Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly
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Referee #1
- The authors should provide more information when...
Responses
- The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A).
- Though this is not stated in the MS
- Figure 6: Why has only...
Response: We expanded the comparison
Minor comments:
- The text contains several...
Response: We added...
Referee #2
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Referee #3
Evidence, reproducibility and clarity
all OK
Significance
This is a valuable paper that make use of the rapid mitotic cycles of the Drosophila syncytial embryo to study the recruitment of proteins in mitotit centrosome maturation. The synchrony of these cycles make this an excellent experimental system in which to follow the relative timing of recruitment of individual molecules to the centrosome and, while the system may have idiosyncrasies that facilitate rapid cycling, it provides valuable information. This is a significant data set that shows the pulsatile recruitment of Spd2 and Polo kinase peaking in mid S-phase in contrast to the continuous recruitment of Cnn.
Th…
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Referee #3
Evidence, reproducibility and clarity
all OK
Significance
This is a valuable paper that make use of the rapid mitotic cycles of the Drosophila syncytial embryo to study the recruitment of proteins in mitotit centrosome maturation. The synchrony of these cycles make this an excellent experimental system in which to follow the relative timing of recruitment of individual molecules to the centrosome and, while the system may have idiosyncrasies that facilitate rapid cycling, it provides valuable information. This is a significant data set that shows the pulsatile recruitment of Spd2 and Polo kinase peaking in mid S-phase in contrast to the continuous recruitment of Cnn.
The authors carry out some interesting modelling to account for the pulsatile activity of Polo through recruitment to the centriole. As they have previously shown Polo recruitment to be dependent upon S-S/t motifs in An1 and Spd, the authors examine the effects of multiple mutations at these potential recruitment sites. Interestingly they show that mutation of 34 such sites in Ana1 has little effect on recruitment of Polo to old-mother centrioles but perturbs recruitment onto ne mothers. Expression of the multiply mutated Spd-2, on the other hand, perturbs the Polo pulse on both old- and new- mothers. Together this would be in line with their previous suggested role for Ana1 in initially recruiting Polo to centrioles and Spd2 having a role in expanding the PCM.
The modelling carried out by the authors is simple but effective. As with almost any cell cycle model, the models have their short-comings and the authors are largely aware of these. I thought it would be worthwhile to have some more discussion of what activates Polo kinase. It could be partially activated by the Polo-box binding to its receptor site but do other kinases carry out its T-loop phosphorylation? There are plenty of mitotic kinases around and so this could be discussed in greater detail. Moreover, although the pulsatile association of Polo with the centrosome does not have to correspond to pulsatile activity, this is likely. In which case, further discussion of the roles of opposing phosphatases would be in order.
All in all, however, this is a useful paper that comes up with a thorough description of the timing of events of centrosome maturation in Drosophila embryos.
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Referee #2
Evidence, reproducibility and clarity
Review of 'Mother centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly' from Wong et al.
In this paper, Wong et al address the mechanisms of centrosome assembly in flies. They start with the interesting observation that Polo localized at centrosomes oscillates before cells enter mitosis, while Cnn (and with it centrosome maturation) either increases or reaches a plateau. The phenomenon is local, since Polo levels at in the cell are high during mitosis. They propose that the oscillation is driven by a negative feedback loop whereby Polo inhibits its own binding to the centrosome, Ana1 being …
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Referee #2
Evidence, reproducibility and clarity
Review of 'Mother centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly' from Wong et al.
In this paper, Wong et al address the mechanisms of centrosome assembly in flies. They start with the interesting observation that Polo localized at centrosomes oscillates before cells enter mitosis, while Cnn (and with it centrosome maturation) either increases or reaches a plateau. The phenomenon is local, since Polo levels at in the cell are high during mitosis. They propose that the oscillation is driven by a negative feedback loop whereby Polo inhibits its own binding to the centrosome, Ana1 being the most likely relevant receptor. Finally, they discuss the possible meaning of this oscillatory behavior, in the light of the rapidity of the early embryonic cell cycles.
