Structural insights into mitotic-centrosome assembly

  1. Nada Mohamad
  2. Siu-Shing Wong
  3. Anupa Majumdar
  4. Alan Wainman
  5. Ingelise Holland-Kaye
  6. Lars Hubatsch
  7. Zsofia Novak
  8. Andrey Pozniakovsky
  9. Martine Ruer-Gruss
  10. Andreas F.M. Haensele
  11. Anna Caballe
  12. Steven Johnson
  13. Susan M. Lea
  14. Anthony Hyman
  15. Jordan W. Raff

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Abstract

Centrosomal material assembles rapidly in mitosis. In Drosophila , the coiled-coil protein Cnn forms a scaffold that recruits PCM clients; in C.elegans , SPD-5 plays an analogous role. Here we show that full-length Cnn and SPD-5 can both form spherical condensates in vitro , but that the interactions driving their assembly into scaffolds inside cells appear to diverge. We show that the Cnn PReM adopts a helical hairpin fold that autoinhibits CM2 binding but that phosphorylation appears to increase hairpin breathing to permit CM2 engagement and robust scaffold assembly. Phospho-blocking mutations prevent PReM–CM2 interactions and scaffold formation, whereas phospho-mimetic substitutions partially restore function. The human homologue CDK5RAP2 contains a CM2 domain that can partially substitute for fly CM2 in vivo and we identify a candidate CDK5RAP2 PReM region that forms macromolecular networks with human CM2 in vitro . By contrast, the putative PReM and CM2 regions of SPD-5 cannot substitute for their equivalent fly domains and they do not interact detectably, suggesting a distinct assembly mechanism in worms despite conserved PLK1-dependent control of PCM growth.

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    Reply to the reviewers

    We thank the Reviewers for their comments on our manuscript “Structural insights into mitotic-centrosome assembly”. As described below, we have substantially revised the manuscript in response to their comments and are hoping you would consider the revised manuscript “Phosphorylation relieves autoinhibition to drive Cnn centrosome scaffold assembly” at The EMBO Journal. Our specific responses (black text) to the Reviewer’s comments (blue text) are detailed below

    Reviewer #1

    Main Points:

    1. From previous studies, it seems to me that for the residues potentially relevant for the hairpin regulation there is direct evidence of phosphorylation only for S567 (mass spec, phospho-antibody). Have the authors tested single site mutants (S567A and E)? Also, have they tested D mutations? If so, this should be commented on and shown. If not, it should be tested, in particular since the 2E phospho-mimetic is not functioning properly in vivo. If S571 is indeed crucial, it should be demonstrated that it is also phosphorylated. Otherwise it is possible that the mutation of this residue simply impairs important interactions (e.g. PReM-CM2, others), independent of phosphorylation.

    As requested, we have now tested individual S567A and S571A mutations and found that they both perturb Cnn scaffold assembly, but to a lesser extent than the 2A double mutant (New Fig.S3A). We also now confirm by MS that recombinant Polo can phosphorylate both S567 and S571 in vitro, and we have examined the behaviour of a 2D mutant and find that it behaves very similarly to the 2E mutant (New Fig.S3B).

    1. It is unclear why in vitro only A mutations have been tested and not phospho-mimetics. This should be tested for the interaction between PReM and CM2. This would allow to probe the model that phosphorylation opens the hairpin to allow interaction. Currently, such proof is missing in the study. Alternatively, the authors could phosphorylate the recombinant protein in vitro. The in vivo data is harder to interpret due to the complexity of the model and the authors should take advantage of the in vitro system.

    As requested, we now show in New Fig.S5 that whereas in vitro WT Cnn490-608 and Cnn-2A490-608 behave as dimers, Cnn-2E490-608 elutes in two major fractions—a tetramer species and a much larger species that elutes in the void volume (meaning that 2E can form very large species even in the absence of CM2) (Figure S5A). In the presence of CM2, Cnn-2E490-608 forms a tetramer (that eluted slightly later than the Cnn-2E490-608 tetramer) and larger complexes that contained CM2 and eluted in the void volume with a profile similar to Cnn-2E490-608 on its own (Figure S5B). These results are consistent with the possibility that the 2E substitutions open the helical hairpin to allow self-interactions that drive homo-tetramer and larger complex assembly* in vitro*.

    1. Regarding the worm PReM and CM2 domains, the authors mention that they have tested in vitro phosphorylation by PLK-1, but I could not find any data showing this. They should demonstrate successful phosphorylation or test candidate site by phospho-mimetic mutation. It is possible that the worm proteins depend more strongly on phosphorylation to relieve autoinhibition compared to the fly proteins.

    This is a good point, and we apologise for this omission. We now state that we confirmed by MS analysis that the recombinant worm PLK-1 we used in these in vitro experiments phosphorylates the putative SPD-5 PReM domain on the three sites (S627, S653 and S658) known to be important for promoting SPD-5 scaffold assembly in vivo (Figure Legend, Figure 6). Thus, the lack of detectable binding between these proteins is not due to the lack of phosphorylation.

