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
Response to Reviewers and Revision Plan
We thank all three reviewers for their time and their comments on our manuscript.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Here Ryan et al. have used localization analysis following induced rapid relocalization of endogenous proteins to investigate the composition and recruitment hierarchy of a clathrin-TACC3-based spindle complex that is important for microtubule organization and stability.
The authors generate different HeLa cell lines, each with one of four complex members (TACC3, CLTA, chTOG and GTSE1) endogenously tagged with FKBP-GFP via Cas9-mediated editing. This tag allows rapid recruitment to the mitochondria upon rapamycin addition ("knocksideways"). They ultimately quantify each of the 4 components' localization to the spindle following knocksideways of each component using fluorescently-tagged transfected constructs. The authors' interpretation of the results of this analysis are summarized in the last model figure, in which a core MT-binding complex of clathrin and TACC3 recruit the ancillary components GTSE1 and chTOG. In addition, the authors investigate the contribution of individual clathrin-binding LIDL motifs in GTSE1 to the recruitment of clathrin and GTSE1 to spindles. Their findings here largely agree with and confirm a recent report regarding the contribution of these motifs to GTSE1 recruitment to the spindle. They further analyzed GTSE1 fragments for interphase and mitotic microtubule localization, and identified a second region of GTSE1 required (but not sufficient) for spindle localization. Finally, the authors report that PIK3C2A is not part of this complex, contradicting (correcting) a previously published study.
**Major comments:**
1.The chTOG-FKBP-GFP cell line the authors generate has only a small fraction of chTOG tagged, and thus should not be used for any conclusions about protein localization dependency on chTOG. Because they were unable to construct a HeLa cell line with all copies tagged, the authors expect that the homozygous knock-in of chTOG-FKBP-GFP is lethal, and thus their experience is appropriate to report. However, the authors should not use this cell line alone to make statements about chTOG dependency. They would have to use similar localization analysis, but after another method to disrupt chTOG (as a second-best approach), such as RNAi. In fact, they have reported this in a previous publication (Booth et al 2011). However, the result was different. There, loss of chTOG resulted in reduced clathrin on spindles, suggesting it may stabilize or help recruit the complex. Alternatively, they could remove their chTOG data, but this would compromise the "comprehensive" nature of the work.
The referee is correct. The point here is to show the results we had using this approach for all four proteins under study. For this reason, we do not want to remove this data and prefer to show our results “warts-and-all”. We feel that the shortcomings of our approach are honestly presented and discussed in the manuscript. While only a fraction of chTOG was tagged, we should expect some co-removal after its induced mislocalization. Since we saw no change, we concluded that chTOG is auxiliary.
The “second best” approach suggested (RNAi of chTOG) is problematic for two reasons. First, chTOG RNAi results in gross changes to spindle structure (multipolar spindles) and it is difficult to pick apart differences in protein partner localization that result from loss of chTOG from those resulting from changes in spindle structure. Second, the paper is about induced mislocalization as a method for determining protein complexes once a normal spindle has formed. So, removing chTOG prior to mitosis is not comparable. If we get the same or different result, does it confirm or conflict with the data we have? Nonetheless, given the discrepancy with our earlier work, we should investigate this further.
To address this concern, we will stain endogenous clathrin, TACC3 and GTSE1 following chTOG RNAi and measure their relative levels at the spindle.
Making the chTOG-FKBP-GFP cell line was difficult. As described in the paper, we only recovered heterozygous clones despite repeated attempts. Since submission, we have been made aware of a HCT116 chTOG-FKBP-GFP cell line that is reported to be homozygously tagged (Cherry et al. 2019 doi: 10.1002/glia.23628).
A note about this cell line has been added to the paper (Results section, final sentence of 1st paragraph).
