Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase

  1. Ana Pimenta-Marques
  2. Tania Perestrelo
  3. Patricia Rodrigues
  4. Paulo Duarte
  5. Mariana Lince-Faria
  6. Mónica Bettencourt-Dias

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Abstract

Centrioles play critical roles in our cells, being part of centrosomes and cilia, which are important microtubule organising centers (MTOC) with a variety of roles. While centrioles are very stable structures, they disappear in certain cell types upon differentiation, such as in oocytes. Little is known about the regulation of centriole structural integrity. We previously uncovered that the pericentriolar material (PCM), and its recruiter Polo kinase, are required for both the maintenance of centriole structural integrity and centrosome MTOC activity. Using an hypothesis driven RNAi screen, we show that both the cartwheel and the centriole wall play an important role in centrosome integrity. In particular, we uncovered that the centriole wall protein ANA1 is critical for the integrity of both new and mature centrioles, in Drosophila oogenesis as well as in cultured cells. Moreover, our results show that the activity of both Polo and the PCM in centriole integrity depends on ANA1. Our work suggests that the structural integrity of centrioles, once thought to be very stable organelles, depends on the turnover of key components, suggesting new perspectives for understanding the dysfunction of those structures in disease.

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  1. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The team explores a previously developed "centriole stability assay" to monitor the disappearance of centrioles after RNAi-dependent depletion of various centrosome components. Important roles in centriole stability are found for the PCM and for cartwheel proteins, in addition to proteins of the centriole wall. The remainder of the study focuses on the centriole wall protein ANA1: induced degradation of ANA1 during Drosophila oogenesis strongly reduces the PCM and other centriole markers, and ANA1-dependent defects cannot be prevented by GFP-Polo-PACT, which is otherwise known to protect from the loss of PCM. In complementary experiments, forced targeting of ANA1 to the PCM, or overexpression of AN1 protects centriole integrity.

    Significance

    The study shows that ANA1 is important for the integrity of centrosomes. Generally, this work is well executed and correctly controlled. The novelty of the results is somewhat limited, since a role of ANA1 in centrosome assembly has already been reported by others. The present work emphasizes aspects of centrosome protein maintenance, but doesn't provide mechanistic details of protein turnover. The manuscript should be of interest to the scientific community working on the centrosome.

    Other comments:

    I wonder whether the results from the centrosome maintenance experiment with GFP-Polo-PACT (Figure 3) are really very telling: since PCM and other centriole markers are lost upon ANA1-depletion, GFP-Polo-PACT cannot target to the PCM, and it is therefore unsurprising that GFP-Polo-PACT fails to provide its protective effect. Would expression of GFP-Polo-PACT prior to addition of ANA1-RNAi have a protective effect?

    Minor point:

    Figure 1H: it is unclear to me how centrioles are identified with the BLD10 marker in samples that have been treated with BLD10 RNAi.

    Referees cross-commenting

    I agree very much with reviewers 1 and 2 that a role of ANA1 "downstream" of the PCM is not really supported by the data.

    I also think that all other points raised in the reviews merit attention.

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper, the authors show that the turnover of centriole components is necessary for proper centriole maintenance within Drosophila cultured cells (during prologued cell cycle arrest) and within Drosophila oocytes, where centrioles are normally degraded prior to fertilisation. They highlight Ana1 as an important player in centriole maintenance. The authors begin with a candidate screen to identify core centriole proteins that are required to properly maintain centrioles. They then focus on Ana1, given that its depletion had the strongest effect, and show that its depletion leads to a reduction in the levels of centriole components in Drosophila oocytes. They show that the previously observed ability of centriole-targeted Polo to counteract centriole loss depends at least in part on Ana1 and that targeting Ana1 to centrioles also counteracts centriole loss. The authors conclude that Ana1 is a component of the PCM-promoted centriole integrity pathway.