Major comments
1- One can imagine different reasons for the fact that the model displays different dynamics for Cnn and Spd-2/Polo. For example, a major difference may be due to the different dissociation rates of the clusters Cstar and Shat. These are governed by different laws and different parameters (kdis vs kidsCstar1/n). If I understand, both parameters and dependency on Cstar^2 are assumptions. Hence, it would be important to pinpoint which component of the model is more directly responsible for the observed behavior. The analysis should not be limited to the dissociation, but should be extended to the whole model. To this aim, one could test the robustness of the model's parameters. The results of this analysis will also be a prediction of the model.
2- The presence of a positive-feedback loop involving Cnn could offer an alternative and more robust explanation for the slower dynamics of Cnn. Such a loop between Cnn and Spd-2 was proposed by the authors (Conduit, eLife, 2014). I think some comment on this point would be interesting (eg, could the Cnn/Spd-2 loop proposed earlier work in this context? If not, why? If yes, should not this option be explored?).
3- The prediction presented in Figure 6 is very relevant. I wonder how robust this behavior is to changes in parameters values.
4- Additional testing of the model would be important to confirm that the negative feedback loop is actually in place, although I understand experiments may be difficult to be performed. Possible examples: constantly high levels of Polo are expected to decrease its centrosomal localization, is that correct and, if so, testable? Is it possible to delay one cycle, and then observe the decay in Cnn values? This latter experiment, for example, could help to distinguish positive feedback vs slow decay rates. If the experiments are not possible, it may be worth anyway to present some predictions worth testing.
5- The difference between Models 2 and 3 is not clear to me. In mathematical terms, they seem to be basically the same thing: reaction (50)=(33), (51)~(34) given (40) and (52)~(35) again given (40). This is precisely since the model comes with the assumption of a well-stirred system, and thus adding P* in solution is not so different from assuming P=Rphat (40). I would have imagined that also Model 2 accounts for the fact that in Spd-2-S16T and Ana1-S347T Polo is recruited slower and for a longer period. Is it not true? If so, is model 3 really needed? More in general, assuming a role for an increase of local concentration of P is quite a jump, especially given the small distances involved, and the fast diffusion occurring within cells.
Minor points
1-Could the authors use the FRAP data to estimate the different kdis? If so, a comparison with the 20-fold difference used in the model would be useful.
2- p. 6, The authors should state clearly for the worm-uneducated like me whether the fusions were done with the endogenous proteins or not.
3- p.7 Figure 1B, in the text it is referred to display 'levels of peaks' and in the figure and legend we find 'growth period'. Not clear how the two refer to the same quantity.
4- Spd2-mCherry is present in both Figure 1C and D, but with very different amplitudes. Why is that the case?
5- The fact that Polo peaks in mitosis is a key observation. Unfortunately, this is often reported as a personal communication. The authors never tried to produce this piece of data?
6- p.11 It is explained that NM and OM differ for their initial values because the OM starts with some PCM from the previous cycle. However in Figure 3A, for example, the values of Polo at the end of the cycle are identical in the two. Is not this in contrast with the explenation?
Still p11, there is reference to Figure 3C,D, but Figure 3D does not exist, I guess it should be 3A,C.
7- In the formulation of the model (page numbers in Suppl Mat are unfortunately missing..), one citation for the total amount of Polo being large is needed.
8- I do not understand this point: scaled c* output is 1, and the initial condition for c*=1 also?
9- It has been shown in different systems (from yeast -- haase winey reed, NCB, 2001-- to worms -- McCLeland O-Farrell CB 2008) that centrosome duplication can occur independently from the cell cycle oscillator. I was wondering whether the proposed negative feedback loop may play a role in this phenomenon. This is only a curiosity, which does not need to be addressed.