2.The authors initially analyze complex member localization after knocksideways experiments by antibody staining, which has the advantage of analyzing endogenous proteins (versus the later transfected fluorescent constructs). Setting aside potential artefacts from fixation, this would seem to be a better method for controlled analysis to take advantage of their setup (short of generating stable cell lines with second proteins endogenously tagged in a second color - a huge undertaking). The authors conclude that antibody specificity problems confounded their analysis and explained unusual results. However, I think is worth investing a little more effort to sort this out, rather than bringing doubt to the whole data set. Verifying and then using another antibody for chTOG localization would be informative. Of course, the negative control should not be their chTOG-FKBP-GFP line, as it does not relocalize most of chTOG.
In the case of GTSE1, an alternative explanation to antibody specificity issues would be that the GTSE1-FKBP-GFP cell line is not in fact homozygously tagged. Given the low expression levels on the western provided, and the detection of GTSE1 on the spindle in the induced GTSE1-FKBP-GFP cell line (but not TACC3-FKBP-GFP), it seems plausible that an untagged copy remains. If there are multiple copies of GTSE1 in Hela cells, one untagged copy could represent a small fraction of total GTSE1. This should thus be ruled out. GTSE1 clones should be analyzed with more protein extracts loaded - dilutions of the extracts can determine the sensitivity of the blot to lower protein levels. In addition, sequencing of genomic DNA can reveal a small percentage with different reads.
We used a two-pronged approach for assessing relocalization of protein partners (staining vs transfected constructs). The staining approach is superior since endogenous proteins are examined, but it is limited by antibody specificity. The transfection approach overcomes this limitation but is in turn limited by effects of overexpression and tagging. Together the two approaches allow us, and anyone employing this method, to get a picture of protein complexes. We didn’t want to create the impression that one or other approach is confounded, but the referee is correct that this analysis would benefit from further work.
Specifically, to address these concerns:
- We will verify and use alternative chTOG antibodies to try to improve this dataset.
- We will test the possibility that an untagged allele of GTSE1 remains. We will use western blotting and a summary of our genomic analysis will be added to the paper.
3.There is a lot of data contained in the small graphs summarizing quantification of localization in Figs 3 and 4. They would be more accessible to the reader if they were larger and/or an "example" of the chart with labels was present explaining it (essentially what is in the figure legends). Furthermore, there is no statistical test applied to this data that I see. This is needed. How do authors determine whether there is an "effect"?
Our aim was to compress a lot of information into a small space, while still showing some example primary data. All reviewers raised the same concern which tells us that we went too far towards “data visualization”.
To address this point, we will rework these figures.
**Minor issues:**
1.The GTSE1 constructs used for mutation and localization analysis are 720 amino acids long. A recent study analyzing similar mutations uses a 739 amino acid construct (Rondelet et al 2020). The latter is the predominant transcript in NCBI and Ensembl databases. It appears the construct used by the authors omits the first 19 a.a.. I do not think using the truncated transcript affects conclusions of the manuscript, but it could generate confusion when identifying residues based on a.a.#s of mutant constructs (Fig 6). This should be somehow clarified.
We were aware of the longer transcript but were using the 720 residue form since it is the canonical sequence in Uniprot (https://www.uniprot.org/uniprot/Q9NYZ3). We did not know that the 739 form is the predominant transcript. We agree this is unlikely to affect our work but that the numbering may cause confusion.
We have added a note to the Methods (Molecular Biology section) to accurately describe what we and Rondelet et al. have used.
2.The labeling of constructs in Fig 6C/D is confusing, and appears shifted by eye at places. Please relabel this more clearly.
Apologies for the error.
We have relabeled Figure 6C,D and also made a similar alteration to Figure 5C.
The recommended new experimental data (Analysis complex member levels on spindles after full perturbation of spindle chTOG; new chTOG antibody stainings in the FKBP lines; reanalysis of GTSE1 DNA/protein in GTSE1-FKBP line) should only require a new antibody/siRNA, plus a few weeks time to repeat the analyses already in the paper with new reagents.
