Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes

  1. Zihan Zhu
  2. Isabelle Becam
  3. Corinne A. Tovey
  4. Eugenie C. Yen
  5. Fred Bernard
  6. Antoine Guichet
  7. Paul T. Conduit

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Abstract

Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 “core” complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila , Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. Here we show that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but that Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologues in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data therefore help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organising centres. They also suggest that the dynamic properties of microtubules are influenced by how they were nucleated.

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    Reply to the reviewers

    We thank all the Reviewers for their highly constructive reviews. Below, I have pasted the Reviewer’s comments in black and my replies in red, for easy reading.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In their study, Zhu and colleagues study how the centrosome proteins Spd-2 and Cnn in Drosophila recruit gamma-tubulin complexes to centrosomes, which is an important step in mitotic spindle formation. The authors make use of mutant flies and RNAi and find that the two factors Spd-2 and Cnn together are responsible for mitotic centrosomal accumulation of gamma-tubulin. By inactivating Spd-2 or Cnn separately, the authors show that Cnn appears to recruit the large share of mitotic gamma-tubulin pool by its CM1-domain. Interestingly, this involves only gamma-TuSCs (subcomplexes of gamma-TuRC) and not gamma-TuRCs. A smaller pool is recruited by Spd-2, and this pool depends on gamma-tubulin complex proteins that are only present in pre-assembled, complete gamma-TuRCs. This suggests that Drosophila makes microtubule nucleation templates in two ways. First, as in yeast, by direct recruitment of gamma-TuSCs to mitotic centrosomes, where additionally oligomerization needs to happen. And second, by recruitment and activation of preassembled gamma-TuRCs. Inactivation of both Cnn- and Spd-2 pathways abolishes mitosis-specific gamma-tubulin recruitment, resulting in low, but not complete loss of gamma-tubulin at centrosomes. The authors show that these low-gamma-tubulin centrosomes are still able to organize microtubules, but these microtubules have different dynamicity. Inspired by existing literature in flies and other model organisms, the authors identify Msps/Xmap215 as an important nucleation factor in this scenario.

    Major points:

    1. The authors use fly embryos with mutant Grip71, Grip75 and Grip163 alleles, which are central to the study. Most conclusions are based on the assumption that some mutants contain only gamma-TuSC, whereas wildtype cells contain a mix of gamma-TuSC and gamma-TuRC. It would be important to show sucrose gradient analyses of extracts to confirm the expected presence/absence of gamma-TuSC/gamma-TuRC.

    We agree that it would be nice to perform sucrose gradient analysis of γ-tubulin mutants in different mutant backgrounds, but unfortunately this is not as easy as the Reviewer may think. To clarify, we have used larval brain cells (not embryos) for the analysis of γ-tubulin recruitment to centrosomes. We cannot use embryos because most mutant combinations are lethal beyond larval stages, meaning that mutant adult females are not available for embryo collection (embryos use maternally loaded proteins and mRNA and so it is the genotype of the mother that is important). Performing sucrose gradients with larval brain extracts would be extremely challenging, if not impossible, because a relatively large amount of starting material is required for sucrose gradient centrifugation, and manually dissecting and preparing hundreds if not thousands of larval brains is unrealistic, especially as mutant larvae are rare.

    Given that we are not able to carry out these experiments, we have modified the text to include the caveat that some higher-order complexes may partially form in certain mutants. For example, in relation to the ability of Grip71 to recruit γ-TuSCs in cnn,grip75,grip163 mutants, the text now reads: “Thus, Spd-2 appears to recruit a very small amount of γ-TuSCs (which may, or may not, be present as larger assemblies due to an association with Grip128-γ-tubulin) via Grip71 (i.e. the recruitment that occurs in cnn,grip75GCP4,grip163 GCP6 cells), but its recruitment of γ-tubulin complexes relies predominantly on the GCP4/5/4/6 core.”

    Nevertheless, the most important conclusion is that Cnn can recruit γ-TuSCs independent of pre-formed cytosolic γ-TuRCs and this is based on the finding from one particular mutant – the spd-2,grip71,grip75,grip128,grip163 mutant – where γ-tubulin levels at mitotic centrosomes are only very slightly reduced compared to single spd-2 mutants (Figure 1B). This conclusion is based on three assumptions that we argue are all very reasonable:

    Assumption 1: flies depleted of 2, if not all 3, GCP4/5/4/6 core components (grip75,grip128,grip163) do not have a functioning GCP4/5/4/6 core. The Grip75GCP4 allele is a null mutant and is combined with a deficiency chromosome that depletes the whole Grip75GCP4 gene, and the Grip163GCP6 allele is a very strong depletion allele and is also combined with a deficiency chromosome that depletes the whole Grip163GCP6 gene. Even if the efficiency of the RNAi against Grip128GCP5 were poor, it would be hard to form a GCP4/5/4/6 core without Grip75GCP4 and in the near absence of Grip163GCP6 (which together provide 3 of the 4 molecules of the complex, including the outermost ones).

    Assumption 2: cells depleted of the GCP4/5/4/6 core cannot assemble cytosolic γ-TuRCs. This is reasonable given that even individual depletion of Grip75GCP4, Grip128GCP5 or Grip163GCP6 already strongly reduces the presence of cytosolic γ-TuRCs (Vogt et al., 2006; Vérollet et al., 2006). In spd-2,grip71,grip75,grip128,grip163 mutant brain cells, the only γ-TuRC protein not targeted, except for the γ-TuSC components, is Actin (Mozart 1 is expressed only in testes (Tovey et al., 2018) and Mzt2 does not exist in flies). In Xenopus and humans, Actin appears to facilitate γ-TuRC assembly via interactions with a GCP6-N-term-Mzt1 module, and so it would be unlikely to allow γ-TuSC assembly into higher-order complexes without GCP6 (i.e Grip163GCP6) and Mzt1.

    Assumption 3: Were Cnn not able to recruit γ-TuSCs independently of pre-formed γ-TuRCs, we would expect a much stronger reduction in γ-tubulin recruitment to centrosomes in spd-2,grip71,grip75,grip128,grip163 mutant cells. It is reasonable to assume, even without sucrose gradients, that the assembly of γ-TuRCs is strongly impeded in spd-2,grip71,grip75,grip128,grip163 mutant cells. Nevertheless, γ-tubulin is still recruited to centrosomes at ~66% compared to ~77% in spd-2 single mutant cells. While statistically significant (as stated in the updated manuscript), this reduction would surely be much greater were Cnn not able to recruit γ-TuSCs.