    Minor Point:

    4). Fig. 6C, D: the labeling of the chimeric constructs using "+" symbols is confusing, since it suggests that separate proteins were expressed. If I understand this correctly, with the current labeling, deltaCM2+DmCM2 means WT? The authors should write the full name of the wildtype or chimeric construct in each case and use a more standard/less confusing nomenclature. Also, I suggest to start the panels and graphs with the WT sample.

    We thank the Reviewer for this suggestion and have re-labelled this Figure to clarify this point. We understand the point about putting the WT panels first in Figure 6C,D (now Figure 5C,D) but think that this is not the correct comparison to emphasise. We are testing the ability of the various CM2 domains to “rescue” the lack of a CM2 domain, so we feel Drosophila Cnn lacking CM2 is the correct baseline for this comparison.

    Reviewer #2

    Main Comments:

    1. The title is too vague. Any number of existing papers could be said to provide "structural insights into mitotic centrosome assembly". The authors need to narrow down to a defined conclusion and state this as the title.
    2. I think the strongest and most novel aspects of this study relate to the mechanism of Cnn assembly via relief of the auto-inhibited PReM. The effort to elucidate assembly mechanisms of SPD-5 and CDK5RAP2 are comparatively light and there are no accompanying experiments in worms or human cells. Without the in vivo experiments, it's hard to know if the in vitro experiments are valid. It's speculative for the authors to say they found the true PReM for CDK5RAP2; they do not demonstrate that PLK-1 phosphorylation potentiates assembly in Figure 8. Thus, I suggest re-writing the paper to focus on Cnn. Experiments in Figure 6 are still valid if reframed. For example, substituting Cnn's CM2 with the CM2 from CDK5RAP2 vs. the C-term of SPD-5 illustrates that a simple coiled-coil with open ends (H.s.CM2) is sufficient to interact with PReM whereas a coiled-coil with a closed end (SPD-5 C-term, predicted by Figure 6A) cannot. We thank the Reviewer for these helpful comments and have re-written and re-organised the manuscript in accord with these suggestions—most importantly providing a more specific title and re-ordering the data to better focus the paper on the relief of Cnn autoinhibition.

    The purpose of Figure 1 is unclear. None of the other figures examine SPD-5 and CNN in the condensate form, which required using 4% PEG in this paper. The other assays look at the network form, which could behave differently and have different dependence on specific domains. I think they should perform the condensate assay for all other figures, otherwise leave it out. Furthermore, CDK5RAP2 is mentioned, yet not examined in Figure 1. It must be noted that CDK5RAP2 will also condense into droplets under crowding conditions or with a synthetic nucleator (Rios et al., 2025 J Cell Sci). Thus, it seems that condensation potential is a universal feature of known PCM scaffold proteins.

    The original Figure 1 has been moved to end of the paper (now Figure 8) and we now more thoroughly explain the logic of these experiments. Briefly, given that the PReM and CM2 domains in flies and worms seem to function in different ways in vivo, we sought here to test whether this was also the case in vitro—where the behaviour of full-length SPD-5 and of these domains of Cnn have been extensively studied, but never directly compared. We believe such a direct comparison will be of some interest to the field (the Woodruff et al., 2017 paper describing these in vitro SPD-5 condensates has been cited >700 times). We now also cite the Rios et al., 2025 paper but note that, despite extensive efforts, we were unable to purify enough well-behaved CDK5RAP2 for our experiments and so could not include it in this analysis. We think Rios et al., used an MBP-fusion of CDK5RAP2 in their experiments, which may explain this difference.

    The study uses different species without doing the same types of experiments on each. Sometimes human CDK5RAP2 is thrown in, sometimes not. They solve crystal structures of PReM from Cnn but not from the other proteins. This gets confusing, especially since the authors state that they seek to test if fly Cnn and worm SPD-5 assemble through different mechanisms (see last sentence of the intro). Also, if the focus is on worm vs. fly PCM assembly mechanisms, why include the human protein, especially Figure 8?

    On re-reading our original manuscript we appreciate this confusion. We hope that in re-writing the manuscript along the lines suggested by the Reviewer the logical flow of our experiments will be clearer.

    The conclusion that SPD-5's narrow PReM and "CM2" domains don't interact is consistent with the cross-linking mass spectrometry data from Rios et al. 2024. They showed only one X-link with low occurrence (1 out of 6 samples) between these two regions, even in the phosphorylated state (Fig. 1G). However, Nakajo et al (2022) claimed the opposite, showing that a larger PReM-containing construct (a.a. 272-732) interacts with a C-terminal construct (a.a. 1061-1198) after PLK-1 phosphorylation. Can the authors comment on this? Perhaps there is another site in SPD-5, outside of a.a. 541-677, that acts like the Cnn PReM?

    These are good points and we now mention this last possibility in the Discussion. We also now mention the supporting cross-linking Mass Spec data from Rios et al., 2024.