Reviewer #1 (Significance (Required)):
While multiple individual components of this complex have been previously characterized, the structure and nature of the complex formation and its recruitment to microtubules/spindles remains a complex problem that has yet to be solved.
Overall this study represents a comprehensive localization-dependency analysis of the Clathrin-TACC3 based spindle complex using a consistent methodology. Although several of the conclusions of the findings echo previous reports, some of the previous literature is contradictory within itself as well as with the conclusions here. Analyzing all components with a single, rapid-perturbation technique thus has great value to present a clear data set, given that the experimental setup conditions and analysis are solid (a goal to which the majority of comments refer).
Beyond the complex localization/recruitment analysis, two novel findings of this study that emerge are:
a)GTSE1 contains a second, separate protein region, distinct from the clathrin-binding motifs that is required for its localization to the spindle, and most likely a microtubule-interaction site. This suggests that GTSE1 recruitment to the spindle is more complex than previously reported.
b)PI3KC2A, which has been reported previously to be a stabilizing member of this complex, is in fact not a member, nor localizes to spindles, nor displays a mitotic defect after loss. This is important conclusion to be made as it would correct the literature, and avoid future confusion.
--
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
In this paper, the authors investigate the nature of interactions between members of the TACC3-chTOG-clathrin-GTSE1 complex on the mitotic spindle. By using a series of HeLa cell lines that they have created by CRISPR/Cas9 editing to enable spatial manipulation (knocksideways) of either TACC3, chTOG, clathrin and GTSE1, they show that on spindle microtubules TACC3 and clathrin represent core complex members whereas chTOG and GTSE1 bind to them respectively but not to each other. Additionally, the authors find that the protein PIK3C2A, which has been implicated in this complex previously is in fact not a component of this complex in mitotic cells. The main advance of the paper in my opinion is the endogenous tagging of the proteins for knocksideways experiments since former experiments depended on RNAi silencing and expression of tagged proteins from plasmids, which introduced issues of protein silencing efficiency and plasmid overexpression problems. This approach seems to alleviate these problems, except in the case of chTOG which seems to be lethal in its homozygous variant.
**Major comments:**
I find the key conclusions regarding the localization of the components of the complex convincing. There are some issues regarding the specificity of antibodies in immunostaining experiments (Fig 3.) and the influence of mCherry-TACC3 expression on distorted localization of the complex prior to knocksideways. However, I think the general conclusion about which complex components (clathrin and TACC3) influence the localization of the other proteins in the complex (chTOG and GTSE1) stands. One thing that I miss from the paper is the data on the consequences on the spindle shape and morphology after knocksideways. I have noticed on images in both Figure 3 and Figure 4 that in some cases distribution of the signal seems to influence quite a bit the spindle morphology. Also, In Figure 3 I have noticed what seems to me a quite big variation in spindle size in tubulin signal in both untreated and rapamycin cells. Since authors have many of these images already, I believe it would be realistic, not costly and of additional value for the paper to provide more data on the consequences of the knocksideways experiments. Change of spindle size, tubulin intensity and DNA/kinetochore misalignment upon knocksideways would be helpful to appreciate more the findings of the paper. More so since the authors on more than one occasion find their motivation in the field of cancer research and spindle stability relation to it. Some data connection to this motivation would be of value. Experiments seem reproducible.
The focus of the paper is on using the knocksideways methodology to understand a protein complex during mitosis, rather than looking at its function. We are not keen to do new experiments that are not part of the central message of the paper. However, the Reviewer is correct that we do already have a dataset that can be mined in the manner described.
To address this point, we will analyze spindle size parameters and also the intensity of tubulin. Our analysis will be limited to the short timeframe of our experiments, but it should reveal or refute any changes in spindle structure that may result from loss of complex members.
**Minor comments:**
I have some problems with the clarity of Figure 3 and 4. For Figure 3. In Figure 3 plots on the right are a bit small and not easy to read. Some reorganization of the figure might be beneficial. In Figure 4 plots to the right are also too small to be clear. Also, I miss the number of cells (n) I can't see the number of individual arrows because of the size of graphs.