    In the absence of experimental data, we have therefore made these arguments in the main text by making some text modifications and adding a new paragraph, as follows:

    *“….the centrosomes in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells had ~66% of the γ-tubulin levels found at wild-type centrosomes, only slightly lower than ~77% in spd-2 mutants alone (Figure 1A,B). Thus, the recruitment of γ-tubulin to mitotic centrosomes that occurs in the absence of Spd-2, i.e. that depends upon Cnn, does not appear to require Grip71 or the GCP4/5/4/6 core. *

    While we cannot rule out that residual amounts of GCP4/5/4/6 core components in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells may support a certain level of γ-TuSC oligomerisation in the cytosol, we favour the conclusion that Cnn can recruit γ-TuSCs directly to centrosomes in the absence of the GCP4/5/4/6 core for several reasons: First, the alleles used for grip71 and grip75GCP4 are null mutants, and the allele for grip163GCP6 is a severe depletion allele (see Methods), and even individual mutations in, or RNAi-directed depletion of, Grip75GCP4, Grip128GCP5 or Grip163GCP6 are sufficient to strongly reduce the presence cytosolic γ-TuRCs (Vogt et al., 2006; Vérollet et al., 2006). Second, spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells are depleted for all structural γ-TuRC components except for γ-TuSCs and Actin (note that Mozart1 (Mzt1) is not expressed in larval brain cells (Tovey et al., 2018) and that Mzt2 does not exist in flies). In human and Xenopus γ-TuRCs, Actin supports γ-TuRC assembly via interactions with a GCP6-N-term-Mzt1 module (Liu et al., 2019; Wieczorek et al., 2019, 2020; Zimmermann et al., 2020; Consolati et al., 2020), and so Actin alone is unlikely to facilitate assembly of γ-TuSCs into higher order structures. Third, our data agree with the observation that near complete depletion of Grip71, Grip75GCP4, Grip128 GCP5, and Grip163GCP6 from S2 cells does not prevent γ-tubulin recruitment to centrosomes (Vérollet et al., 2006). Fourth, given the strength of mutant alleles used, one would have expected a much larger decrease in centrosomal γ-tubulin levels in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells were Cnn not able to directly recruit γ-TuSCs to centrosomes. Thus, our finding that Cnn can still robustly recruit γ-tubulin to centrosomes in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells strongly suggests that Cnn can recruit γ-TuSCs to centrosomes without a requirement for them to first assemble into higher-order complexes.”

    1. Given the advantage of the CnnΔCM1 separation of function mutant, I do not understand why it is not used throughout the study. Instead, full Cnn loss is used, which results in strongly reduced Spd-2 levels (Figure 2C,D). Are the observed differences between wild-type and mutants in Figure 2-5 dependent on defective PCM or do they also occur in a CnnΔCM1 background?

    This is a good point, and we agree that it would have been “cleaner” to use the CnnΔCM1 mutant in these experiments. The reason that the CnnΔCM1 mutant was not used is that this mutant allele was made only after we had already generated the multi-allele stocks and performed most of the other experiments in Figures 2-5. It would have taken a long time to go back and generate fly stocks containing the CnnΔCM1 allele instead of the cnn null mutant allele. As we have shown that the CnnΔCM1 mutant cannot recruit any γ-tubulin, we don’t believe that using this mutant would change the results regarding recruitment of γ-tubulin by the spd-2 pathway i.e. when we have examined γ-tubulin recruitment in the cnn mutant background (Figure 2). Nevertheless, in terms of the efficiency to which microtubules can be nucleated in the absence of γ-tubulin complexes, which was examined in a cnn,grip71,grip163 mutant background, it is likely that using a cnnΔCM1,grip71,grip163 mutant background would better maintain Spd-2 in the PCM and thus better allow Msps and Mei-38 to stimulate microtubule nucleation. We may therefore find that microtubules can be nucleated even more efficiently in the absence of γ-TuRCs. Note that we do state this caveat in the paper. That said, performing the experiments would not be essential to conclude that microtubules can be nucleated independent of γ-TuRCs, which is the main point of this part of the paper.

    Should the Reviewer and Editor deem it necessary, we will generate CnnΔCM1,grip71,grip163 lines to test whether or not γ-tubulin can be recruited to mitotic centrosomes under these conditions, and, if no γ-tubulin is recruited, we will generate CnnΔCM1,grip71 ,grip163,Jup-mCherry lines to test the ability of these centrosomes to nucleate microtubules (using the CherryTemp). Please note, however, that this would be several months of difficult fly genetics and data collection and we would therefore appreciate it if you consider the cost/benefit ratio when making your decision on whether you expect this data or not.

    1. Statistical tests should support the conclusions in the text. If the authors claim differences between different genetic backgrounds (e.g. that spd2-mutants only have ~77% of gamma-tubulin at mitotic centrosomes compared to wild-type), statistical tests must compare mutant mitosis vs. wild-type mitosis.

    We agree. We have now carried out the appropriate statistical tests and included them in the new version of the paper. For more detail, see the response to Reviewer 2 point 2.

    1. While Cnn, grip71, grip163 mutants do not accumulate gamma-tubulin at centrosomes in mitosis, they still have low levels of centrosomal gamma-tubulin. It is therefore misleading to refer to "gamma-tubulin negative centrosomes".

    This is a fair point. While we suspect this small fraction of γ-tubulin is non-functional in regard to microtubule nucleation i.e. it is the interphase pool of γ-tubulin and interphase centrosomes do not organise microtubules, we agree that referring to them as "gamma-tubulin negative centrosomes" is misleading. We have now changed the text to refer to them simply as “cnn,grip71,grip163 mutant centrosomes” or “cnn,grip71,grip163 centrosomes”.

    Minor points:

    1. The abstract states that gamma-TuRC is a catalyst of microtubule nucleation. By definition, a catalyst takes part in a reaction but is not part of the final product. Although our knowledge of the nucleation mechanism is still incomplete, mechanistic studies suggest a non-catalytical mechanism since gamma-TuRC was found to stay attached to the microtubule end after nucleation (Consolati et al. 2020, Wieczorek et al. 2020).

    We have now removed any reference to the γ-TuRC being a catalyst.

    1. CnnΔCM1 flies: genotyping data should be provided besides describing gRNAs.

    We are not entirely sure what the Reviewer means here. We had already stated in the main text and methods that the deletion region spanned from R98 to D167. For further clarity, we now included the word “inclusive” in both the main text and the methods: main text: “We therefore used CRISPR combined with homology-directed repair to delete the CM1 domain (amino acids 98-167, inclusive) from the endogenous cnn gene…”; Methods:“R98 to D167, inclusive”. Please do let us know if further information is required.

    1. Is it important to combine spd-2 with all four mutants, grip75 grip128 grip163 and grip71? What about spd-2 grip71 cells and spd-2 grip75 grip128 grip163 cells? Should that not have the same effect?

    This comment relates to Major point 1, as our main conclusion (that Cnn can recruit γ-TuSCs) is only possible when combining spd2 with all four mutants i.e. targetting all γ-TuRC specific proteins is the most likely way to deplete as many pre-formed γ-TuRCs as possible. Depleting only Spd-2 and Grip71 would leave fully assembled γ-TuRCs in the cytosol, as assembly does not require Grip71. Depleting Spd-2, Grip75, Grip128, and Grip163 would prevent cytosolic γ-TuRC assembly, but there is a possibility that Grip71 may still act as a link between γ-TuSCs and Cnn. It was therefore necessary to deplete Spd-2, Grip75, Grip128, and Grip163, and Grip71.

    1. CM1-containing factors are the only known factors able to directly bind and activate gamma-TuRC. How do the authors envision activation of gamma-TuRC in the absence of Cnn?

    This is a good question but remains unanswered. Phosphorylation of γ-TuRCs is the most obvious possibility. For example, Aurora A phosphorylates NEDD1 (homologue of Grip71) to promote microtubule nucleation (Pinyol et al., 2013). NME7 kinase has been shown to increase the activity of purified γ-TuRCs (Liu et al., 2014). Other γ-TuRC components are also phosphorylated, but the consequences on γ-TuRC activity are not known. Another possibility is that TOG proteins indirectly promote the closing of the γ-TuRCs while adding tubulin dimers onto γ-tubulin (Thawani et al., 2020).

    1. Do the authors think that each identified pathway to microtubule nucleation (i.e. Spd-2/gamma-TuRC, Cnn/gamma-TuSC, Msps/mei38) as revealed by mutant genetic backgrounds contributes to a similar extent to overall nucleation capacity also in an unperturbed genetic background?

