Cdk/Cyclin activity helps set mitotic centrosome size by influencing the centrosome growth rate and growth period

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Mitotic centrosomes assemble when centrioles recruit large amounts of pericentriolar material (PCM) around themselves in preparation for cell division. How the mitotic PCM grows to the correct size is unclear. In Drosophila syncytial embryos, thousands of mitotic centrosomes assemble in a common cytoplasm as the embryo proceeds through 13 rounds of near-synchronous nuclear division. During nuclear cycles (NCs) 11-13 these divisions gradually slow, and we find that mitotic centrosomes respond by reciprocally slowing their growth rate and increasing their growth period so that they grow to a consistent size at each cycle. This size homeostasis is enforced, at least in part, by the Cdk/Cyclin cell cycle oscillator (CCO). Moderate levels of CCO activity appear to initially promote centrosome growth by stimulating Polo/PLK1 recruitment to centrosomes, while higher levels of activity subsequently inhibit centrosome growth by phosphorylating centrosome proteins to decrease their centrosomal recruitment and/or maintenance as the embryos enter mitosis. Thus, the CCO initially promotes, and subsequently restricts, mitotic centrosome growth to help ensure that centrosomes grow to a consistent size.

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    Reply to the reviewers

    We thank the Reviewers for their helpful and constructive comments. In response to these suggestions we have performed new experiments and amended the manuscript, as we describe in our detailed response below.

    Reviewer #1:

    1. The Reviewer notes that while our analysis of centrosome size was comprehensive, we provided no analysis of centrosomal MTs, pointing out that while centrosome size declines as the embryos enter mitosis, the ability of centrosomes to organise MTs might not. This is a good point, and we now provide an analysis of centrosomal-MT behaviour (Figure 2). We find that there is a dramatic decline in centrosomal MT fluorescence at NEB, although the pattern of centrosomal MT recruitment prior to NEB is surprisingly complex.

    2. The Reviewer questions how PCM client proteins can be recruited in different ways by the same Cdk/Cyclin oscillator. We apologise for not explaining this properly. It is widely accepted that Cdk/Cyclins drive cell cycle progression, in part, by phosphorylating different substrates at different activity thresholds (e.g. Coudreuse and Nurse, Nature, 2010; Swaffer et al., Cell, 2016). Moreover, it is also clear that Cdk/Cyclins can phosphorylate the same protein at different sites at different activity thresholds (e.g. Koivomagi et al., Nature, 2011; Asafa et al., Curr. Biol., 2022; Ord et al., Nat. Struct. Mol. Biol., 2019). Thus, we hypothesise that rising Cdk/Cyclin cell cycle oscillator (CCO) activity phosphorylates multiple proteins at different times and/or at different sites to generate the complicated kinetics of centrosome growth. We now explain this point more clearly throughout the manuscript.

    3. The Reviewer is puzzled as to how we conclude that Cdk/Cyclins phosphorylate Spd-2 and Cnn at all the potential Cdk/Cyclin phosphorylation sites we mutate in our study. The Reviewer is right that we cannot make this conclusion, and we did not intend to make this claim. As we now clarify (p11, para.1), although it is unclear if Cdk/Cyclins phosphorylate Spd-2 or Cnn on all, some, or none of these sites, if either protein can be phosphorylated by Cdk/Cyclins, then these mutants should not be able to be phosphorylated in this way—allowing us to address the potential significance of any such phosphorylation. We now also note that several of these sites have been shown to be phosphorylated in embryos in Mass Spectroscopy screens (Figure S6).

    4. The Reviewer highlights differences in how Spd-2 and Cnn help recruit γ-tubulin to centrosomes (Figure 6). They ask for a more detailed description, and are puzzled as to how this is compatible with direct regulation by a single oscillator. We now explain our thinking on this important point in much more detail. It appears that Spd-2 helps recruit γ-tubulin throughout S-phase, while Cnn has a more prominent role in late S-phase (Figure 6). This is consistent with our overall hypothesis of CCO regulation, as we postulate that low-level CCO activity promotes the Spd-2/γ-tubulin interaction in early S-phase, while higher CCO activity promotes the Cnn/γ-tubulin interaction in late-S-phase, potentially explaining the increase in the rate of γ-tubulin (but not γ-TuRC) recruitment we observe at this point (see minor comment #1, below, for an explanation of the various γ-tubulin complexes in flies). This is consistent with recent literature showing that CCO activity promotes γ-tubulin (but not γ-TuRC) recruitment by Cnn/SPD-5 in worms and flies (Ohta et al., 2021; Tovey et al., 2021).

    5. The Reviewer was not convinced by our model (Figure 8, now Figure 9), raising two major concerns. First, they were unsure how a single oscillator could generate different patterns of protein recruitment. We addressed this in point #2 and #4, above, where we explain how different thresholds of CCO activity trigger different events, so there is no expectation that we should observe steady changes in recruitment over time as CCO activity rises. Second, they questioned how modest levels of Cdk/Cyclin activity can promote recruitment, while high levels of activity can inhibit recruitment. In point #1, above, we cite several examples where such positive and negative regulation by different Cdk/Cyclin activity levels have been described. We also now explain throughout the manuscript why this hypothesis provides a plausible explanation for our results: with moderate CCO activity promoting Spd-2-dependent PCM-client recruitment in early S-phase; higher CCO activity promoting a decrease in Spd-2 recruitment in mid-late-S-phase (so centrosomal Spd-2 levels decline); and even higher levels of CCO activity leading to a decrease in the interactions between the client proteins and the Spd-2/Cnn scaffold as the embryos enter mitosis (so the client proteins are rapidly released from the centrosome).