Our aim was to compress a lot of information into a small space, while still showing some example primary data. All reviewers raised the same concern which tells us that we went too far towards “data visualization”.
To address this point, we will rework these figures.
Reviewer #2 (Significance (Required)):
I find that the biggest significance of the paper is in the creation of new tools (cell lines) to study the localization of proteins TACC3, chTOG, clathrin and GTSE1. Cell lines where endogenous proteins can be delocalized rapidly will be of value for scientist working not only in mitosis but such as in the case of clathrin research, vesicle formation and trafficking or p53-dependent apoptosis in the case of GTSE1. In the field of mitosis it will surely help and speed up the research concerning the role of these proteins in spindle assembly and stability.
Field of expertise: mitotic spindle
--
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
**Summary:**
This papers analyses the chTog/TACC3/clathrin/GTSE1 complex that crosslinks and stabilises microtubule bundles in the mitotic spindle. The authors have developed an elegant knock sideways approach to specifically analyse the effects of removing individual components of the complex from the spindle and study the effect this has on the other interactors. They report, based on these assays that the core of the complex is formed by TACC3 and Clathrin while GTSE1 and chTog are auxiliary interactors. They also refute previous evidence that this complex also incorporates PIK3C2A. Overall, this is an interesting study that distinguishes itself predominantly by its methodology. However, some of the reported results need more thorough analysis to allow convincing conclusions.
**Major comments:**
1)The knockside way method is the main highlight if this paper. Unlike previous studies by the PI, this time endogenous genes are tagged which is a key advance and allows much better interpretation of the results. I am not sure why the authors have chosen HeLa cells as their model here, given the messed up genome of these cells. A non-transformed cell line would have been preferable, but as a proof of principle study, I think HeLa are acceptable, and I wouldn't expect the authors to repeat all the experiment in another system.
Figure 1,2 and S1 are describing and validating this approach in some detail, but this will require some more work.
The authors state that gene targeting was validated using a combination of PCR, sequencing, Western blotting, but show only the results for westerns. PCR analysis that demonstrates homozygous or heterozygous gene targeting should be shown here.
Another issue is the penetrance of the phenotypes induced by Rapamycin. The authors show nice data of the system working in individual cells but do not give us an idea if this happens in all cells. The localisation of the individual tagged genes should be quantified (ideally with line plots) in 50 randomly chosen mitotic cells with 3 repeats before and after rapamycin treatment. Moreover, the analysis of mitotic duration (Figure S1D) should be extended to include a plus Rapamycin cohort and this should be moved in the main Figure.
If the system works only in a small proportion of cells, this should be clearly stated. I don't think this would prevent publication, but it is an important piece of information that is missing.
The Reviewer raises two issues here.
- PCR analysis should be shown. This issue was also partly raised by Reviewer 1. A summary of our PCR analysis was actually included in Table 1, since the analysis we did is pretty unwieldy. We agree though that presenting our evidence for homozygosity of the cell lines would be useful. To address this point, we will add more detail of the PCR and sequencing work done to validate these cell lines.
- Does knocksideways happen in all cells? The answer to this depends on the transient expression of MitoTrap and sufficient application of rapamycin. We agree that this will be a useful piece of information to add to the manuscript. A related issue is whether knocksideways of complex members affects mitotic progression. We have established through other experiments that rapamycin application to wild-type cells alters mitotic progression, although application of Rapalog does not have this effect. Our plan to address these points is 1) to analyze the efficacy of knocksideways that readers can expect to achieve using these, or similar cells, and 2) analyze mitotic duration in rapalog-treated cells expressing a rapalog sensitive MitoTrap.
2)Apart from a simple quantification of mitotic duration, I believe a more detailed mitotic phenotype analysis for each knock-side way gene, especially the homozygous targeted clones, should be included. This can involve more high-resolution live cell imaging of mitotic progression with SiR-DNA and GFP-tubulin, using the dark mitotrap.