    Another good question, but it is very difficult to answer. Our view is that when γ-TuRCs are present and active they will likely dominate microtubule nucleation, out-competing the ability of TOG domain proteins to stimulate microtubule nucleation independently of γ-TuRCs. Nevertheless, TOG proteins will likely help promote microtubule nucleation from γ-TuRCs when both are present, as has been previously shown in vitro (Thawani et al., 2018; King et al., 2020; Consolati et al., 2020) and in fission yeast (Flor-Parra et al., 2018). We also believe that both Spd-2 and Cnn γ-TuRC recruitment pathways will contribute simultaneously. Another question is whether Cnn recruits γ-TuRCs instead of γ-TuSCs when γ-TuRCs are present in the cytosol. We assume this will depend on Cnn’s affinity of γ-TuRCs versus γ-TuSCs and on the relative levels of γ-TuRCs and γ-TuSCs in the cytosol.

    1. How does CM1 mediate binding to gamma-TuRC? Using recombinant Cnn fragments, the authors find that a Cnn triple mutant (R101Q, E102A and F115A) no longer binds gamma-tubulin, suggesting these residues together mediate binding to gamma-tubulin complexes. However, it is not tested to what extent R101, E102 and F115 individually contribute to gamma-tubulin binding. Does the binding mode in Drosophila resemble more the one in humans or in budding yeast? Also, was this done with extracts from Grip71, Grip75, Grip128RNAi, Grip163 embryos or normal embryos?

    In future, we will test the relative contributions of R101, E102 and F115, but for this study we wanted only to show that the CM1 domain was necessary for Cnn binding (hence why we directly mutated all three residues). We apologise for not stating that the IPs were carried out using wild-type embryos extracts – we have now included this information in the main text and methods.

    1. Figure 2C: Should the green channel not correspond to Spd-2?

    Thank you for pointing out this mistake – now corrected.

    1. I suggest to reconsider the color-coding of graphs. While the colored background of the dot plots in Figure 1 and 2 are a matter of taste, the coloring of graphs in Figure 4F-H is confusing. Here, genetic backgrounds of fly lines are colored in the same way as the microscopy channels in Figure 4A-E, but they do not belong together.

    We have now modified the colour-coding of images/graphs in Figure 4A-E as suggested.

    1. A tacc mutant allele is used in experiments, but is not further described. Please provide the necessary background information.

    We thank the reviewer for pointing this out. We had also forgotten to include the msps alleles used. The information for msps and tacc are now included in the methods.

    1. The authors assess spindle quality in Cnn, grip71, grip163 cells and show that spindle quality worsens with ectopic msps. For comparison it would be good to compare spindle quality side by side with a wild-type situation.

    This data is now included in Figure S4A,B.

    1. Introduction: "[...], however, as they depend upon each other for their proper localisation within the PCM and act redundantly." - Sentence is incomplete.

    I think this was just to do with how we had phased the sentence (the position of “however” was confusing). We have now rephrased the sentence: “It is complicated, however, to interpret the individual role of these proteins in the recruitment of γ-tubulin complexes, as they depend upon each other for their proper localisation within the PCM and act redundantly”.

    1. Introduction: "Cnn contains the highly conserved CM1 domain (Sawin et al., 2004), which binds directly to γ-tubulin complexes in yeast and humans (Brilot et al., 2021; Wieczorek et al., 2019)". - Choi et al 2010 should also be cited here.

    This citation has been added.

    1. Results: "Typically, interphase centrosomes have only ~5-20% of the γ-tubulin levels found at mitotic centrosomes, [...]". - Citation is needed

    We now cite our Conduit et al., 2014 paper.

    1. The authors should discuss that Msps was found to act non-redundantly with gamma-tubulin in interphase nucleation (Rogers, MBC, 2008), contrary to the conclusions in the current manuscript.

    Thank you for pointing this out. We have now modified the relevant part of the discussion to read:

    “TOG domain and TPX2 proteins have been shown to work together with γ-TuRCs (or microtubule seed templates) to promote microtubule nucleation (Thawani et al., 2018; Flor-Parra et al., 2018; Gunzelmann et al., 2018b; Consolati et al., 2020; King et al., 2020; Wieczorek et al., 2015). Consistent with this, co-depletion of γ-tubulin and the Drosophila TOG domain protein Msps did not delay non-centrosomal microtubule regrowth after cooling compared to single depletions in interphase S2 cells (Rogers et al., 2008). Nevertheless, several studies, mainly in vitro, have shown that TOG and TPX2 proteins can also function independently of γ-TuRCs to promote microtubule nucleation (Roostalu et al., 2015; Woodruff et al., 2017; Schatz et al., 2003; Slep and Vale, 2007; Ghosh et al., 2013; Thawani et al., 2018; King et al., 2020; Zheng et al., 2020; Tsuchiya and Goshima, 2021; Imasaki et al., 2022). Our data suggest that, unlike from non-centrosomal sites in interphase S2 cells, Msps can promote γ-TuRC-independent microtubule nucleation from centrosomes in mitotic larval brain cells. This difference may reflect the ability of centrosomes to concentrate Msps at a single location.”

    **Referees cross-commenting**

    This is a good paper in my opinion, they need to add some controls though, to determine the expected presence/absence of gTuSC/gTuRC in the different mutants. An important advance is the finding that gTuSC can function as nucleator in parallel to gTuRC, depending on the recruitment mechanism. Different recruitment mechanisms, nucleation templates, and regulatory strategies co-exist and provide complex regulation and robustness to nucleation/spindle assembly. We thank the Reviewer for their thorough and constructive review. We hope they will agree to allow publication without us having to perform the sucrose gradient experiments that, as discussed above, will be very difficult, if not impossible, to carry out.

    Reviewer #1 (Significance (Required)):

    This is a very well-executed study and the data is presented clearly. However, some findings would benefit from additional experiments to substantiate the main interpretations. If these points are addressed, the study would provide an important conceptual advance in the field, namely that animal cells may rely on two different gamma-tubulin complexes for nucleation at mitotic centrosomes, gamma-TuSC and gamma-TuRC, which differ not only in their composition of GCP proteins but also the mode of recruitment to the centrosome. The findings will be of interest to all cell biologists.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary This paper sets out to further our understanding of how two proteins, Cnn and Spd-2, independently recruit g-Tubulin ring complexes(g-TuRC) to mitotic centrosomes in Drosophila cells. It uses some robust classical genetics to generate mutants to reduce/remove GCP4/5/6, Dgrip71 and Cnn and Spd-2 from cells, monitoring the consequences using live imaging.

    It begins by showing that Cnn can recruit g-Tubulin independently of the core g-TuRC components or Dgrip71, and that a mutant Cnn lacking the CM1 domain cannot, strongly suggesting that, similarly to other organisms, the CM1 domain is essential for this function.

    It then demonstrates that Spd-2, in contrast, cannot localise g-Tubulin in the absence of the g-TuRC components or Dgrip71.

    In the second half of the paper, then use this tool as a proxy for centrosomes that completely lack mitotic g-Tubulin recruitment, in order to explore spindle assembly in the absence of centrosomal g-Tubulin. The show that microtubules and spindle are still nucleated but do so with different dynamics. This section is particularly convincing, given the use of the live de/repolymerisation assays using the CherryTemp device.

    Finally, the authors visualise spindle formation in the absence of centrosomal g-Tubulin, alongside a number of other MT associated proteins, including Msps.