    Major comments

    1. The authors say that Plk4 depletion does not lead to centriole loss, but there are significant differences in centriole number between the control and Plk4 depletion cells in Fig 1F and S1D. Please comment.
    2. One of the main results is that depletion of centriole components leads to a reduction in centrosome numbers when measured 8 days after S-phase arrest. I wonder whether a restriction of centriole duplication could add to this effect? Any cells that were in G2 or M phase when the drugs were added would presumably progress into the following S-phase and duplicate their inherited centrioles, but not if centriole duplication proteins had been depleted. It's true that Plk4 depletion leads to a relatively mild centriole loss phenotype, but can the authors be sure that this is not due to variations in the efficiency of different RNAi constructs? Perhaps the authors can show that Plk4 depletion efficiently prevents centriole duplication under otherwise normal conditions.
    3. The authors show that Ana1 depletion has the strongest effect, but this could in theory be due to differences in RNAi efficiency. I don't expect the authors to show the efficiency of all RNAi constructs, but they could state in the text that this is a caveat e.g. "...although we cannot rule out the possibility that differences in RNAi efficiency lead to the observed differences in severity of phenotype..."
    4. A key conclusion is that core centriole components turnover to some extent and that the incorporation of new molecules is necessary for centriole maintenance. This is a very interesting and important point and so it would be nice to have more direct data to support it. This could be done in different ways, including transfecting fluorescently tagged centriole components after S-phase arrest and showing that some molecules become incorporated into the centrioles, or by performing FRAP experiments. Of course, it is possible that the turnover is so low that the incorporated fluorescent molecules cannot be detected...
    5. The authors show that depletion of Ana1 from oocytes leads to a reduction in the intensity of centriole markers. They do not measure centrosome numbers, as the centrosomes cluster too tightly. The authors therefore can't be certain that Ana1 depletion leads to a reduction in centrosome numbers. The authors could show this by inhibiting centrosome clustering while depleting Ana1. There is a recent BioRxiv paper showing that centrosome clustering can be inhibited by depletion of Kinesin-1.
    6. In Figure 3B the authors show that expression of GFP-Polo-PACT partially rescues the effect of "all PCM" depletion, but this seems strange given that Polo's role is presumably to recruit PCM (which has been depleted). Can the authors comment? Also, it would make sense to test whether GFP-Polo-PACT can rescue centriole loss after the depletion of Ana1 alone (not Ana1 and all PCM). If Ana1 has a role in recruiting Polo (either directly or indirectly), which has been shown previously in mitotic cells, then there should be a rescue to some extent.
    7. In Fig4A,C, the authors say that γ-tubulin levels at centrosomes increase when GFP-Polo is forced onto the centrosomes - the graph seems to show a big increase, but the pictures do not...? Are the authors measuring total levels at all centrosomes? If so, I think they should be measuring the average at individual centrosomes. Also, why is the level of GFP alone not much higher when expressed with GFPnanoPACT (Fig 1B)? Presumably GFP should be recruited to the centrosomes by GFPnanoPACT.
    8. The authors show that tethering Ana1-GFP to the centrioles counteracts centriole loss in oocytes (Fig4G). They say that the centrosomes are most likely inactive because they don't recruit PCM, but they have only looked at γ-tubulin, which is a downstream component of the PCM. I think it is important to check whether Polo is recruited, given that tethering Polo to centrioles also counteracts centriole loss and that a recent paper showed that Ana1 has a role in recruiting Polo to centrosomes (Alvarez-Rodigo et al., 2021). The authors also say that these centrosomes do not organise microtubules but do not show the data.
    9. The authors propose that Ana1 is downstream of the PCM, and so over-expressing Ana1 should at least partially rescue centriole loss after PCM depletion. But I don't really agree with this. If Ana1 relies on the PCM then how would its overexpression manage to rescue the phenotype in the absence of the PCM? The finding that over-expressing Ana1 partially rescues centriole loss may instead suggest that Ana1 is either upstream of the PCM or part of an independent pathway. Indeed, the authors show that depletion of both the PCM and Ana1 has a stronger effect than either depletions individually - this is indicative of two independent pathways.