    The Reviewer also raised the important point here that our model does not explain why the mutant forms of Spd-2 and Cnn accumulate to higher levels at the start of S-phase, and not just at the end of S-phase/entry into mitosis. We apologise for not explaining this properly. The accumulation of the mutant proteins (particularly Spd-2, Figure 5C) in early-S-phase occurs because the excess mutant protein that accumulates at centrosomes in _late-_S-phase/mitosis is not removed properly from centrosomes during mitosis (presumably because there is insufficient time). Thus, centrosomes still have too much mutant Spd-2 at the start of the next S-phase. We show this in Reviewer Figure 1 (attached to this letter), which tracks Spd-2 behaviour further into mitosis, and now explain this in more detail in the text (p12, para.1).

    1. The Reviewer questions how the CCO can both induce centrosome growth and also switch it off, as it is unclear how an oscillator that only phosphorylates sites to decrease centrosome binding could also promote growth. They ask if we can identify and mutate any Cdk/Cyclin sites in centrosome proteins that promote centrosome recruitment. As we now clarify, we did not intend to claim that the CCO only phosphorylates sites that decrease the centrosome binding of proteins, although we do hypothesise that such phosphorylation is important for switching off centrosome growth in mitosis. In addition, we hypothesise that moderate levels of CCO initially promote centrosome growth, and our data suggests that the CCO does this, at least in part, by promoting Polo recruitment (Figure 8). We speculate that the CCO phosphorylates specific Polo-box-binding sites in Ana1 and Spd-2, the main proteins that recruit Polo to centrioles. We agree that identifying these sites is an important next step, but it is complicated as our studies indicate that multiple sites contribute in a complex manner. Importantly, it is well established that the CCO triggers centrosome growth as cells prepare to enter mitosis, so our hypothesis that moderate levels of CCO activity initiate centrosome growth is not new or controversial.

    Minor Comments

    1. The reviewer asks how we explain the different incorporation profiles we observe for the different subunits of the γ-tubulin ring complex. We apologise for not discussing this point. In flies there is a “core” γ-tubulin-small complex (γ-TuSC) and a larger γ-tubulin-ring complex (γ-TuRC) that contains the Grip71, Grip75 and Grip128 subunits we analyse here (Oegema et al., JCB, 1999). The γ-TuSC functions independently of the γ-TuRC so γ-tubulin and γ-TuRC components can behave differently.

    2. The Reviewer questions why we claim an “inverse-linear” relationship between S-phase length and the centrosome growth rate when the relationship is not linear (Figure 3, now Figure S3). I was originally confused by this as well but, mathematically, a linear relationship means y is proportional to x, whereas an inverse-linear relationship means y is proportional to 1/x. Thus, an inverse-linear relationship between x and y does not plot as a straight line, but rather as the curves we show on the graphs. We now explain this in text (p9, para.2).

    Reviewer #2:

    This Reviewer found the manuscript hard to follow, so we are very grateful that they took the time to try to understand it. We agree that the subject matter is complicated, and that our presentation was not always helpful. The Reviewer’s comments have been very useful in helping us to identify (and hopefully improve) areas of particular difficulty.

    Major points:

    1. The Reviewer highlights that the two experimental approaches underpinning our main conclusions are problematic: (1) Experiments with mutants of Spd-2 and Cnn that theoretically cannot be phosphorylated by Cdk/Cyclins are hard to interpret as these mutations may have other effects; (2) It is unclear whether reducing Cyclin B levels reduces peak CDK activity or simply slows the time it takes to reach peak levels. They suggest a more direct test of our model would be to analyse PCM recruitment in embryos arrested in S-phase or mitosis. (1) We agree that the mutations designed to prevent Cdk/Cyclin phosphorylation could perturb function in other ways, but this is true for any such mutation, and there are many papers that infer a function for Cdk/Cyclin phosphorylation from such experiments. Importantly, the centrosomal accumulation of the phospho-null mutants actually slightly increases compared to WT (Figure 5C and I), and we now show that the centrosomal accumulation of a phosphomimicking Spd-2-Cdk20E mutant slightly decreases (Figure S8). We now acknowledge the potential caveat of a non-specific perturbation of protein function, but feel that the reciprocal behaviour of the phospho-null and phospho-mimicking mutants somewhat mitigates this concern (p12, para.2). (2) Fortunately, and as we now clarify, it has recently been shown that reducing Cyclin levels does not reduce peak Cdk activity, but rather slows the time it takes to reach peak activity (Figure 2A, Hayden et al., Curr. Biol., 2022). Thus, the cyclin half-dose experiments provide an excellent alternative test of our hypothesis as they show that the WT proteins can exhibit similar behaviour to the mutants if the rate of Cdk/Cyclin activation is slowed. We feel the evidence supporting our hypothesis is strong enough that it warrants serious consideration.