    Major Comments

    1. The claims and conclusions relating to the first half of the paper are supported by the data, but they need to be caveated by a clear explanation of the alleles used. Some are well-characterised mutant lines but have they been previously shown to completely remove the associated protein products? For the RNAi lines, do the authors have evidence (via Western blots) that these remove the protein products? It is not necessary that they show Western blots for all the lines, and it does not invalidate the major conclusions that the fly line carrying mutations in cnn, grip71, grip163 completely fails to localise g-Tubulin to mitotic centrosomes. However, they need to help the reader understand much more clearly whether these lines are complete nulls and, consequently this may impact the strength of their interpretation of the relationship between Grip163 versus Grip75, discussed both at the end of the relevant section and in the Discussion.

    We appreciate the reviewer’s concern and have now included a detailed description in the Methods section of the alleles we use and their known effect on protein levels (pasted below for convenience). We have also included western blots for cnn and spd2 mutants to show the absence of detectable protein in larval brains. Unfortunately, we could not provide western blots for the other mutants, as we don’t have working antibodies for these proteins (although for Grip71 we did make an antibody and did western blots that showed the absence of protein in grip71 mutants, but this antibody has now been commercialised and so the western blot is published on the CRB website: https://crbdiscovery.com/polyclonal-antibodies/anti-grip71-antibody/). Nevertheless, protein levels for the grip75, grip163, msps and tacc mutants have been shown previously (now cited in the new text). We have also modified the main text to allow the reader to better understand whether proteins are completely absent or strongly reduced. In response to the specific comment about interpreting the relationship between Grip163 and Grip75, as we mention in the new methods section, the Grip75 allele is a null mutant while the Grip163 mutant is a severe depletion; thus, the fact that the Grip163 mutant has a stronger effect on γ-tubulin recruitment is not due to a stronger depletion.

    New text in methods: “For spd-2 mutants, we used the dspd-2Z35711 mutant allele, which carries an early stop codon resulting in a predicted 56aa protein. Homozygous dspd-2Z35711 mutant flies lack detectable Spd-2 protein on western blots and so the allele is therefore considered to be a null mutant (Giansanti et al., 2008). This allele no longer produces homozygous flies (which is common for mutant alleles kept as balanced stocks for many years), which combined dspd-2Z35711 with a deficiency that includes the entire spd-2 gene (dspd-2Df(3L)st-j7). On western blots, there was no detectable Spd-2 protein in extracts from dspd-2Z35711 / dspd-2Df(3L)st-j7 hemizygous mutant brains (Figure S4B). For cnn mutants, we combined the cnnf04547 and cnnHK21 mutant alleles. The cnnf04547 allele carries a piggyBac insertion in the middle of the cnn gene and is predicted to disrupt long Cnn isoforms, including the centrosomal isoform (Cnn-C or Cnn-PA) (Lucas and Raff, 2007). This mutation is considered to be a null mutant for the long Cnn isoforms (Lucas and Raff, 2007; Conduit et al., 2014). The cnnHK21 allele carries an early stop codon after Cnn-C’s Q78 (Vaizel-Ohayon and Schejter, 1999) and affects both long and short Cnn isoforms – it is considered to be a null mutant (Eisman et al., 2009; Chen et al., 2017a). On western blots, there was no detectable Cnn-C protein in cnnf04547 / cnnHK21 hemizygous mutant brains (Figure S4A). For Grip71, we used the grip71120 mutant allele, which is a result of an imprecise p-element excision event that led to the removal of the entire grip71 coding sequence except for the last 12bp; it is considered to be a null mutant (Reschen et al., 2012). We combined this with an allele carrying a deficiency that includes the entire grip71 gene (grip71Df(2L)Exel6041). On western blots, there is no detectable Grip71 protein in grip71120 / grip71df6041 hemizygous mutant brains (see blots on CRB website, which were performed by us). For Grip75GCP4, we used the grip75175 mutant allele, which carries an early stop codon after Q291. Homozygous grip75175 mutant flies lack detectable Grip75GCP4 protein on western blots and so the allele is therefore considered to be a null mutant (Schnorrer et al., 2002). We combined this with an allele carrying a deficiency that includes the entire grip75GCP4 gene (grip75Df(2L)Exel7048). In the absence of a working antibody, we have not confirmed the expected absence of Grip75GCP4 protein in grip75175 / grip75Df(2L)Exel7048 hemizygous mutant flies on western blots. For Grip128GCP5, we used the UAS-controlled grip128-RNAiV29074 RNAi line, which is part of the VDRC’s GD collection, and drove its expression using the Insc-Gal4 driver (BL8751), which is expressed in larval neuroblasts and their progeny. In the absence of a working antibody, we have not confirmed the absence or reduction of Grip128GCP5 protein on western blots. RNAi was used for grip128GCP5 as its position on the X chromosome made generating stocks with multiple alleles technically challenging. For Grip163GCP6, we used the grip163GE2708 mutant allele, which carries a p-element insertion between amino acids 822 and 823 (total protein length is 1351aa) and behaves as a null or strong hypomorph mutant (Vérollet et al., 2006). We combined this with an allele carrying a deficiency that includes the entire grip163GCP6 gene (grip163Df(3L)Exel6115). In the absence of a working antibody, we have not confirmed the absence or reduction of Grip163GCP6 protein in grip163GE2708 / grip163Df(3L)Exel6115 hemizygous mutant flies on western blots. For Msps, we used the mspsp and mspsMJ15 mutant alleles. The mspsp allele carries a p-element insertion within, or close to, the 5’ UTR of the msps gene and results in a strong reduction, but not elimination, of Msps protein on western blots (Cullen et al., 1999). The mspsMJ15 allele was generated by re-mobilisation of the p-element (the genetic consequence of which has not been defined) and also results in a strong reduction, but not elimination, of Msps protein on western blots (Cullen et al., 1999; Lee et al., 2001). For TACC, we used the taccstella allele which contain a p-element insertion of unknown localisation but that results in no detectable TACC protein on western blots (Barros et al., 2005). For Mei-38, we used the UAS-controlled mei-38-RNAiHMJ23752 RNAi line, which is part of the NIG’s TRiP Valium 20 collection, and drove its expression using the Insc-Gal4 driver (BL8751). In the absence of a working antibody, we have not confirmed the absence or reduction of Mei-38 protein on western blots. RNAi was used for mei-38 as its position on the X chromosome made generating stocks with multiple alleles technically challenging. Moreover, the only available mutant of mei-38 affects a neighbouring gene.”

    I have an issue with the statistics in Figure 1 &2. I realise the t-tests in Figure 1 show the significant differences between g-Tubulin recruitment to centrosomes in interphase and mitosis, in order to demonstrate the difference between the Spd-2;Grip combination line in (B) and the Spd-2; CnnCM1 double mutant in (D). But in doing so, it draws attention to the fact that there is no similar t-test between mitotic g-Tubulin recruitment to centrosomes in WT, Spd-2 and the Spd-2;Grip combination lines. This lack of stats between conditions is further confused by the language used in the text: In the Figure legend, the authors claim mitotic centrosomal g-Tubulin levels between are WT, Spd-2 and the Spd-2;Grip combination lines "similar", and in the text they say: the spd-2 Grip combination line had g-Tubulin "similar to the levels found at spd-2 mutants alone". But then they give numbers - an average of 77% of wild type for spd2 and 66% of wild type for the spd-2 Grip combination. I'm sure if they did a t-test they would find a significant difference between these conditions. This doesn't invalidate the thrust of what they're claiming, but they do need to be consistent in language, analysis and interpretation.