    I have serious doubts that the C-terminus of SPD-5 has a CM2 domain. To me, there is no real sequence homology with the traditional CM2's from humans and flies, and the AF3 predictions support this. Ohta et al. (2021) called this region "CM2-like" based on very poor homology, which a is questionable practice. Any coiled-coil region will appear somewhat homologous due to the heptad repeat pattern that defines them (e.g., leucines line up quite nicely). Thus, is it fair to say that SPD-5 doesn't assemble through a PReM-CM2 interaction? There may be a different region in SPD-5 that looks more like the canonical CM2. I think the authors have compelling evidence to give the C-terminal coiled-coil region in SPD-5 its own name rather than calling it CM2.

    This is a fair point, although the literature is already quite confusing on the nomenclature for the C-terminal region of SPD-5 (e.g., Ohta et al., JCB, 2021; Nakajo et al., JCS, 2022), so we are reluctant to add another name to the mix. Given that we draw comparisons with the fly and human CM2 domains (that are clearly related by sequence), we think it is easiest for readers if we use the “CM2” nomenclature throughout, although making clear our conclusion that SPD-5 “CM2” does not appear to function in the same way as fly/human CM2.

    Figure 3E. Would measuring scaffold mass be more appropriate? The PReM(deltaH1,NTH2) leads to more compact scaffolds, but maybe they assemble just as well as the deltaH1 mutant. As it stands, there is a discrepancy between panel E and F in terms of what is measured (area vs. intensity) and the outcome.

    In several previous papers we use fluorescence intensity to measure the “amount” of protein at centrosomes in vivo but, in our original paper (Feng et al., Cell, 2017), we quantified PReM::CM2 scaffold assembly in vitro by measuring the area of scaffold assembly. Thus, we prefer to present the current data in this way for consistency across publications, and we believe either measure is valid. We could measure the area and intensity of the PReM∆H1 and PReM∆H1∆NTH2 scaffolds to compare scaffold density, but we think this would unnecessarily complicate this data. The main point is not how much or how dense each scaffold is, but rather that the PReM∆H1∆NTH2 protein doesn’t really make a scaffold at all—but rather makes smaller “blobs” that tend to bunch together (further characterised in Fig.S2).

    Minor Comments:

    1. In one version of the PDF there are images missing in Fig 1F, 4C, 4D. I opened another version (source version) and the images were there. Just FYI.
    2. Figure 4A. The blue coloration makes it difficult to read the black letters.
    3. Figure 4A. Why is part of the protein colored in green? This coloration isn't defined, nor does it show up again in panel B.
    4. The layout of Figure 4 is confusing. It took me a few minutes to realize that the big red box inset belonged to panel B and not panel A.
    5. Figure 4C,D. The sample size is not mentioned in the legend.
    6. The title for Figure 4 seems too speculative. How can the authors say that phosphorylation relieves the autoinhibition without structural data?
    7. Figure 5B. The sample size is not mentioned in the legend.
    8. Figure 6B,D. The sample size is not mentioned in the legend.
    9. The text in Figure 7B is hard to read because it is too small. Please make this bigger.
    10. Figure 8C. What is colored in magenta? Is there an additional labeled protein besides mNG-CM2?
    11. Figure 8C. What is the sample size? How many images were taken? Also, why are there data points off to the right of the last column?
    12. The wording of these sections needs improving. I found them complicated and difficult to understand. We thank the Reviewer for taking the time to make these helpful comments. We have addressed all these points in the revised manuscript. On point 10, the magenta objects were fiduciary beads that were inadvertently included on this panel (and are no longer shown).

    Reviewer #3

    Major Comments:

    1. The title, "Structural Insights into Mitotic-Centrosome Assembly," is overly broad. The study primarily focuses on CM2-PReM intramolecular interactions in D. melanogaster Cnn and does not comprehensively address mitotic centrosome assembly across species. A more specific title reflecting the fly-centric and structural focus would better align with the manuscript's scope and conclusions.

    As described at the start of our response to Reviewer #2, the title and focus of the manuscript have been extensively revised along these lines.

    The authors analyze condensate formation by Cnn and SPD-5 but overlook condensate formation by CDK5RAP2, which was recently reported by Rios et al. (2025, PMID: 40454523). Including CDK5RAP2 would enable a more balanced and informative comparison across fly, worm, and human homologs.

    As described in point 3 of our response to Reviewer #2, we now cite Rios et al., 2025 but note that, despite extensive efforts, we were unable to purify enough well-behaved CDK5RAP2 for our experiments and so could not include it in this analysis. We believe Rios et al., used a full-length MBP-fusion of CDK5RAP2 in their experiments, which may explain this difference as MBP is very good at keeping proteins soluble (but would not be appropriate in our experiments where we compare full-length untagged proteins).

    In Figure 3, reconstitution of Cnn scaffolds using purified CM2 and PReM fragments yields "macromolecular scaffolds," but their physical properties are not defined. It remains unclear whether these assemblies are ordered or amorphous, and whether they exhibit solid- or gel-like behavior. Moreover, the heterogeneous, scattering particles observed by negative-stain EM (Figure S3B), likely corresponding to the Cnn490-608-CM2 complex, raise the possibility of nonspecific aggregation rather than organized scaffold formation. Appropriate controls lacking CM2 are needed to exclude spontaneous aggregation of PReM fragments. In addition, testing shorter truncations of the PReM H2 helix could help define the minimal requirements for scaffold assembly. Finally, the rationale for including the CnnΔExPReM construct only in vivo (Figure 3F), but not in the in vitro assays (Figure 3A-E), should be clarified.