    The suggestion to look at PCM recruitment in embryos arrested in either S-phase or M-phase is a good one, but these experiments produce complicated data. In M-phase arrested embryos, for example, Cnn levels continue to rise (see Figure 1G, Conduit et al., Dev. Cell, 2014), but the other PCM proteins do not (unpublished); in S-phase arrested embryos (arrested by mitotic cyclin depletion) centrosomes continue to duplicate, but now do so asynchronously, greatly complicating the analysis (McCleland and O’Farrell, Curr. Biol.., 2008; Aydogan et al., Cell, 2020). The centrosomes that don’t duplicate, however, reach a constant steady-state size (where the rate of centrosome protein addition is balanced by the rate of loss). These observations are consistent with our recent mathematical modelling of mitotic PCM assembly (Wong et al., 2022) if we additionally account for cell cycle regulation (which was not considered in our original model). We believe such analyses are beyond the scope of the current paper and we plan to publish a second paper incorporating our new hypothesis into our mathematical modelling.

    1. The Reviewer questions whether our methods accurately measure centrosomal protein accumulation, pointing out that γ-tubulin and Grip128 occupy different centrosomal areas—which should not be possible if they are part of the same complex. They suspect that our use of different transgenes with different promotors could explain these differences. As we should have described (see point #1 in our response to the minor comments of Reviewer #1), γ-tubulin exists in two complexes in flies, only one of which contains Grip128, so γ-tubulin and Grip128 exhibit different localisations. Moreover, as we now show (Figure S2), using different promotors does not seem to make a difference to overall recruitment kinetics. Thus, we are confident that our methods measure centrosome protein recruitment dynamics accurately.

    2. The Reviewer is concerned that our measurements of centrosome size based on fluorescence intensity (Figure 1) and centrosomal area (Figure S1) do not always match. They suggest a potential reason for this is that proteins are not uniformly distributed within centrosomes, and this may impact our ability to measure protein accumulation based on 2D projections (noting, for example, that Polo and Spd-2 are concentrated at centrioles and in the PCM, potentially explaining the different shape of their growth curves compared to the client proteins). When the centrosome-fluorescence-intensity and centrosome-area recruitment profiles of a protein do not match, the average “centrosome-density” of that protein must be changing over time. In some cases, we understand why density changes. Cnn, for example, stops flaring outwards on the centrosomal MTs during mitosis so its centrosomal area decreases even as its fluorescence intensity increases (leading to an increase in its centrosomal-density). We agree (and now discuss—p19, para.3) that the prominent accumulation of Spd-2 and Polo at centrioles could help to explain why Spd-2 and Polo accumulation dynamics differ from the client proteins.

    Other points:

    1. The Reviewer suggests it would be good to know how much Polo at the centrosome is active****. We agree, but although commercial antibodies against PLK1 phosphorylated in its activation loop work in cultured fly cells, we cannot get them to work in embryos. Moreover, the recruitment of Polo/PLK1 to its site of action by its Polo-Box Domain is sufficient to partially activate the kinase independently of phosphorylation (Xu et al., NSMB, 2013). Thus, it seems likely that all the Polo/PLK1 recruited to centrosomes will be at least partially activated, even if it is not necessarily phosphorylated on its activation loop.

    2. The Reviewer asks if it is clear that less Spd-2 and Cnn are recruited to centrosomes in the half gene-dosage embryos. We apologise for not mentioning that this is indeed the case. We showed this previously for Cnn (Conduit et al., Curr. Biol., 2010) and we now state that this is also the case for Spd-2. We do not show the Spd-2 data as we plan to publish a comprehensive dose-response curve of Spd-2 (and Cnn) recruitment in our next modelling paper.

    3. Would it not be relevant to examine Polo ½ dosage embryos? We do have this data (Reviewer Figure 2), attached to this letter, but it is quite complicated to interpret (as we explain in the legend). We feel it would be more appropriate to include this in our next modelling paper where we can properly explain the behaviours we observe. Publishing this data here would distract from our main message without changing any of our conclusions.

    4. The Reviewer asks why the non-phosphorylatable Spd-2 protein is also present at higher levels on centrosomes at the start of S-phase (not just the end of S-phase). This was also raised by Reviewer #1 (point #5), so please see the second paragraph of our response there.

    Minor/Discussion Points:

    1. We thank the Reviewer for highlighting that absolute and relative centrosome size control are different things and we have amended the manuscript accordingly.

    2. The Reviewer questions whether it is accurate to describe Spd-2 and Polo as scaffold proteins, noting that only Cnn has been shown to have scaffolding properties. There is strong evidence that Spd-2 has Cnn-independent scaffolding properties in flies (e.g. Conduit et al., eLife, 2014), but this is a fair point for Polo. We think it is justified to separate Polo from other client proteins as Polo is essential for scaffold assembly, whereas other client proteins are not. We now define our scaffold/client terminology to avoid confusion (p4, para.3).

    3. The Reviewer highlights several points related to differences in recruitment kinetics (also touched on in points #2 and #3, above), noting we don’t discuss properly the idea of two different modes of PCM recruitment. These are all good points, largely addressed in our response to points #2 and #3, above. We now discuss much more prominently the two different modes of client protein recruitment throughout the manuscript.

    4. As we now clarify, in all our experiments we use centrosome separation and nuclear envelope breakdown (NEB) to define the start and end of S-phase, respectively.