    We agree that we should have performed a statistical comparison between the γ-tubulin levels for “WT mitosis” vs “spd2 mitosis” and for “spd-2 mitosis” vs “spd2,grip71,grip75,grip128,grip163 mitosis” (Figure 1B). We have now done this and found statistically significant differences in both cases. We have included the new p-values in the figure and modified the main text to read: “In fact, the centrosomes in these spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells had ~66% of the γ-tubulin levels found at wild-type centrosomes, only slightly lower than the levels found at spd-2 mutants alone (Figure 1A,B).”; and we have modified the legend to read: “A one-way ANOVA with a Sidak’s multiple comparisons test was used to make the comparisons indicated by p values in the graph. Note that there is only a small reduction in mitotic centrosomal γ-tubulin levels in spd-2 mutants and in spd-2, grip71,grip75GCP4, grip128GCP5-RNAi,grip163GCP6 mutants, showing that Cnn can still efficiently recruit γ-tubulin complexes to mitotic centrosomes when only γ-TuSCs are present.” Note that due to performing comparisons multiple times with the same data sets, it was necessary to use a one-way ANOVA with a Sidak’s multiple comparisons test (rather than paired t-tests).

    For Figure 1D, we did not compare WT mitosis vs cnn∆CM1,spd-2 mitosis, as the point here was to test whether there was an increase from interphase to mitosis in cnn∆CM1,spd-2 mutants and we wanted to maintain the statistical power of using a paired t-test (one is more likely to detect differences with a paired t-test than with a multiple comparisons ANOVA, making the conclusion that there is no difference between interphase and mitotic cnn∆CM1,spd-2 centrosomes even more solid).

    Similarly, in Figure 2, it would be better to assess any statistically significant difference between mitotic accumulation of g-Tubulin between fly lines, rather than accumulation between interphase and mitosis (which is pretty clear cut). This would help to clarify whether differences between loss of grip subunits are merely additive or synergistic. Again, this doesn't invalidate the overall result that concomitant loss of cnn, grip71 and grip163 completely abolishes mitotic centrosomal accumulation of g-Tubulin, but it is a more complete analysis of the extant data.

    As for Figure 2, we respectfully disagree that we should make comparisons between genotypes instead of, or in addition to, making comparisons between interphase and mitotic centrosomes within the same genotype. This is because we will lose statistical power by performing a multiple comparisons test. Indeed, if we were to compare both within and between selected genotypes (14 comparisons in total), then we lose the statistically significant differences between interphase and mitotic centrosomes in cnn,grip75,grip163 (p=0.04) and cnn,grip71,grip75 (p=0.08) genotypes, when there clearly appears to be a difference (as stated by the Reviewer). Given that the point of this experiment is to elucidate which proteins are required to allow maturation from interphase to mitosis, rather than which combination of mutations has the stronger effect, we feel that maintaining the paired t-test analysis is more appropriate.

    One OPTIONAL experiment that would significantly improve the study would be similar CherryTemp live imaging of the cells lacking both centrosomal g-Tubulin and Msps. Currently the manuscript finishes with a fixed analysis of MT de/repolymerisation in these cells, which provides evidence that Msps has a role in MT nucleation in the absence of centrosomal g-Tubulin-nucleated MTs, but very little else can be concluded.

    We would love to do this experiment but the genetics are complicated. We would have to generate stocks containing a cnn,grip71,GFP-PACT triple allele chromosome II and a grip163,msps,Jupiter-mCherry triple allele chromosome III. While live data would provide interesting insights into the dynamics of microtubules nucleated in the absence of γ-TuRCs and reduction of Msps, our fixed analysis is at least sufficient to implicate Msps in γ-TuRC-independent microtubule organisation.

    1. There is, perhaps surprisingly, no mention of Augmin in the paper. Augmin has been shown to recruit g-TuRC to pre-existing MTs, through the grip71 subunit (Chen et al., 2017). So, presumably, in cnn, grip71, grip163, g-Tubulin cannot be recruited to pre-existing MTs either? This could add impact to the results - in that it implies the MT nucleation seen in the absence of cnn, grip71 and grip163 actually reflects, not just loss of centrosome function, but also loss of Augmin function. Mentioning this in the discussion could help increase the impact of the paper.

    We apologise for this oversight. Indeed, it is perfectly possible that Grip71/Augmin-mediated amplification of microtubules during microtubule re-growth from centrosomes could influence the difference in recovery rates between control and mutant centrosomes. We have now modified the results section to read:

    “Our data suggest that microtubules are more resistant to cold-induced depolymerisation when they have been nucleated independently of γ-TuRCs, but that microtubules are nucleated more efficiently when γ-TuRCs are present. However, it must be considered that, due to the loss of Cnn from centrosomes in the cnn,grip71,grip163 mutant cells, general PCM levels are reduced, likely reducing the levels of any protein involved in γ-TuRC-independent microtubule nucleation. Moreover, Grip71 is necessary for γ-TuRC recruitment to microtubules, most likely via the Augmin complex (Reschen et al., 2012; Chen et al., 2017b; Dobbelaere et al., 2008; Vérollet et al., 2006), enabling microtubules to be nucleated from the sides of pre-existing microtubules. Thus, the potential for Augmin-mediated amplification of centrosome-nucleated microtubules in control cells may also contribute to the increased microtubule recovery speed in control cells. Importantly, however, both of these caveats make it even clearer that microtubules can be nucleated independently of γ-TuRCs from mitotic centrosomes in Drosophila.”

    Minor comments

    1. The cnn, grip71, grip163 mutant image in Fig3 B after 40 min cooling appears to have 4 centrioles. Is this a cell that exited and re-entered mitosis?

    Cnn mutant cells often have centrosome segregation problems, resulting in cells with variable numbers of centrioles (Conduit et al., 2010b, Current Biology). We have now mentioned this in the legends for Figure 3, Figure 4, and Figure S4.

    Methods should contain more detail on the de/repolymerisation live imaging analysis (including the numbers of cells contributing to the analysis) and techniques such exponential curve fitting.

    We have now included this information in the methods and updated this information in the figure legend (to include cell numbers, not just centrosome numbers, and to indicate that GraphPad Prism was used to generate the models.

    P5 para 2 - "GPC4/5/4/6" should read "GCP4/5/6"

    We actually use the GCP4/5/4/6 nomenclature throughout as it represents the 2 copies of GCP4 to one copy of GCP5 and GCP6 in the complex, as well as the order of these molecules.

    Fig legend 1 - "error bar" should read "scale bar"

    Thanks, now corrected

    Reviewer #2 (Significance (Required)):

    The experimental approach (genetics and cell biology) taken in this manuscript is very appropriate and the experiments are of high quality. It uses the strengths of Drosophila to cleverly engineer flies to pull apart the relationship between two different ways to recruit the main MT nucleator, g-Tubulin, to mitotic centrosomes. This is an important advance for the specific research field of centrosome biology.

    By generating a fly that completely fails to localise g-Tubulin to mitotic centrosomes, the paper is able to explore whether MTs and the mitotic spindle can form in its absence. Again, there is very high quality imaging and image analysis, using a commercially available (but very cool) fast heating/cooling apparatus - the CherryTemp to explore the dynamics of MT generation. The limitation to this approach, though, is that g-Tubulin itself is still present and presumably able to nucleate MTs in the cytosol or elsewhere, albeit inefficiently. As such, it adds to a body of centrosomal and cell division research, rather than adding a highly significant conceptual advance.

    Similarly, the finding that Msps is involved in nucleating MTs in the absence of centrosomal g-Tubulin, via fixed analysis, supports other work, rather than moving the field forwards.