    We apologise, as our presentation of this data has clearly led to some confusion on these points.

    First, as we now clarify, the amorphous solid-like physical properties of the PReM::CM2 scaffolds were described in our previous paper where we also showed that these scaffolds are not simply non-specific aggregates—as several single point mutations that disrupt the LZ::CM2 tetramer also prevent PReM::CM2 scaffold assembly in vitro as well as Cnn scaffold assembly *in vivo *(see Fig.5, Feng et al., Cell, 2017). Also, in all in vitro scaffolding experiments we always perform a negative control (-CM2) to confirm that none of the scaffolds are aggregates of the PReM domain being tested. We don’t usually show this control now as there would be lots of empty black boxes on the Figures. We do, however, show this control for the human putative PReM domain (Figure 7C), as we are testing this here for the first time.

    Second, the request to test shorter truncations of the PReM H2 helix to define the minimal requirements for scaffold assembly is unnecessary as PReM∆H1∆NTH2 already cuts H2 at the start of the LZ, and we previously showed the LZ is required for PReM::CM2 scaffold assembly in vitro (Feng et al., Cell, 2017). Thus, any further truncation of H2 will start to remove the LZ, which we already know is essential. We have now made this point more clearly.

    Finally, the Cnn∆ExPReM construct the Reviewer mentions was tested in both the in vitro (now Figure 2B) and in vivo (now Figure 2F) assays, but the labelling was confusing so this was not clear. We have now clarified this point.

    The coarse-grained (CG) simulation methodology is insufficiently described. Given that CG approaches sacrifice atomic detail and may oversimplify interactions, readers require more information to evaluate the model's reliability and limitations. A comparison with the framework used by Ramirez et al. (2024, PMID: 38356260) would be informative. It is also unclear why available crystal structures of WT and 2A Cnn (Figure 2C; Figure S4) were not used as simulation inputs, or why the structure of Cnn490-579 2E was not determined to complete the structural comparison.Furthermore, mutation of Ser567 and Ser571 to alanine markedly stabilizes the PReM domain (Figure 5C, D), implying that these residues maintain domain flexibility. Back-mapping CG models to atomic resolution could reveal the interactions altered by these mutations. The exclusive focus on double mutants (2A and 2E) is also limiting; analysis of single-point mutants at S567 or S571 would clarify whether both residues contribute equally or play distinct roles.

    We performed coarse-grained simulations because although they simplify atomic interactions and capture overall conformational dynamics, which is what we are trying to assess here (Fig.4C,D). We now clarify this point and provide more detail of our simulation methodology in the main text and Materials and Methods. We used the full helical hairpin (i.e., H2+H3+H4) prediction in these simulations—rather than the crystal structure of the partial helical hairpin (i.e., H2+most of H3)—as we reasoned that the presence of the full H3 and H4 might influence breathing, and the full helical hairpin (see Video S1) seems likely to be the relevant biological fold. As we now show (new Figure S5), and as discussed above, the 2E mutants do not behave well in vitro so we were unable to solve their structure. We agree that we could perform atomic resolution simulations to better understand how the 2A/E and single A/E mutations might suppress/enhance breathing, but we believe such an analysis is beyond the scope of the current manuscript and would distract from our main conclusions.

    The discussion lacks sufficient integration with prior studies and often presents conclusions without adequate citation. For example, the claim that flies and humans rely on related PReM-CM2 interactions whereas worms use distinct phosphorylation-regulated mechanisms is not supported by appropriate references. In addition, limited cross-referencing to the manuscript's own data weakens the connection between results and conclusions. Expanding and better grounding the discussion in existing literature would significantly enhance its depth and clarity. We thank the Reviewer for this general point and have tried to better integrate our results with prior studies—particularly in the Discussion section.

    Minor Comments:

    1. In Figure 1B, the molecular weight units for the protein marker are missing and should be included. Fixed.

    In Figures 1E and 1F, readability would be improved by including x-axis labels on all graphs, rather than only on the bottom panels.Fixed. The protein structures shown in Figures 2C and 2D sh7w b b∫ybb ould be explicitly labeled as dimers to avoid confusion. Fixed. In Figures 3A-D, using fluorescently labeled CM2 would help validate both the interaction with the PReM domain and its localization within the scaffold.We have previously tried fluorescently tagging the CM2 domain, but scaffold formation is much less robust. We do not think this invalidates this assay, as the evidence supporting the PReM::CM2 interaction is very strong—including assessing the physiological influence of multiple point mutations in both domains in residues at the heart of the interaction interface identified by crystallography (e.g., see Fig.4, Feng et al., Cell, 2017).