Significance
The new observation and hypotheses presented in the paper provide a sizeable advance. The presence of an oscillation in Polo, uncoupled from cellular levels, is new, and the model proposes a testable hypothesis to explain it. Some additional experiments to verify the model would strengthen the manuscript.
The work is probably more appropriate for experts in the centrosome field. My primary expertise for this review was in mathematical models.
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Referee #1
Evidence, reproducibility and clarity
Summary:
Embryogenesis is characterized by rapid cell divisions without gap phases. How these cells achieve successive rounds of chromosome segregation in dozens of minutes without failure is of great interest to cell and developmental biologists. A key aspect of rapid divisions is the oscillatory nature of centrosome assembly, which aids in building the mitotic spindle during mitosis, and centrosome disassembly during mitotic exit. Polo kinase activation and localization to the centriole is essential for centrosome dynamics, but its molecular targets, timescales of activation and deactivation, and overall mechanism of action is …
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
Summary:
Embryogenesis is characterized by rapid cell divisions without gap phases. How these cells achieve successive rounds of chromosome segregation in dozens of minutes without failure is of great interest to cell and developmental biologists. A key aspect of rapid divisions is the oscillatory nature of centrosome assembly, which aids in building the mitotic spindle during mitosis, and centrosome disassembly during mitotic exit. Polo kinase activation and localization to the centriole is essential for centrosome dynamics, but its molecular targets, timescales of activation and deactivation, and overall mechanism of action is still not fully determined.
This paper aims to build a mathematical model to tease out the mechanism of Polo recruitment to centrioles and transformation of centrosome scaffold proteins (Spd2/Cep192 and Cnn/CDK5RAP2) from inactive forms to functional, multimeric platforms. The authors posit that several features are critical to describe the dynamics of Polo, Spd-2, and Cnn: 1) a negative feedback loop that releases Polo from a centriole receptor, 2) a kinetic relay that allows Spd-2 to assemble, followed by Cnn, and 3) a disassembly mechanism driven by de-phosphorylation. They validate their model in several ways, most notably by introducing an Ana1 mutant that inhibits Polo binding to centrioles: their model predicts a delay in Polo accumulation which bears out in vivo.
The cell biology experiments of this paper are of high quality and well quantified, and I have no concerns there. However, the mathematical model elevates this study to the next level, and thus deserves greater scrutiny. I'm not concerned that the model doesn't get everything right, or that all of the parameters are correct. This is new territory. I think the value of models is their power to predict, rather than their power to explain existing data. The authors are giving the field a great hypothesis generator which we can use to plan experiments for the next 5 years. Then the model will be updated to be more accurate. Thus, this work represents a significant achievement.
Still, some key validations regarding phosphorylation rates are missing that could be easily tested. Furthermore, the study would be strengthened by greater understanding of the PCM disassembly. mechanism. Addressing these two points will improve my confidence in the mathematical model.
Major Comments.
- This study builds a model that relies heavily on rates of phosphorylation and de-phosphorylation. Further, de-phosphorylation is assumed to be the key disassembly mechanism, but this has not been rigorously studied in fly embryos. Thus, two critical aspects of the model remain unverified.
Surely, the authors could test how changing phosphorylation rate (kcat S and kcat C) and de-phosphorylation rate (kdis) affects the recruitment and departure of Spd-2, Polo, and Cnn in vivo. This could be achieved by 1) titrating an inhibitor of Polo (e.g., BI-2536) or introducing a mutation in the T-loop of Polo (the equivalent of T210D or T210V in flies; T210D should raise kcat, while T210V should lower kcat; https://doi.org/10.1021/bi602474j), and 2) inhibiting a phosphatase such as PP2A, which is the presumed antagonist of Polo according to several C. elegans studies.
If their model adequately predicts the outcome of these two experiments (changing phosphorylation and dephosphorylation rates), I will be more convinced.
- The models focus on Polo and Spd-2 pulses during mitosis, but ignore the disassembly phase of Cnn. Do Cnn levels drop during mitotic exit? Can this drop in Cnn be described by any of the authors' proposed models?