    5. The Reviewer quotes the landmark Woodruff paper (Cell, 2017) as showing that the ability to concentrate client proteins (including ZYG-9, the worm homologue of Msps) is an intrinsic property of the PCM scaffold, so how do we explain that Msps departs prior to NEB while Cnn continues to accumulate? It is indeed a striking observation of our study that all PCM client proteins (not just Msps) start to leave the centrosome prior to NEB, even as Cnn levels continue to accumulate. Our hypothesis is that this ‘leaving’ event is triggered by a threshold level of Cdk/Cyclin activity—explaining why these client proteins all start to leave the PCM at the same time (just prior to NEB) irrespective of nuclear cycle length. This is not incompatible with the Woodruff paper, which did not attempt to reconstitute any potential regulation by Cdk/Cyclins in their in vitro studies.

    6. The Reviewer questions why Spd-2 that cannot be phosphorylated by Cdk/Cyclins (Spd-2-Cdk20A) accumulates abnormally at centrosomes in late S-phase, yet γ-tubulin (which is recruited by Spd-2) seems to leave centrosomes more slowly in the presence of the mutant protein. As we now explain more clearly, there is no contradiction here. Spd-2-Cdk20A accumulates to abnormally high levels in late-S-phase/early mitosis (Figure 5C), and this reduces the γ-tubulin dissociation rate, as we would predict (Figure 7B, right most graph). It does not “prevent” dissociation, however, (as the Reviewer seems to suggest it should?), but this is probably because these experiments have to be performed in the presence of large amounts of the WT Spd-2 (Figure 5A).

    7. The referencing error has been corrected.

    8. The Reviewer asks why in Figure 1 not all of the centrosome proteins could be followed for the full time period (as we mention in the legend, but do not explain). There are different reasons for different proteins: (1) Polo cannot be followed in mitosis as it binds to the kinetochores, making it impossible to accurately track centrosomes (so the data for mitosis is missing for Polo); (2) Cnn exhibits extensive flaring at the end of mitosis/early S-phase (Megraw et al., JCS, 1999), so we cannot track individual separating centrosomes labelled with NG-Cnn in early S-phase until they have moved sufficiently far-apart (so the early S-phase time-points are missing for Cnn); (3) In addition, several of the client proteins bind to the mitotic spindle, so although we can still track and measure the centrosomes in late mitosis in the graphs, we don’t show pictures of these late mitosis centrosomes in the montage in Figure 1A as the images look a bit odd. We now explain these reasons in the Materials and Methods.

    9. We now indicate that nuclear cycle 12 (NC12) is being analysed in Figures 4-8.

    10. The reviewer questions why we don’t show the decrease rate for γ-tubulin in Figure 6 (the Spd-2 and Cnn half-dose experiments), when we do show it in Figure 7 (the Spd-2 and Cnn Cdk-mutant experiments), suspecting that it is slowed in both cases. The reviewer is correct and we now show this data for both sets of experiments.

    11. We have corrected the labelling error in Figure S1.

    12. The Reviewer suggest moving some of the data from the main Figures, and the entirety of Figures 2 and 3 to the Supplemental Information. We understand this point, and agree that the amount of data presented in Figures 1-3 is somewhat overwhelming. We have played around with the Figures a lot—in particular trying to show a few examples of the data and moving the rest to Supplementary—but it is hard to pick a “typical” example, and the power of comparing the behaviour of so many different centrosome proteins is somewhat lost. We have tidied up several Figures and, as a compromise, we keep Figure 2 (now Figure 3) in the main text, but have moved Figure 3 to Supplementary (now Figure S5).

    13. The Reviewer suggests that we should repeat the analysis of Spd-2, Polo and Cnn dynamics that we show here, as we already presented this data in a previous publication (Wong et al., EMBO. J, 2022). We understand this point, but feel this would be a less accurate comparison, as essentially all of the data shown in Figure 1 was obtained several years ago during a contiguous ~6month period. Since then, the lasers and software on our microscope system have been updated, so it would probably be less fair of a comparison to obtain new data for a subset of these proteins (and it seems overkill to perform the entire analysis again). We clearly state that this data has been presented previously, so we hope the Reviewer will agree that it is acceptable to present it again here so readers can more easily compare the data.

    Reviewer #3:

    This Reviewer is broadly supportive of the manuscript, but to publish in a prestigious journal they think additional experimental evidence will be required to support our hypothesis.

    The Reviewer notes that our only evidence that Cdk/Cyclins directly phosphorylate Spd-2 comes from our analysis of the Spd-2-Cdk20A mutant, as the effect of reducing Cyclin B dosage on WT Spd-2 behaviour is very modest. They request that we analyse the behaviour of a Spd-2-Cdk20E phospho-mimicking mutant. The effect of halving the dose of Cyclin B on Spd-2 behaviour is modest, but this is what we would predict as all we are doing in this experiment is slowing S-phase by ~15%, so Spd-2 should accumulate at centrosomes for a slightly longer time and to a slightly higher level (as we observe, Figure 5E). A great advantage of the early fly embryo system is that we can compare the behaviour of many hundreds of centrosomes, so even subtle differences like this are usually meaningful. To illustrate this point, we have now repeated the Spd-2 analysis in WT and CycB1/2 embryos (but now using a CRISPR/Cas9 Spd-2-NG knock-in line) and we see the same subtle differences (Figure S9). In addition, as requested, we have now analysed the behaviour of a Spd-2Cdk20E mutant protein using an mRNA injection assay (as it would have taken too long to generate and test new transgenic lines). In this assay we injected embryos with mRNA encoding either WT Spd-2-GFP, Spd-2-Cdk20A-GFP or Spd-2-Cdk20E-GFP. The mRNA is quickly translated, and we computationally measured the fluorescence intensity of the centrosomes in mid-S-phase (i.e. at the Spd-2 peak) (Figure S8). This analysis confirms that Cdk20A accumulates to slightly higher levels, and reveals that Cdk20E accumulates to slightly lower levels, than the WT protein. Together, these new experiments strongly support our original conclusions.