    Overall, assuming the caveats mentioned in the major comments are dealt with, I see this as a robust and very well carried out piece of research, that will be of interest to those investigating the broad field of cell division

    My field of expertise is Drosophila cell division

    We thank the Reviewer for their thorough and constructive review. We hope that the reviewer may agree with us and the other Reviewers that revealing the complexity of γ-TuRC recruitment and microtubule nucleation at centrosomes, particularly the finding that different types of γ-tubulin complexes are recruited to centrosomes by different tethering proteins, provides an important conceptual advance.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Centrosomes are complex and it has been appreciated for some time that they likely nucleate microtubules by more than one mechanism. However, what these mechanisms exactly are, and which are the most significant has not been clear. A major contributor to centrosomal microtubule nucleation the tubulin isoform gamma-tubulin (g-tubulin), which is present in two complexes, a smaller gTuSC that contains gamma tubulin along with GCP2 and 3 and a larger g-tubulin ring complex (gTuRC) whose assembly additionally requires GCP4/5/6. A second high-level question has been whether the centrosome has any g-tubulin-independent microtubule nucleation mechanisms. In this manuscript, the authors use a collection of mutants and RNAi conditions in the Drosophila brain to generate a picture of centrosomal microtubule nucleation pathways. They show that there are two g-tubulin-dependent and a third g-tubulin-independent microtubule nucleation pathways. They show that the first g-tubulin-dependent pathway depends on the CM1 domain of the centrosomal PCM matrix protein, Centrosomin (Cnn) and on the gTuSC components GCP2/3, but not on the components specifically required for gTuRC assembly. The second g-tubulin-dependent pathway depends on Spd-2 (and not Cnn) and requires the gTuRC-specific components and NEDD1/Grip71. By inhibiting both of these pathways, the authors also show that there is a robust g-tubulin-independent microtubule nucleation pathway. Overall, has the potential to be an impactful contribution from a conceptual point-of-view. I would be excited to recommend publication if the major comments below, particularly points 1 and 2, could be addressed.

    1. The experiment in Fig. 2B examines what is required for Spd-2 to recruit g-tubulin to mitotic centrosomes that lack Cnn. This panel should include a cnn mutant-only control, for which the readers are currently referred to an older paper from 2014. Without repeating this control in parallel to one of the conditions in this panel, it is impossible to say whether the addition of the grip71 mutation has any effect on g-tubulin levels.

    This is a good point. We will perform a cnn vs cnn,grip71 experiment and include this data in the new version of the paper. This will take a couple of months, as this will involve growing up fly lines, performing the necessary crosses, microscopy, data analysis, and manuscript updating.

    1. The experiment in Fig. 2B is in the background of a Cnn loss-of-function mutation in which centrosomal Spd-2 is at just under 40% of its levels in brains with Cnn (according to Fig. 2D). So the Spd-2 doing the recruiting-is the non-Cnn-dependent population. The authors should also do one experiment in the background of their Cnn-CM1delete mutant or their Cnn CM1 g-tubulin recruitment mutant, because these backgrounds would be expected to have normal amounts of Cnn matrix and normal levels of Spd-2. Comparing the amount of g-tubulin recruitment in a cnn loss-of-function mutant to that in a cnn-CM1delete mutant would reveal whether the Cnn-bound Spd-2 can contribute to g-tubulin recruitment in the same way that the Cnn-independent Spd-2 can. These two populations could easily differ in their ability to recruit g-tubulin. Also, is it clear that these two pathways can act in parallel (i.e. that assembly of the Cnn matrix around the centriole does not mask the ability of Cnn-independent Spd-2 to recruit g-tubulin)? Thus, there are three possibilities- all interesting- for the outcome of this experiment. The Cnn-CM1delete mutant/Cnn-CM1 g-tubulin recruitment mutants could: (1) recruit less g-tubulin than the cnn loss-of function mutant (if Cnn matrix assembly inhibits the Cnn-independent Spd-2 pathway), (2) recruit the same amount of g-tubulin as the cnn loss-of-function mutant (if the Cnn matrix does not inhibit the Cnn-independent Spd-2 pathway but Cnn-dependent Spd-2 does not itself recruit g-tubulin), or (3) recruit more g-tubulin than the cnn loss-of-function mutant (if both the Cnn-dependent and Cnn-independent Spd-2 can recruit g-tubulin).

    These are very interesting points that we have not considered before. As the reviewer suggests, we will perform an analysis of γ-tubulin levels at centrosomes in cnnnull vs cnn∆CM1 to test the ability of Cnn-dependent and Cnn-independent populations of Spd-2 to recruit γ-tubulin. This should take ~2 months.

    1. The paper needs a summary model figure that the field can understand. The current model in Fig. 2E does not suffice in this regard. It would be nice to have this model appear at the end of the paper to outline the 3 pathways for centrosomal microtubule nucleation outlined by the work. Maybe have an arc for the centrosome at the bottom of the figure and show arrows from the gTuSC to the Cnn CM1 domain from the gTuRC to the Cnn CM1 domain and the gTuRC to Spd-2 or something like this. How you draw this could be impacted by the experiment outlined above in point 2. Also, there would be a g-tubulin-independent pathway in the figure. Not everyone reads papers carefully, and you want people to be able to get the takeaway message at a glance.

    We have now completely modified the Figure and moved it to the end of the paper (new Figure 5). We thank the Reviewer for this suggestion as it really does provide a clearer message for the reader.

    1. The authors show that this pathway is modulated by loss of Minispindles (Msps)-but as this is a critical microtubule assembly factor, it seems likely that Msps loss might modulate all of the pathways. From the data in Figure 4, my main takeaway would be that Msps is not the central player in the g-tubulin independent nucleation pathway. It might make the paper more impactful to end the story after Fig. 4, move the current Fig. 5 to the supplement and add a nice model figure at the end.

    We agree that Msps may play a role beyond microtubule nucleation, including plus end growth, and that this may also influence the efficiency of spindle formation in cnn,grip71,grip163,msps mutants. Nevertheless, our microtubule regrowth data in original Figure 5A clearly show that Msps is a key player in the g-tubulin independent nucleation pathway at centrosomes. Perhaps the Reviewer missed this point as the data was in Figure 5 and not Figure 4. Moreover, the original Figure 5E shows that the effect of depleting Msps in addition to cnn, grip71 and grip163 is specific to cells containing centrosomes i.e. if Msps played a significant role in microtubule regulation beyond its role at centrosomes, then one would expect spindle formation to be worse when comparing mutant cells that lack centrosomes. Nevertheless, we now realise it would be better to include the microtubule regrowth from centrosomes data for cnn,grip71,grip163 vs cnn,grip71,grip163,msps in Figure 4, and move the spindle assembly data from original Figure 5C-E to a new supplementary Figure (Figure S4). We then end the paper on a model figure in new Figure 5.

    Minor comments:

    1. In Fig. 1E the sequence labels are confusing. Please label each sequence on the left with the residue numbers in the corresponding endogenous protein that are shown in the alignment.

    You are absolutely right, I’m not sure why our labelling was like that. Now corrected.

    In Fig. 1F, please label with location of molecular weight markers

    Now added.