    In Figure 3E, no statistical comparisons are presented between the original PReM construct and other samples. In addition, information regarding sample size and the number of experimental replicates is missing from the figure legend. Fixed. In Figure 3F, the absence of a pixel intensity scale bar makes the data difficult to interpret, as color values corresponding to high and low signal intensities are unclear. Moreover, no additional centrosome marker is included, nor is there evidence that PReM fragment expression levels are comparable across samples. These concerns also apply to Figures 4C and 4D.We now include pixel intensity scales in all relevant Figures. We think we do not need to show additional centrosome markers in our images as centrosomes exhibit a very reproducible behaviour in these embryos so we can be very confident that the objects we show here are genuine centrosomes. Considering expression levels, the images in Fig.4C,D (now 3C,D) are derived from stable transgenic lines so we can measure protein expression levels and show that the 2A and 2E mutants are expressed at similar levels to WT (new Figure S6). The images in 2F are from mRNA injections, so cannot be quantified in this way. However, we have vast experience with this assay (used in >15 publications since 2014) and can tell when, very occasionally, an injected mRNA is not expressed well (as this leads to a lack of general fluorescence in the cytoplasm). In addition, we know that deletions in Cnn do not generally destabilise the protein as we have analysed many such transgenic lines (see, for example, Reviewer Figure 1). Thus, the differences in centrosomal levels observed and quantified in 2F are almost certainly not caused by differences in the stability of the proteins being generated from the injected mRNAs.

    In Figure 4A, the interacting residues of PReM and CM2 shown in the red inset would be clearer if residue annotations for each domain were displayed in distinct colors. Additionally, the legends for Figures 4C and 4D do not specify the scale bar length.Fixed. The authors state that interactions between CM2 and PReM-2A462-608 could not be detected in vitro based on SEC chromatograms (Figure 5A), yet the figure does not clearly show this result. The accompanying SDS-PAGE images are too small and lack lane labels, making interpretation difficult (a similar issue applies to Figure 7B). Furthermore, the SEC chromatogram x-axis lacks volume annotations, hindering correlation between chromatographic peaks and SDS-PAGE results (in contrast to Figure 7B, which provides an appropriate example).We thank the reviewer for these points, all of which have now been fixed/adjusted.

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    Referee #3

    Evidence, reproducibility and clarity

    This study by Mohamad et al. builds on prior work by Conduit et al. (2014, PMID: 24656740) and Feng et al. (2017, PMID: 28575671), which established the essential role of intramolecular interactions between the phospho-regulated multimerization (PReM) domain and centrosomin motif 2 (CM2) of Drosophila Cnn in pericentriolar matrix (PCM) expansion during mitosis. Extending these studies, the authors investigate the structural properties of Cnn's PReM and CM2 domains and compare them with homologous proteins in C. elegans (SPD-5) and humans (CDK5RAP2). Their analyses suggest a phosphorylation-dependent mechanism that relieves Cnn autoinhibition, with particular emphasis on Ser567 and Ser571 within the PReM domain. The authors further propose that, whereas Cnn and CDK5RAP2 share conserved CM2-PReM interactions, SPD-5 has diverged to employ distinct mechanisms for PCM scaffold assembly.

    Although these conclusions rely heavily on AlphaFold3-predicted models (Abramson et al., 2024, PMID: 38718835), they are supported by a combination of in vitro and in vivo experiments, including live-cell imaging and molecular dynamics simulations. However, inconsistencies between in vitro and in vivo observations weaken some interpretations and warrant more careful discussion. Addressing the concerns below would substantially strengthen the manuscript.

    Major Comments

    1. The title, "Structural Insights into Mitotic-Centrosome Assembly," is overly broad. The study primarily focuses on CM2-PReM intramolecular interactions in D. melanogaster Cnn and does not comprehensively address mitotic centrosome assembly across species. A more specific title reflecting the fly-centric and structural focus would better align with the manuscript's scope and conclusions.
    2. The authors analyze condensate formation by Cnn and SPD-5 but overlook condensate formation by CDK5RAP2, which was recently reported by Rios et al. (2025, PMID: 40454523). Including CDK5RAP2 would enable a more balanced and informative comparison across fly, worm, and human homologs.
    3. In Figure 3, reconstitution of Cnn scaffolds using purified CM2 and PReM fragments yields "macromolecular scaffolds," but their physical properties are not defined. It remains unclear whether these assemblies are ordered or amorphous, and whether they exhibit solid- or gel-like behavior. Moreover, the heterogeneous, scattering particles observed by negative-stain EM (Figure S3B), likely corresponding to the Cnn490-608-CM2 complex, raise the possibility of nonspecific aggregation rather than organized scaffold formation. Appropriate controls lacking CM2 are needed to exclude spontaneous aggregation of PReM fragments. In addition, testing shorter truncations of the PReM H2 helix could help define the minimal requirements for scaffold assembly. Finally, the rationale for including the CnnΔExPReM construct only in vivo (Figure 3F), but not in the in vitro assays (Figure 3A-E), should be clarified.
    4. The coarse-grained (CG) simulation methodology is insufficiently described. Given that CG approaches sacrifice atomic detail and may oversimplify interactions, readers require more information to evaluate the model's reliability and limitations. A comparison with the framework used by Ramirez et al. (2024, PMID: 38356260) would be informative. It is also unclear why available crystal structures of WT and 2A Cnn (Figure 2C; Figure S4) were not used as simulation inputs, or why the structure of Cnn490-579 2E was not determined to complete the structural comparison.