    Minor comments

    1. When the authors say that the centriole wall and cartwheel components are "dynamic" I think that they need to make it clear that this "dynamicity" is not very fast. Using the term dynamic tends to suggest rapid turnover (like in the PCM). Perhaps the authors could use the term "slow exchange" or something similar.
    2. The authors currently use a 0 or 1 centriole categorisation - it would be nice to see the breakdown of what percentage of cells have 0, 1, 2, or >2 centrioles, perhaps in a supplementary excel file.

    Significance

    How centrioles are eliminated in certain cells is an interesting question and the data presented is also relevant to understanding centriole biology in general, because it seems that some apparently very stable structural proteins actually turnover. It is widely known that PCM proteins turnover relatively quickly, but core centriole proteins are considered to be stably incorporated. The data will therefore raise interest in the centrosome field. I do, however, feel that for the authors to make this point more strongly it would be good to show this more directly. Overall, this is a very interesting paper that is well written. The data is well presented and supports the conclusions that centriole components turnover and that Ana1 is involved in maintaining centriole integrity.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript Pimenta-Marques build on their previous work addressing how centrioles are stabilized and maintained or destabilized and disassembled, depending on the cell type and developmental context. Using Drosophila cell culture and oogenesis as an in vivo model for centriole destabilization, they identify the centriole wall protein Ana1 as a central player in centriole stability. Its presence is required for the maintenance even of mature centrioles, suggesting that there is continued turnover of centriole structural components.

    Major comments:

    1. The experiments and results are very well described and most of the conclusions are supported by the data. One aspect needs clarification though. It is not clear to this reviewer how the authors envision the regulation and mechanism by which Ana1 functions in centriole stability. The data suggest that it can stabilize centrioles independent of PCM (Fig. 3B, 5B), yet the authors claim in the results and discussion that it functions downstream of PCM. As presented, this does not make sense. I would argue the opposite, it may function upstream or in parallel to the PCM. Related to the above, the last sentence of the intro states: "Finally, we found that both Polo and the PCM require ANA1 to promote centriole structural integrity." This is shown for Polo, but where is the data showing that PCM requires ANA1 for promoting centriole stability?
    2. I have a concern regarding the number n used for statistics in the quantifications. In many cases it seems that the number n of cells etc. was used (e.g. n>100 cells) rather than the number of experiments (e.g. n=3). The statistics should measure variability between experimental repetitions, not between cells etc. If statistics were indeed not done on experiments and would have to be changed, some of the observed effects may not be statistically significant and would require additional experimental replicates, which would increase the time needed for revision.

    Minor comments:

    1. I would advice the authors to improve the presentation of the figures. In particular the labels are in many cases very small and difficult to read. Readability is also reduced by the use of bold font in the labels and a mix of various font sizes within single figure panels.
    2. The result section could be shortened/become more readable by moving several paragraphs to the intro or discussion.
    3. The introduction is quite long and some parts read more like an introduction of a review on the topic.

    Significance

    This is a nice, focused study on the requirements underlying centriole stability and maintenance. The first part identifies the cartwheel, the centriole wall, and the PCM as important for centriole maintenance. The remaining parts identify and focus on the essential role of ANA1 in this process. This is an important finding, since the mechanisms underlying centriole stability and maintenance are poorly understood, yet highly relevant. Some cell types inactivate and/or disassemble centrioles during differentiation and this is likely important to their function. Providing more mechanistic insight, for example, regarding the relationship between ANA1 and PCM recruitment or the regulation of ANA1's centriole function by Polo, would have further strengthened the study. The audience interested in this work will be cell and developmental biologists. My expertise is in centrosome biology and microtubule organization.

    Referees cross-commenting

    I agree with the additional points raised by the other reviewers. I still think that overall the paper is fine and most things could be addressed in a reasonable time frame. The work does not provide much mechanism though. In this regard, the confusing placement of ANA1 downstream of PCM, would be the only mechanistic aspect, and it seems the authors got it wrong, at least based on the provided data. Here, additional experiments could elucidate these relationships further, but if this is not the goal, text changes could also address this and it would remain a smaller, more focused study.

  5. Version published to 10.1101/2022.04.06.487296v1 on bioRxiv