    Reviewer #3 (Significance (Required)):

    Repeating my text from above. Centrosomes are complex and it has been appreciated for some time that they likely nucleate microtubules by more than one mechanism. However, what these mechanisms exactly are, and which are the most significant has not been clear. A major contributor to centrosomal microtubule nucleation the tubulin isoform gamma-tubulin (g-tubulin), which is present in two complexes, a smaller gTuSC that contains gamma tubulin along with GCP2 and 3 and a larger g-tubulin ring complex (gTuRC) whose assembly additionally requires GCP4/5/6. A second high-level question has been whether the centrosome has any g-tubulin-independent microtubule nucleation mechanisms. In this manuscript, the authors use a collection of mutants and RNAi conditions in the Drosophila brain to generate a picture of centrosomal microtubule nucleation pathways. They show that there are two g-tubulin-dependent and a third g-tubulin-independent microtubule nucleation pathways. They show that the first g-tubulin-dependent pathway depends on the CM1 domain of the centrosomal PCM matrix protein, Centrosomin (Cnn) and on the gTuSC components GCP2/3, but not on the components specifically required for gTuRC assembly. The second g-tubulin-dependent pathway depends on Spd-2 (and not Cnn) and requires the gTuRC-specific components and NEDD1/Grip71. By inhibiting both of these pathways, the authors also show that there is a robust g-tubulin-independent microtubule nucleation pathway. Overall, has the potential to be an impactful contribution from a conceptual point-of-view. I would be excited to recommend publication if the major comments, particularly points 1 and 2, could be addressed.

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    Referee #3

    Evidence, reproducibility and clarity

    Centrosomes are complex and it has been appreciated for some time that they likely nucleate microtubules by more than one mechanism. However, what these mechanisms exactly are, and which are the most significant has not been clear. A major contributor to centrosomal microtubule nucleation the tubulin isoform gamma-tubulin (g-tubulin), which is present in two complexes, a smaller gTuSC that contains gamma tubulin along with GCP2 and 3 and a larger g-tubulin ring complex (gTuRC) whose assembly additionally requires GCP4/5/6. A second high-level question has been whether the centrosome has any g-tubulin-independent microtubule nucleation mechanisms. In this manuscript, the authors use a collection of mutants and RNAi conditions in the Drosophila brain to generate a picture of centrosomal microtubule nucleation pathways. They show that there are two g-tubulin-dependent and a third g-tubulin-independent microtubule nucleation pathways. They show that the first g-tubulin-dependent pathway depends on the CM1 domain of the centrosomal PCM matrix protein, Centrosomin (Cnn) and on the gTuSC components GCP2/3, but not on the components specifically required for gTuRC assembly. The second g-tubulin-dependent pathway depends on Spd-2 (and not Cnn) and requires the gTuRC-specific components and NEDD1/Grip71. By inhibiting both of these pathways, the authors also show that there is a robust g-tubulin-independent microtubule nucleation pathway. Overall, has the potential to be an impactful contribution from a conceptual point-of-view. I would be excited to recommend publication if the major comments below, particularly points 1 and 2, could be addressed.

    1. The experiment in Fig. 2B examines what is required for Spd-2 to recruit g-tubulin to mitotic centrosomes that lack Cnn. This panel should include a cnn mutant-only control, for which the readers are currently referred to an older paper from 2014. Without repeating this control in parallel to one of the conditions in this panel, it is impossible to say whether the addition of the grip71 mutation has any effect on g-tubulin levels.
    2. The experiment in Fig. 2B is in the background of a Cnn loss-of-function mutation in which centrosomal Spd-2 is at just under 40% of its levels in brains with Cnn (according to Fig. 2D). So the Spd-2 doing the recruiting-is the non-Cnn-dependent population. The authors should also do one experiment in the background of their Cnn-CM1delete mutant or their Cnn CM1 g-tubulin recruitment mutant, because these backgrounds would be expected to have normal amounts of Cnn matrix and normal levels of Spd-2. Comparing the amount of g-tubulin recruitment in a cnn loss-of-function mutant to that in a cnn-CM1delete mutant would reveal whether the Cnn-bound Spd-2 can contribute to g-tubulin recruitment in the same way that the Cnn-independent Spd-2 can. These two populations could easily differ in their ability to recruit g-tubulin. Also, is it clear that these two pathways can act in parallel (i.e. that assembly of the Cnn matrix around the centriole does not mask the ability of Cnn-independent Spd-2 to recruit g-tubulin)? Thus, there are three possibilities- all interesting- for the outcome of this experiment. The Cnn-CM1delete mutant/Cnn-CM1 g-tubulin recruitment mutants could: (1) recruit less g-tubulin than the cnn loss-of function mutant (if Cnn matrix assembly inhibits the Cnn-independent Spd-2 pathway), (2) recruit the same amount of g-tubulin as the cnn loss-of-function mutant (if the Cnn matrix does not inhibit the Cnn-independent Spd-2 pathway but Cnn-dependent Spd-2 does not itself recruit g-tubulin), or (3) recruit more g-tubulin than the cnn loss-of-function mutant (if both the Cnn-dependent and Cnn-independent Spd-2 can recruit g-tubulin).
    3. The paper needs a summary model figure that the field can understand. The current model in Fig. 2E does not suffice in this regard. It would be nice to have this model appear at the end of the paper to outline the 3 pathways for centrosomal microtubule nucleation outlined by the work. Maybe have an arc for the centrosome at the bottom of the figure and show arrows from the gTuSC to the Cnn CM1 domain from the gTuRC to the Cnn CM1 domain and the gTuRC to Spd-2 or something like this. How you draw this could be impacted by the experiment outlined above in point 2. Also, there would be a g-tubulin-independent pathway in the figure. Not everyone reads papers carefully, and you want people to be able to get the takeaway message at a glance.
    4. The authors show that this pathway is modulated by loss of Minispindles (Msps)-but as this is a critical microtubule assembly factor, it seems likely that Msps loss might modulate all of the pathways. From the data in Figure 4, my main takeaway would be that Msps is not the central player in the g-tubulin independent nucleation pathway. It might make the paper more impactful to end the story after Fig. 4, move the current Fig. 5 to the supplement and add a nice model figure at the end.

    Minor comments:

    1. In Fig. 1E the sequence labels are confusing. Please label each sequence on the left with the residue numbers in the corresponding endogenous protein that are shown in the alignment.
    2. In Fig. 1F, please label with location of molecular weight markers

    Significance

    Repeating my text from above. Centrosomes are complex and it has been appreciated for some time that they likely nucleate microtubules by more than one mechanism. However, what these mechanisms exactly are, and which are the most significant has not been clear. A major contributor to centrosomal microtubule nucleation the tubulin isoform gamma-tubulin (g-tubulin), which is present in two complexes, a smaller gTuSC that contains gamma tubulin along with GCP2 and 3 and a larger g-tubulin ring complex (gTuRC) whose assembly additionally requires GCP4/5/6. A second high-level question has been whether the centrosome has any g-tubulin-independent microtubule nucleation mechanisms. In this manuscript, the authors use a collection of mutants and RNAi conditions in the Drosophila brain to generate a picture of centrosomal microtubule nucleation pathways. They show that there are two g-tubulin-dependent and a third g-tubulin-independent microtubule nucleation pathways. They show that the first g-tubulin-dependent pathway depends on the CM1 domain of the centrosomal PCM matrix protein, Centrosomin (Cnn) and on the gTuSC components GCP2/3, but not on the components specifically required for gTuRC assembly. The second g-tubulin-dependent pathway depends on Spd-2 (and not Cnn) and requires the gTuRC-specific components and NEDD1/Grip71. By inhibiting both of these pathways, the authors also show that there is a robust g-tubulin-independent microtubule nucleation pathway. Overall, has the potential to be an impactful contribution from a conceptual point-of-view. I would be excited to recommend publication if the major comments, particularly points 1 and 2, could be addressed.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This paper sets out to further our understanding of how two proteins, Cnn and Spd-2, independently recruit g-Tubulin ring complexes(g-TuRC) to mitotic centrosomes in Drosophila cells. It uses some robust classical genetics to generate mutants to reduce/remove GCP4/5/6, Dgrip71 and Cnn and Spd-2 from cells, monitoring the consequences using live imaging.