    The Reviewer notes that we propose that the CCO initially promotes centrosome growth by stimulating Polo recruitment to centrosomes, but states that we only provide indirect evidence for this by showing that centrosomal Polo levels are strongly reduced in Cyclin B half-dose embryos. They suggest we determine Spd-2 levels in Polo half-dose embryos, and/or the centrosome levels of mutant forms of Spd-2 that cannot be phosphorylated by Polo. We believe the Cyclin B half-dose experiment provide direct support for our hypothesis that Cdk/Cyclin activity influences Polo recruitment (Figure 8), although, clearly, we have not identified the mechanism. We do, however, suggest a plausible mechanism: Ana1 and Spd-2 are largely responsible for recruiting Polo to centrosomes, and we have previously shown that several of the potential phosphorylation sites in these proteins that help recruit Polo to centrosomes are Cdk/Cyclin or Polo phosphorylation sites (Alvarez-Rodrigo et al., eLife, 2020 and JCS, 2021; Wong et al., EMBO J., 2022). We are currently testing this hypothesis, but progress is slow as it is clear that multiple sites in both proteins can influence this process.

    As the Reviewer requests, we have now also examined how Spd-2 and Cnn behave in Polo half-dose embryos (Reviewer Figure 2, attached to this letter). As we describe in the Figure legend, this data is informative, but is complicated. With relatively minor, but mechanistically important, tweaks to our previous mathematical modelling we can explain these behaviours, but introducing such a significant mathematical modelling element would be beyond the scope of this paper. As described above, these findings will form the basis of a follow-up paper that is more mathematically oriented.

    It is a great idea to look at mutant forms of Spd-2 that cannot be phosphorylated by Polo, but the consensus Polo phosphorylation site (N/D/E-X-S, with the N/D/E at -2 and the S at 0 being preferences, rather than a strict rule) is less well-defined than the consensus Cdk/Cyclin phosphorylation site (where the Pro at -1 is essentially invariant). Thus, we cannot accurately predict which sites would need to be mutated to generate such a mutant.

    The Reviewer requests that we analyse the behaviour of TACC in embryos expressing the Spd-2-Cdk20A and Cnn-Cdk6A (as we do in Figure 7 for γ-tubulin). This is a reasonable request, but we prefer not to show this data as we have recently identified an interesting interaction between TACC, Spd-2 and Aurora A that will be the subject of another paper we hope to submit shortly. This data is hard to interpret without explaining these interactions properly, which is beyond the scope of the current manuscript.

    We hope the Reviewers will agree that these changes have improved the manuscript substantially, and that it is now suitable for publication. We would like to thank them again for taking the time to read this rather complicated paper so thoroughly.

    We look forward to hearing from you.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors investigated growth control of PCM at the mitotic centrosomes in late stages of the Drosophila syncytial embryos. They observed that mitotic centrosomes reach to the correct sizes through 13 rounds of nuclear division by reciprocally slowing their growth rate and increasing their growth period. They assumed that the Cdk/Cyclin cell cycle oscillator (CCO) is a main controller, based on their previous works (Aydogan et al., 2018, 2020; 2022). They determined the recruitment dynamics of the key mitotic PCM scaffolding proteins (Spd-2, Polo and Cnn) and PCM-client proteins (γ-tubulin, Msps, TACC, GFP, Grip75, Grip128 and Aurora A) in living embryos, and proposed that moderate levels of the CCO activity promote centrosome growth by stimulating Polo recruitment to centrosomes, while higher levels of activity subsequently inhibit centrosome growth by phosphorylating centrosome proteins, such as Spd-2, to decrease their centrosome recruitment and/or maintenance as the embryos enter mitosis.

    Experiments were cleverly designed and carefully executed. The results are nicely presented, the manuscript is clearly written, and their proposal draws a strong attention. However, in order to publish the manuscript in a prestigious journal, the authors may provide additional experimental evidence to support their proposal.

    • It is very significant that the centrosome levels of Spd-2-Cdk20A-NG is stronger than Spd-2-NG throughout the cell cycle (Figure 5B,C). However, this is only an experimental evidence to support that Cdk/Cyclins directly phosphorylate Spd-2 in the run-up to mitosis to help reduce Spd-2's centrosome recruitment and/or maintenance. As the authors confessed, recruitment of Spd-2-NG to the centrosomes in CycB1/2 embryos (Figure 5D,E) may be moderate or not significant at least in this reviewer's eyes. It is worth to perform the same experiments with a phospho-mimetic Spd2-Cdk20E-NG mutant.
    • The authors proposed that moderate levels of CCO activity promote centrosome growth by stimulating Polo recruitment to centrosomes. They provided an indirect evidence that centrosome levels of polo were strongly reduced in CycB1/2 embryos (Figure 4E,F). It is worth to determine the centrosome levels of Spd-2 in the Polo1/2 embryos and/or the centrosome levels of Polo phospho-resistant Spd-2 (Spd-2-Polo#A-NG).
    • TACC may be an ideal PCM-client protein, apart from its importance in spindle formation in comparison to γ-tubulin (Figure 4C,D). Therefore, it is worth to perform the Figure 7 experiments with TACC.