- These models are described as the "simplest possible" yet have many unknown parameters. For example, Model 1 has 12 parameters, none of which have been determined experimentally. How did the authors land on these values? Is it possible that one could alter any combination of these parameters and achieve a similar outcome? Or, if the Kcat of Polo is changed two-fold, does the whole model fall apart (see above)?
Experimentally determining these parameters would greatly strengthen this paper, but I think that would require gargantuan effort that is beyond the scope of the current work. Instead, it is therefore critical to test how robust the model is by probing the parameter space. For example, could the authors show us what the model predicts (e.g., as in Figure 2C) when each parameter is changed by 2-fold? Presumably the authors have already done this, but I would like to see the outcomes.
Minor Comments.
-Figure 1. The authors should include representative images of centrosomes for the plots in panels A,C and D. The x-axes could have more informative labels (e.g., time relative to NEB).
- Figure 1A. Much of Figure 1 has already been performed in C. Elegans, yet this fact is not mentioned until the discussion. For example, the pulsed nature of Polo and SPD-2 appearance and disappearance has been reported in C. elegans in Mittasch et al. 2020 and Magescas et al., 2019. These findings, and their implications for evolutionary conservation, should be mentioned in the main text (e.g. page 6 or 7).
- Figure 2. It's hard to envision how a scaffold can both flux outward and be structurally strong. The mere fact that there is outward movement of scaffold chunks implies breaking of bonds, which indicates overall structural weakness. Are the authors talking about strength of the entire PCM, or just strength of the chunks? It would be great if the authors could clarify this. -Figure 2. One would think that scaffold flux and strength are anti-correlated. Perhaps this is the case? As far as I'm aware, previous studies of Cnn flux were performed primarily in S-phase, when there is presumably less need for PCM strength. What about during mitosis during chromosome segregation? Does the PCM become stronger during mitosis? Does Cnn flux decrease during mitosis?
- Figure 2B. I would prefer a legend in the actual figure indicating what the different symbols mean. I found it difficult glancing back and forth between the text and the figure.
- "We also allow the rate of 𝐶∗ disassembly to increase as the size of the 𝐶∗ scaffold increases, which appears to be the case in these embryos (Conduit et al, 2010)."
I can't find any analysis of PCM disassembly in this study. What are the authors referring to as "disassembly"? Do they mean departure of Cnn from the PCM in S-phase? Or, disassembly of the whole PCM during mitotic exit?
-"If the centriole and PCM receptors (Ana1 and Spd-2, respectively) recruit less Polo, the centriole receptor (Ana1) will be inactivated more slowly."
Is Ana1 a known substrate of Polo? This seems highly speculative. The authors should note that deactivation of Ana1 could be through various other mechanisms. Furthermore, Polo could be locally degraded as shown in human cells doi: 10.1083/jcb.200309035.
-"We note that our mathematical models are purposefully minimal to reduce the number of parameters and test possible mechanisms rather than to mimic experimental data." I appreciate this statement.
Significance
This paper aims to build a mathematical model to understand the cyclic nature of centrosome assembly and disassembly in fruit flies. Due to the conserved nature of the components (proteins in the system, such as Polo Kinase, Spd-2, and Cnn/CDK5RAP2), this model could likely be extended to a broad swath of eukaryotes. This approach is quite unique in the centrosome field, as only one other study (Zwicker et al., PNAS 2014) has tried seriously to model the growth kinetics of PCM, the outermost part of a centrosome. The field has been dominated by genetics and cell biology approaches, so implementing a mathematical model will advance the field and generate hypotheses, even if the model is not yet fully fleshed out. This paper represents a significant advance.
This study will be of broad interest to the centrosome field.
Expertise: centrosome biogenesis, mitosis, biophysics
Note: I am not sufficiently qualified to evaluate the mathematics underlying the model.
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