    Furthermore, mutation of Ser567 and Ser571 to alanine markedly stabilizes the PReM domain (Figure 5C, D), implying that these residues maintain domain flexibility. Back-mapping CG models to atomic resolution could reveal the interactions altered by these mutations. The exclusive focus on double mutants (2A and 2E) is also limiting; analysis of single-point mutants at S567 or S571 would clarify whether both residues contribute equally or play distinct roles.

    1. The discussion lacks sufficient integration with prior studies and often presents conclusions without adequate citation. For example, the claim that flies and humans rely on related PReM-CM2 interactions whereas worms use distinct phosphorylation-regulated mechanisms is not supported by appropriate references. In addition, limited cross-referencing to the manuscript's own data weakens the connection between results and conclusions. Expanding and better grounding the discussion in existing literature would significantly enhance its depth and clarity.

    Minor Comments

    1. In Figure 1B, the molecular weight units for the protein marker are missing and should be included.
    2. In Figures 1E and 1F, readability would be improved by including x-axis labels on all graphs, rather than only on the bottom panels.
    3. The protein structures shown in Figures 2C and 2D should be explicitly labeled as dimers to avoid confusion.
    4. In Figures 3A-D, using fluorescently labeled CM2 would help validate both the interaction with the PReM domain and its localization within the scaffold.
    5. In Figure 3E, no statistical comparisons are presented between the original PReM construct and other samples. In addition, information regarding sample size and the number of experimental replicates is missing from the figure legend.
    6. In Figure 3F, the absence of a pixel intensity scale bar makes the data difficult to interpret, as color values corresponding to high and low signal intensities are unclear. Moreover, no additional centrosome marker is included, nor is there evidence that PReM fragment expression levels are comparable across samples. These concerns also apply to Figures 4C and 4D.
    7. In Figure 4A, the interacting residues of PReM and CM2 shown in the red inset would be clearer if residue annotations for each domain were displayed in distinct colors. Additionally, the legends for Figures 4C and 4D do not specify the scale bar length.
    8. The authors state that interactions between CM2 and PReM-2A462-608 could not be detected in vitro based on SEC chromatograms (Figure 5A), yet the figure does not clearly show this result. The accompanying SDS-PAGE images are too small and lack lane labels, making interpretation difficult (a similar issue applies to Figure 7B). Furthermore, the SEC chromatogram x-axis lacks volume annotations, hindering correlation between chromatographic peaks and SDS-PAGE results (in contrast to Figure 7B, which provides an appropriate example).

    Significance

    This work will be of interest not only to cell biologists studying centrosomes, but also to molecular biologists investigating how protein modifications regulate protein behavior.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Mohamed et al. set out to compare the assembly mechanisms of pericentriolar material (PCM) in flies and nematodes. They reveal that the main PCM scaffold protein in each species (Cnn in flies, SPD-5 in nematodes) are sufficient to form supramolecular droplets (with a crowding agent) or networks (without a crowding agent). However, they diverge in one key aspect: Cnn scaffold assembly relies on the interaction between a C-terminal CM2 domain and a central phospho-regulated domain (PReM), whereas SPD-5 does not. The authors solve the crystal structure of a region within Cnn's PReM. With the help of modeling, they speculate that this region is auto-inhibited through backfolding of alpha helices, thus preventing its interaction with the CM2 domain. This auto-inhibition would be relieved by phosphorylation, which modeling suggests would increase "breathing" of the backfolded structure. The author end by presenting evidence to suggest that the human PCM scaffold protein CDK5RAP2 may assemble through a PReM-CM2 interaction.

    Major Comments:

    1. The title is too vague. Any number of existing papers could be said to provide "structural insights into mitotic centrosome assembly". The authors need to narrow down to a defined conclusion and state this as the title.
    2. I think the strongest and most novel aspects of this study relate to the mechanism of Cnn assembly via relief of the auto-inhibited PReM. The effort to elucidate assembly mechanisms of SPD-5 and CDK5RAP2 are comparatively light and there are no accompanying experiments in worms or human cells. Without the in vivo experiments, it's hard to know if the in vitro experiments are valid. It's speculative for the authors to say they found the true PReM for CDK5RAP2; they do not demonstrate that PLK-1 phosphorylation potentiates assembly in Figure 8. Thus, I suggest re-writing the paper to focus on Cnn. Experiments in Figure 6 are still valid if reframed. For example, substituting Cnn's CM2 with the CM2 from CDK5RAP2 vs. the C-term of SPD-5 illustrates that a simple coiled-coil with open ends (H.s.CM2) is sufficient to interact with PReM whereas a coiled-coil with a closed end (SPD-5 C-term, predicted by Figure 6A) cannot.
    3. The purpose of Figure 1 is unclear. None of the other figures examine SPD-5 and CNN in the condensate form, which required using 4% PEG in this paper. The other assays look at the network form, which could behave differently and have different dependence on specific domains. I think they should perform the condensate assay for all other figures, otherwise leave it out. Furthermore, CDK5RAP2 is mentioned, yet not examined in Figure 1. It must be noted that CDK5RAP2 will also condense into droplets under crowding conditions or with a synthetic nucleator (Rios et al., 2025 J Cell Sci). Thus, it seems that condensation potential is a universal feature of known PCM scaffold proteins.
    4. The study uses different species without doing the same types of experiments on each. Sometimes human CDK5RAP2 is thrown in, sometimes not. They solve crystal structures of PReM from Cnn but not from the other proteins. This gets confusing, especially since the authors state that they seek to test if fly Cnn and worm SPD-5 assemble through different mechanisms (see last sentence of the intro). Also, if the focus is on worm vs. fly PCM assembly mechanisms, why include the human protein, especially Figure 8?
    5. The conclusion that SPD-5's narrow PReM and "CM2" domains don't interact is consistent with the cross-linking mass spectrometry data from Rios et al. 2024. They showed only one X-link with low occurrence (1 out of 6 samples) between these two regions, even in the phosphorylated state (Fig. 1G). However, Nakajo et al (2022) claimed the opposite, showing that a larger PReM-containing construct (a.a. 272-732) interacts with a C-terminal construct (a.a. 1061-1198) after PLK-1 phosphorylation. Can the authors comment on this? Perhaps there is another site in SPD-5, outside of a.a. 541-677, that acts like the Cnn PReM?
    6. I have serious doubts that the C-terminus of SPD-5 has a CM2 domain. To me, there is no real sequence homology with the traditional CM2's from humans and flies, and the AF3 predictions support this. Ohta et al. (2021) called this region "CM2-like" based on very poor homology, which a is questionable practice. Any coiled-coil region will appear somewhat homologous due to the heptad repeat pattern that defines them (e.g., leucines line up quite nicely). Thus, is it fair to say that SPD-5 doesn't assemble through a PReM-CM2 interaction? There may be a different region in SPD-5 that looks more like the canonical CM2. I think the authors have compelling evidence to give the C-terminal coiled-coil region in SPD-5 its own name rather than calling it CM2.
    7. Figure 3E. Would measuring scaffold mass be more appropriate? The PReM(deltaH1,NTH2) leads to more compact scaffolds, but maybe they assemble just as well as the deltaH1 mutant. As it stands, there is a discrepancy between panel E and F in terms of what is measured (area vs. intensity) and the outcome.

    Minor Comments

    1. In one version of the PDF there are images missing in Fig 1F, 4C, 4D. I opened another version (source version) and the images were there. Just FYI.
    2. Figure 4A. The blue coloration makes it difficult to read the black letters.
    3. Figure 4A. Why is part of the protein colored in green? This coloration isn't defined, nor does it show up again in panel B.
    4. The layout of Figure 4 is confusing. It took me a few minutes to realize that the big red box inset belonged to panel B and not panel A.
    5. Figure 4C,D. The sample size is not mentioned in the legend.
    6. The title for Figure 4 seems too speculative. How can the authors say that phosphorylation relieves the autoinhibition without structural data?
    7. Figure 5B. The sample size is not mentioned in the legend.
    8. Figure 6B,D. The sample size is not mentioned in the legend.
    9. The text in Figure 7B is hard to read because it is too small. Please make this bigger.
    10. Figure 8C. What is colored in magenta? Is there an additional labeled protein besides mNG-CM2?
    11. Figure 8C. What is the sample size? How many images were taken? Also, why are there data points off to the right of the last column?
    12. The wording of these sections needs improving. I found them complicated and difficult to understand.

    "Fly and worm Spd-2/SPD-2 and Polo/PLK-1 are clear homologues, but Cnn and SPD-5 share little sequence homology-although they are both predicted to be large coiled-coil-rich proteins. Thus, it remains unclear whether these two, largely unrelated, molecules form mitotic-PCM scaffolds that assemble and function in a similar manner"

    "We first focused on Drosophila Cnn as, although the full structure of the original PReM domain (Cnn403-608) is unknown, this domain contains an internal leucine-zipper (LZ) dimer (Cnn490-544) whose crystal structure, in a tetrameric complex with a CM2 dimer, had been solved (Figure 2A) (Feng et al., 2017)."

    "When the full PReM and CM2 domains are mixed in vitro, they form large micron-scale assemblies and point mutations that perturb the LZ::CM2 tetramer perturb PReM::CM2 scaffold assembly in vitro and Cnn scaffold assembly in vivo."