    Experiments were cleverly designed and carefully executed. The results are nicely presented, the manuscript is clearly written, and their proposal draws a strong attention. However, in order to publish the manuscript in a prestigious journal, the authors may provide additional experimental evidence to support their proposal.

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    Referee #2

    Evidence, reproducibility and clarity

    Control of organelle size has been an active field of research for many years for a large variety of cellular structures and in a range of experimental models. Here, Jordan Raff and colleagues examine the mechanisms underlying centrosome (PCM) size control in Drosophila syncytial embryos, building on their previous work (Wong, EMBOJ 2022) to propose a role for CDK in both promoting (at intermediate levels) and inhibiting (at high levels) PCM expansion.

    I found this a difficult manuscript to review, not only because the subject matter is complicated, but so is the writing. Having read and re-read the manuscript some clarity eventually emerges, but it shouldn't be that inaccessible. As for the authors' model I find it intriguing, but not fully supported by the data currently presented.

    Major points

    1. Central to the authors' model is the proposed dual function of Cdk (or CCO in the authors' terminology) in both promoting and inhibiting centrosomal protein accumulation. This the authors test by reducing the gene dosage of cyclin B and using putatively non-phosphorylatable versions of Spd-2 and Cnn. Both approaches to me appear quite problematic. The latter perturbation is hard to interpret given that whether these are indeed Cdk phosphosites that they have mutated is unknown and there are plenty of other possibilities how this might perturb protein function, as the authors' lack of success doing the same for gamma-tubulin illustrates. The former perturbation also lacks context. Does reducing cyclin B gene dosage reduce peak CDK activity or does it merely take longer to reach the same maximum, as appears to occur naturally as the cell cycle slows between embryonic cycles 11 and 13 (Edgar, Genes Dev 1994)? A more direct way to test their model would be to arrest the embryo in S phase (which in their model should lead to indefinite growth) or mitosis using suitable drugs/genetic perturbations. Is this not feasible in the fly system?
    2. Similarly critical is that centrosomal protein accumulation is accurately measured. I am not entirely convinced that this is so. If one takes their estimations of centrosome size at face value, then the space occupied by gamma-tubulin (slightly over 1 µm2 peak area according to Fig. S3) is significantly smaller than that occupied by Grip128 (4µm2). How is this possible if these form part of the same gamma-tubulin complex? This likely reflects the fact that the dynamics of many proteins is being assessed using transgenic reporters under the control of heterologous regulatory sequences (not all of which are fully functional, eg Polo), which could result in wildly inappropriate centrosomal protein levels. It may then not be a coincidence that the centrosomal domain for Grip128 (endogenously tagged) is larger than that for gamma-tubulin (transgene).
    3. Another concern is that centrosome size and integated signal intensity do not always match, as demonstrated by Grip71 (increasing as expected during centrosome maturation in cycle 13 based on fluorescence intensity but not area
    • compare Figs. 1B and S1). A potential reason for this is that proteins are not uniformly distributed within centrosomes. For example, Polo and Spd2 are highly concentrated at centrioles. This impacts the ability to accurately measure protein accumulation based on 2D projections. Such inaccuracies likely will not affect estimation of when peak protein accumulation occurs, but may explain apparent differences in the kinetics of recruitment/dissociation of different components. Thus, the differences in the shape of the PCM client growth curves compared to those of Polo and Spd-2 (p6) may simply reflect the centriole concentration of the latter.

    Other points

    1. In C. elegans much of Polo at centrosomes is apparently inactive, particularly in the vicinity of centrioles (Cabral, Dev Cell 2019). Knowing whether this is also the case in flies would seem like important information to have, particularly when comparing signal intensities across the cell cycle.
    2. Is it clear that there is less Spd2/Cnn at centrosomes in Spd-2/Cnn 1/2 gene dosage embryos, as the authors assume?
    3. Would it not be relevant to also examine Polo 1/2 dosage embryos?
    4. Based on the authors model, Cdk phosphorylation first drives PCM accumulation, then at higher levels inhibits. Yet, their non-phosphorylatable Spd2 mutant exhibits not only a delayed decline in centrosomal levels, but also higher initial levels (Fig. 5B). If Cdk initially promotes Spd2 activity what is their explanation for this?