We don’t agree that such an analysis should be included. The focus of this paper is on using the knocksideways methodology to understand a protein complex during mitosis, and not looking at its function. There are several papers on the mitotic phenotypes of these genes probed using RNAi in different cellular systems (examples for chTOG: 10.1101/gad.245603; TACC3/clathrin: 10.1038/emboj.2011.15, 10.1242/jcs.075911, 10.1083/jcb.200911091, 10.1083/jcb.200911120; GTSE1: 10.1083/jcb.201606081). Moreover, our 2013 paper used knocksideways (with RNAi and overexpression) and has a detailed analysis of mitotic progression, microtubule stability, checkpoint activity and kinetochore motions (Cheeseman et al., 2013 doi: 10.1242/jcs.124834).
New experiments that are not part of the central message of the paper and are unlikely to give new insight are not the best use of our revision efforts for this paper (especially during the pandemic). Having said this, Reviewer 2’s suggestion to use our existing dataset to investigate mitotic phenotypes, will largely answer Reviewer 3’s request.
We will analyze spindle size parameters and also the intensity of tubulin. Our analysis will be limited to the short timeframe of our experiments, but it should reveal or refute any changes in spindle structure that result from the loss of complex members.
3)Overall, the quantitative analysis in Figure 3 ,4 and 7 is not good enough and sometimes doesn't fully support the conclusions. In Figure 3,4 a convoluted way of demonstrating the change in localisation is shown and this panel is so small that is almost impossible to read. Also, there is no statistical analysis, and the sample size seems very small . At least 25 cells should be analysed here in 3 repeats. I would suggest to unify the quantification in the MS and use the line plots shown in Figure 5 and 6 and compare each protein before and after rapamycin addition. This is much easier to read and more convincing. The images of the cells panels can be moved to a supplement as they contain very little information. This would generate space to expand the size and depth of the quantitative analysis. Instead of Anova tests, I would recommend using a simple t-test comparing each condition to its relevant control since this is the only relevant comparison in the experiment. Statistical significance should be calculated for each experiment with sufficient sample size. It would also be better to show the individual data points from the three repeats in different colours so that the reproducibility between repeat can be judged.
This type of statistical analysis should be uniformly done throughout the MS and also extended to Figure 7.
The referee raises several issues here with our data presentation and statistical analysis.
- Our aim in Figures 3 and 4 was to compress a lot of information into a small space, while still showing some example primary data. All reviewers raised the same concern about these figures which tells us that we went too far towards “data visualization”. To address this point, we will rework Figures 3 and 4 to provide more clear data presentation.
- The Reviewer’s comments about statistical analysis however are not sound. First, it is incorrect to state that simple t-tests can be applied (this is a form of p-hacking). Correction for multiple testing must be done on these datasets. Second, the reviewer arbitrarily states numbers for cells and experimental repeats without considering the effect size or it seems, understanding the structure of the data that we have collected. Sample sizes are small but they are taken from many independent replicates. Third, and related to the previous point, the fixed and live cell data are structured differently which means that a uniform data presentation is not possible. The live data has a paired design and each cell is an independent replicate (with replicates done over several trials). The fixed data is unpaired and we have taken measures from several experiments (independent replicates). The point about applying statistical tests to the data is also made by Reviewer 1 and we will use appropriate tests (NHST or estimation statistics) as we re-work the figures.
Reviewer #3 (Significance (Required)):
In my opinion, the most interesting aspect of the MS is the methodology. Based on this, publication is justified and will be of interest to a wider audience. That is why a more detailed analysis of the penetrance of this manipulation across the cell population will be critical.
The application of this method to analyse the composition of the TACC3/Clathrin complex on the spindle is the main biological advance, and the novel information is rather limited but not unimportant.
Overall, if these results can be properly quantified I would recommend publication.