    Significance

    Overall Assessment:

    While I find the premise of this study to be interesting, its execution and presentation are not fully convincing. The study is a collection of experiments connected by a thread that can be difficult to follow. One concern is the lack of focus and a clearly stated conclusion, which is ultimately embodied by the vague title. For example, the research question at the beginning doesn't match with the outcome in the end. At the end of the introduction, the authors state they wish to compare assembly mechanisms of Cnn and SPD-5. However, at the end of the results, they present data on CDK5RAP2 and speculate on its assembly. Why introduce the human protein here? Another concern is the lack of symmetry in the experiments. There is much more in vitro characterization of Cnn than SPD-5 or CDK5RAP2, and all in vivo work is performed in flies. Finally, this study does not address if the best-established model for SPD-5 assembly-multimerization via specific, multivalent coiled-coil interactions-applies to fly Cnn. Thus, to me, this is study is a deeper dive into the mechanism of Cnn assembly, not necessarily a fair cross-species comparison. I do not have major issues with the results, but I recommend that this paper undergo significant re-writing before being re-reviewed. There are also issues with data display and reporting of experimental details (e.g., sample sizes) that should be easily fixed.

    Advance: this study provides new insight into how two specific domains interact within PCM scaffold proteins to promote scaffold assembly. It provides some new structural insight into the mechanism of Cnn auto-inhibition. However, there is limited conceptual advance, as the bigger ideas (e.g., auto-inhibition as a regulatory control, PCM scaffold assembly through condensation of coiled-coil proteins) were already established.

    Audience: this study will be of interest to cell biologists studying centrosome assembly, mitosis, and evolution.

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    Referee #1

    Evidence, reproducibility and clarity

    The study by Mohamad et al. investigates the structural basis and regulatory role of phosphorylation in the assembly of the mitotic pericentriolar material (PCM) scaffold, which nucleates microtubules and organizes the poles of the mitotic spindle. They use structure determination, biochemical reconstitution and in vivo experiment in flies to address how fly, worm, and human homologs of a key scaffold protein (Cnn, SPD-5, and CDK5RAP2, respectively) are relieved from auto-inhibition in a phosphorylation-dependent manner to form extended scaffolds through interactions between PReM and CM2 domains. An important discovery is a helical hairpin structure in the PReM domain that is the basis of autoinhibition and is regulated by phosphorylation. The work addresses the fundamental question how the centrosome matures in preparation for mitosis, by increasing the size and activity of the PCM scaffold that surrounds the centrioles. It also addresses how conserved the underlying molecular mechanism are among flies, worms, and humans. The study is overall of high quality, building on previous works by the authors and other groups, and adding new structural and biochemical insight. Most of the conclusions are supported by the data. I have a few concerns though that should be addressed. An important issue is the analysis of phosphorylation sites, which appears incomplete. For example, it lacks demonstration that both of the two studied phosphorylation sites are indeed phosphorylated. Kinase motif identification and mutation is not sufficient, considering that phosphorylation is integral to the proposed model of how autoinhibitory intra-molecule interactions are relieved, and considering that phospho-mimetics have not been tested in vitro and function poorly in vivo.

    Main:

    1. From previous studies, it seems to me that for the residues potentially relevant for the hairpin regulation there is direct evidence of phosphorylation only for S567 (mass spec, phospho-antibody). Have the authors tested single site mutants (S567A and E)? Also, have they tested D mutations? If so, this should be commented on and shown. If not, it should be tested, in particular since the 2E phospho-mimetic is not functioning properly in vivo. If S571 is indeed crucial, it should be demonstrated that it is also phosphorylated. Otherwise it is possible that the mutation of this residue simply impairs important interactions (e.g. PReM-CM2, others), independent of phosphorylation.

    2. It is unclear why in vitro only A mutations have been tested and not phospho-mimetics. This should be tested for the interaction between PReM and CM2. This would allow to probe the model that phosphorylation opens the hairpin to allow interaction. Currently, such proof is missing in the study. Alternatively, the authors could phosphorylate the recombinant protein in vitro. The in vivo data is harder to interpret due to the complexity of the model and the authors should take advantage of the in vitro system.

    3. Regarding the worm PReM and CM2 domains, the authors mention that they have tested in vitro phosphorylation by PLK-1, but I could not find any data showing this. They should demonstrate successful phosphorylation or test candidate site by phospho-mimetic mutation. It is possible that the worm proteins depend more strongly on phosphorylation to relieve autoinhibition compared to the fly proteins.

    Minor:

    4). Fig. 6C, D: the labeling of the chimeric constructs using "+" symbols is confusing, since it suggests that separate proteins were expressed. If I understand this correctly, with the current labeling, deltaCM2+DmCM2 means WT? The authors should write the full name of the wildtype or chimeric construct in each case and use a more standard/less confusing nomenclature. Also, I suggest to start the panels and graphs with the WT sample.

    Significance

    The study's strength is the use of a combination of structural and biochemical approaches with in vivo model testing. Its main limitation is that the analyses of the role of phosphorylation lacks depth and is not fully conclusive, despite its importance for centrosomal scaffold assembly. The study advances our understanding of centrosomal scaffold assembly and maturation at a molecular level, and how specific molecular aspects of these processes are conserved or differ among different organisms. The findings are of interest to cell biologists. My expertise is in centrosome and microtubule biology.

  5. Version published to 10.1101/2025.10.03.680336 on bioRxiv