    Minor/discussion points

    1. p4 "In typical somatic cells the two mitotic centrosomes need to grow to approximately the same size, as mitotic centrosome size asymmetry can lead to asymmetric spindle assembly and so to defective chromosome segregation. How centrosome growth is regulated in somatic cells is unclear, but in early C. elegans embryos, mitotic centrosome size appears to be set by a limiting pool of the PCM-scaffolding protein SPD-2."
      The authors here conflate absolute and relative size. Relative size matters to avoid spindle asymmetries, and centriole involvement in PCM recruitment helps to prevent this (Zwicker et al., PNAS 2014). Absolute size, which is what the authors are concerned with in this manuscript, may be important for spindle scaling, but this is not the same thing.
    2. p5 "The centrosomal levels of Polo, Spd-2 and Cnn all started to increase at the start of S-phase, but whereas Cnn levels continued to rise and/or plateau as the embryos entered mitosis, the centrosomal levels of Polo and Spd-2 started to decrease before the entry into mitosis (Wong et al, 2021) (Figure 1A,B). Thus, the components of the mitotic PCM scaffold exhibit different growth kinetics, making it hard to use these proteins to define centrosome "size" at any particular point in the cell cycle."
      It is misleading and confusing for the reader to describe Polo and Spd2 as scaffold proteins as opposed to regulators of scaffold assembly. Presently Cnn is the only PCM protein demonstrated to have self-assembly/scaffolding properties based on the authors' own work (conduit, Dev Cell 2014; Feng, Cell 2017). There is little evidence that Polo and Spd2 form anything other than a nucleus for PCM growth.
    3. p7 "The centrosomal levels of Grip71, Grip75, Grip128, and Aurora A tended to increase steadily through most of NC13, whereas TACC, Msps and γ-tubulin exhibited a noticeable increase in their recruitment rate towards the end of S-phase, shortly before their recruitment levels peaked (compare NC13 graphs in Fig. 1B). This difference was also obvious if we used centrosome area as a measure of centrosome size (Fig. S1). We conclude that PCM client proteins can be recruited to centrosomes in at least two different ways."
      As discussed above apparent differences in kinetics may reflect limitations in the way protein accumulation is measured. It is hard to conceive of a reason why the Grips would display a different mode of protein accumulation from gamma-tubulin, nor is the idea of two different modes of protein accumulation picked up again later in the manuscript.
    4. Since the authors mention that the duration of S phase increases between cycles 11 and 13 (p9), are there any measures for the timing of the beginning/end of S phase in each cycle?
    5. One of the main findings in the landmark Woodruff paper from 2017 Cell paper was that PCM scaffold polymer could dynamically concentrate client proteins in the absence of any other factors, to an extent similar to that observed in vivo. This list did not include gamma-tubulin, which was later shown to require PLK1 phosphorylation of SPD-5 (Ohta, JCB 2021). However, it did include ZYG-9, the C. elegans ortholog of Msps. If client protein accumulation is an intrinsic property of the PCM scaffold, how do the authors explain that Msps departs prior to NEBD while Cnn continues to accumulate?
    6. p13 "The expression of the mutant proteins did not appear to dramatically perturb the centrosomal recruitment of γ-tubulin-GFP, except that the rate at which γ-tubulin-GFP left the centrosome as the embryos entered mitosis was reduced in both mutants compared to WT (Figure 7). This phenotype was subtle, but it was statistically significant, and it seems likely that the presence of large amounts of WT Spd-2 and Cnn in the mutant embryos (Figure 5A,F) would help to mask the potential severity of this phenotype."
      This does not quite make sense. Fig. 5 shows that Spd2 dissociation is significantly slowed in the mutant condition. If Spd2 drives gamma-tubulin accumulation (as Fig 6 shows), then the continued presence of Spd2 should prevent dissociation. Yet it apparently does not. Why?


    1. p3 and following. The reference for the authors' prior work on PCM recruitment (Wong et al, 2021) should probably be for the final, published article in EMBO J, not the 2021 preprint.
    2. Fig. 1. legend "Note that for technical reasons not all of the centrosome proteins could be followed for the full time period." Why not?
    3. Figs 4-6. Which cycle is being assessed here?
    4. Fig 6. Not plotted here is the rate of dissociation of gamma-tubulin, unlike eg in Fig 7. It is notable that both accumulation and dissociation appear to be slowed in the Spd2 1/2 gene dosage condition.
    5. Fig S1B. Some of the graphs in this figure are not labeled (based on Fig.1 presumably gamma-tubulin and Msps).
    6. Some of the data in the main figures, including the entirety of Figs. 2 and 3, could be moved to Supplemental to present a more crisp and accessible manuscript.
    7. While I sympathize with the authors needing to repeat entire sets of experiments I am not entirely sure it is appropriate to recycle entire sets of data from a previous publication of theirs (Cnn, Spd-2 and Polo recruitment kinetics, reproduced from Wong et al., EMBOJ 2022), since this manuscript is largely concerned with apparent differences between the kinetics of those components and the PCM client proteins now being analysed.


    Control of organelle size has been an active field of research for many years for a large variety of cellular structures and in a range of experimental models. Here, Jordan Raff and colleagues examine the mechanisms underlying centrosome (PCM) size control in Drosophila syncytial embryos, building on their previous work (Wong, EMBOJ 2022) to propose a role for CDK in both promoting (at intermediate levels) and inhibiting (at high levels) PCM expansion.

    I found this a difficult manuscript to review, not only because the subject matter is complicated, but so is the writing. Having read and re-read the manuscript some clarity eventually emerges, but it shouldn't be that inaccessible. As for the authors' model I find it intriguing, but not fully supported by the data currently presented.

