantify centrosome numbers upon expression of different Trim37 variants as shown in Figure 4 - figure supplement 1. To make the identification of the cell easier it would help to include a DNA signal or indicate the outline of the cell.
• The generation of stable and dox-inducible cell lines is missing in the material and methods
Significance
Centrosome loss in mammalian cells triggers a somewhat mysterious p53-dependent irreversible cell cycle arrest that bears similarities with senescence. A key modulator of this arrest is the E3 ubiquitin ligase TRIM37; TRIM37-overexpressing cells show increased sensitivity to centrosome loss whereas TRIM37 deletion restores normal growth to cells lacking centrosomes. The precise function of TRIM37 in this process is still not clear.
The authors here report a two-pronged approach to improve our understanding; first, they perform several genome-wide Crispr/Cas9 screens in two cell lines to identify new players that modulate growth arrest following inhibiton of centrosome duplication, and second, they analyse the function of TRIM37, their top candidate, in this process. Whereas the screens recapitulate previous reports by identifying a near identical set of genes, the functional work of TRIM37 provides interesting new data that go beyond (and at places contradict) published work. They describe a complex relationship between TRIM37 function, PLK4 inhibition and growth arrest, and suggest that TRIM37 acts via modulating PLK4 phosphorylation/stability and perhaps its role in autophagy also contributes to the overall phenotype. These possibilities will need to be tested in the future but the current manuscript contains enough interesting and potentially important data that it is worthy of publication following revision.
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