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Wong et al. investigates how cells regulate the increase in the size of the centrosomes (more specifically the size of the pericentriolar material or PCM) that occurs during preparation for mitosis. They use the Drosophila syncytial embryo as a model, focusing on nuclear cycles 11-13, during which cell cycle progression gradually slows. The authors find that centrosomes grow to a consistent size at each cycle by adjusting to the slowed cell cycle, reducing the growth rate and increasing the growth period. This adjustment is proposed to be regulated by the Cdk/Cyclin cell cycle oscillator. Curiously, Cdk/Cyclin activity seems to both promote and inhibit the increase in centrosome size, depending on whether its activity is moderate or very high, respectively. Both effects are proposed to depend on the phosphorylation of centrosome proteins by Cdk/Cyclin.

    1. While being comprehensive in the number and type of markers that are being analyzed, there is no analysis of the centrosome's MTOC activity. In my opinion this is missing since centrosome size alone is not necessarily indicative of its MTOC activity, but MTOC activity is what ultimately matters for its role during mitosis. For example, it was observed that centrosome size declines already before mitotic entry, but it is possible that centrosome MTOC activity does not (similar to differences in the timing of the decline of PCM scaffold vs PCM client proteins). While not strictly related to size control, centrosome activity is biologically more relevant than solely size. I would consider it optional, if the authors decide to talk only about centrosome size, but then it should be made clear that size here may not be the most relevant factor.
    2. The authors say that during NC13 PCM client proteins can be recruited in "at least two different ways" (p. 7), including a way (rapid increase before peak) that does not resemble PCM scaffold recruitment (steady increase during NC13). How can these two different ways and kinetics be determined by the same Cdk/Cyclin oscillator?
    3. I am puzzled by the conclusion that Cdk/Cyclin directly phosphorylates Spd-2 or Cnn at the sites used for mutagenesis. This cannot be concluded based on the presented data.
    4. Fig. 6: Doesn't the data show that Cnn does not affect the initial rate of g-tub recruitment, but only the later rapid recruitment shortly before mitosis? In contrast Spd-2 seems to affect the initial phase. This should be described more precisely. Again, I am wondering how this is compatible with direct regulation by a single oscillator, as suggested by the authors (see also point 2 above.
    5. I don't find the proposed model very convincing and not fully supported by the presented data.
      First, the recruitment kinetics of different centrosome proteins are not all the same, arguing against a simple relationship based on phosphorylation by Cdk/Cyclin. For example, kinases (or phosphatases) may be recruited (or displaced) by Cdk/Cylclin at the centrosome and then locally regulate binding or maintenance of certain centrosome proteins. This could explain profiles that do not display a steady change over time, as would be expected by direct regulation by Cdk/Cyclin.
      Second, it is not clear from the description in the text or from Fig. 8 how moderate Cdk/Cyclin activity can promote recruitment and high activity induce loss of proteins at centrosomes. In fact, the experiments with Spd-2 and Cnn phospho-mutants suggest that phosphorylations at the mutated sites also reduce centrosome binding during S phase (at moderate activity) and not only shortly before mitosis (at high activity), since alanine mutants of both Spd-2 and Cnn are increased at centrosomes also during S phase. The model seems to ignore this observation. If these sites are already phosphorylated to decrease centrosome binding in S phase, then what triggers the rapid decrease shortly before mitosis?
    6. Can the authors identify and mutate CdK/Cyclin dependent phospho-sites in centrosome proteins that promote centrosome recruitment at moderate Cdk/Cyclin activity? As an alternative to the "protein availability" model for regulation of centrosome size, the proposed model needs to explain how a steadily increasing activity (Cdk/Cyclin) can first induce growth and then turn growth off, when the desired size is reached. This is obvious in the "protein availability" model, where the available protein steadily decreases as centrosomes grow, but this is not at all obvious for an oscillator that behaves in the opposite way during the same period and that can only phosphorylate sites that decrease centrosome binding.


    1. The authors observe differences in the intensity profiles for different subunits of the gamma-tubulin complex. How do they explain this? Are they not in the same complex? The authors should mention and comment on this.
    2. The authors refer at various points in the manuscript to an "inverse-linear" relationship between S phase length and centrosome growth rate, but according to the graphs the rate does not change linearly.


    This is an interesting manuscript that reaches somewhat different conclusions regarding centrosome size control when compared to previous studies in other organisms. In particular, work in C. elegans has proposed that centrosome growth regulation is controlled by the limited cytoplasmic availability of PCM building blocks, whereas the current study proposes a different model based on the activity of a cell cycle oscillator. The model system and approaches are well presented and the data is of good quality. The authors monitor a large number of centrosome markers, each with detailed quantifications of intensity and distribution over time during the different cycles. They also employ two different ways of quantifying centrosome size with similar results, making their quantifications more robust. While the authors include phospho-mutants in their analyses that presumably cannot be phosphorylated by Cdk/Cyclin, the study is largely descriptive. Still, the authors present interesting observations and propose the "oscillator model" an alternative to the "limited availability model" for the regulation of centrosome size, and perhaps that of other organelles. Assuming the authors can clarify inconsistencies and/or provide additional data to support the proposed model, this could be an important finding that expands cell biologists' understanding of organellar size control.

    I have expertise in centrosome biology and the role of centrosomes as MTOCs, as well as more general expertise regarding the function of the microtubule cytoskeleton in cell division and differentiation.