    It begins by showing that Cnn can recruit g-Tubulin independently of the core g-TuRC components or Dgrip71, and that a mutant Cnn lacking the CM1 domain cannot, strongly suggesting that, similarly to other organisms, the CM1 domain is essential for this function.

    It then demonstrates that Spd-2, in contrast, cannot localise g-Tubulin in the absence of the g-TuRC components or Dgrip71.

    In the second half of the paper, then use this tool as a proxy for centrosomes that completely lack mitotic g-Tubulin recruitment, in order to explore spindle assembly in the absence of centrosomal g-Tubulin. The show that microtubules and spindle are still nucleated but do so with different dynamics. This section is particularly convincing, given the use of the live de/repolymerisation assays using the CherryTemp device.

    Finally, the authors visualise spindle formation in the absence of centrosomal g-Tubulin, alongside a number of other MT associated proteins, including Msps.

    Major Comments

    1. The claims and conclusions relating to the first half of the paper are supported by the data, but they need to be caveated by a clear explanation of the alleles used. Some are well-characterised mutant lines but have they been previously shown to completely remove the associated protein products? For the RNAi lines, do the authors have evidence (via Western blots) that these remove the protein products? It is not necessary that they show Western blots for all the lines, and it does not invalidate the major conclusions that the fly line carrying mutations in cnn, grip71, grip163 completely fails to localise g-Tubulin to mitotic centrosomes. However, they need to help the reader understand much more clearly whether these lines are complete nulls and, consequently this may impact the strength of their interpretation of the relationship between Grip163 versus Grip75, discussed both at the end of the relevant section and in the Discussion.
    2. I have an issue with the statistics in Figure 1 &2. I realise the t-tests in Figure 1 show the significant differences between g-Tubulin recruitment to centrosomes in interphase and mitosis, in order to demonstrate the difference between the Spd-2;Grip combination line in (B) and the Spd-2; CnnCM1 double mutant in (D). But in doing so, it draws attention to the fact that there is no similar t-test between mitotic g-Tubulin recruitment to centrosomes in WT, Spd-2 and the Spd-2;Grip combination lines. This lack of stats between conditions is further confused by the language used in the text: In the Figure legend, the authors claim mitotic centrosomal g-Tubulin levels between are WT, Spd-2 and the Spd-2;Grip combination lines "similar", and in the text they say: the spd-2 Grip combination line had g-Tubulin "similar to the levels found at spd-2 mutants alone". But then they give numbers - an average of 77% of wild type for spd2 and 66% of wild type for the spd-2 Grip combination. I'm sure if they did a t-test they would find a significant difference between these conditions. This doesn't invalidate the thrust of what they're claiming, but they do need to be consistent in language, analysis and interpretation. Similarly, in Figure 2, it would be better to assess any statistically significant difference between mitotic accumulation of g-Tubulin between fly lines, rather than accumulation between interphase and mitosis (which is pretty clear cut). This would help to clarify whether differences between loss of grip subunits are merely additive or synergistic. Again, this doesn't invalidate the overall result that concomitant loss of cnn, grip71 and grip163 completely abolishes mitotic centrosomal accumulation of g-Tubulin, but it is a more complete analysis of the extant data.
    3. One OPTIONAL experiment that would significantly improve the study would be similar CherryTemp live imaging of the cells lacking both centrosomal g-Tubulin and Msps. Currently the manuscript finishes with a fixed analysis of MT de/repolymerisation in these cells, which provides evidence that Msps has a role in MT nucleation in the absence of centrosomal g-Tubulin-nucleated MTs, but very little else can be concluded.
    4. There is, perhaps surprisingly, no mention of Augmin in the paper. Augmin has been shown to recruit g-TuRC to pre-existing MTs, through the grip71 subunit (Chen et al., 2017). So, presumably, in cnn, grip71, grip163, g-Tubulin cannot be recruited to pre-existing MTs either? This could add impact to the results - in that it implies the MT nucleation seen in the absence of cnn, grip71 and grip163 actually reflects, not just loss of centrosome function, but also loss of Augmin function. Mentioning this in the discussion could help increase the impact of the paper.

    Minor comments

    1. The cnn, grip71, grip163 mutant image in Fig3 B after 40 min cooling appears to have 4 centrioles. Is this a cell that exited and re-entered mitosis?
    2. Methods should contain more detail on the de/repolymerisation live imaging analysis (including the numbers of cells contributing to the analysis) and techniques such exponential curve fitting.
    3. P5 para 2 - "GPC4/5/4/6" should read "GCP4/5/6"
    4. Fig legend 1 - "error bar" should read "scale bar"

    Significance

    The experimental approach (genetics and cell biology) taken in this manuscript is very appropriate and the experiments are of high quality. It uses the strengths of Drosophila to cleverly engineer flies to pull apart the relationship between two different ways to recruit the main MT nucleator, g-Tubulin, to mitotic centrosomes. This is an important advance for the specific research field of centrosome biology.

    By generating a fly that completely fails to localise g-Tubulin to mitotic centrosomes, the paper is able to explore whether MTs and the mitotic spindle can form in its absence. Again, there is very high quality imaging and image analysis, using a commercially available (but very cool) fast heating/cooling apparatus - the CherryTemp to explore the dynamics of MT generation. The limitation to this approach, though, is that g-Tubulin itself is still present and presumably able to nucleate MTs in the cytosol or elsewhere, albeit inefficiently. As such, it adds to a body of centrosomal and cell division research, rather than adding a highly significant conceptual advance.

    Similarly, the finding that Msps is involved in nucleating MTs in the absence of centrosomal g-Tubulin, via fixed analysis, supports other work, rather than moving the field forwards.

    Overall, assuming the caveats mentioned in the major comments are dealt with, I see this as a robust and very well carried out piece of research, that will be of interest to those investigating the broad field of cell division

    My field of expertise is Drosophila cell division

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    Referee #1

    Evidence, reproducibility and clarity

    In their study, Zhu and colleagues study how the centrosome proteins Spd-2 and Cnn in Drosophila recruit gamma-tubulin complexes to centrosomes, which is an important step in mitotic spindle formation. The authors make use of mutant flies and RNAi and find that the two factors Spd-2 and Cnn together are responsible for mitotic centrosomal accumulation of gamma-tubulin. By inactivating Spd-2 or Cnn separately, the authors show that Cnn appears to recruit the large share of mitotic gamma-tubulin pool by its CM1-domain. Interestingly, this involves only gamma-TuSCs (subcomplexes of gamma-TuRC) and not gamma-TuRCs. A smaller pool is recruited by Spd-2, and this pool depends on gamma-tubulin complex proteins that are only present in pre-assembled, complete gamma-TuRCs. This suggests that Drosophila makes microtubule nucleation templates in two ways. First, as in yeast, by direct recruitment of gamma-TuSCs to mitotic centrosomes, where additionally oligomerization needs to happen. And second, by recruitment and activation of preassembled gamma-TuRCs. Inactivation of both Cnn- and Spd-2 pathways abolishes mitosis-specific gamma-tubulin recruitment, resulting in low, but not complete loss of gamma-tubulin at centrosomes. The authors show that these low-gamma-tubulin centrosomes are still able to organize microtubules, but these microtubules have different dynamicity. Inspired by existing literature in flies and other model organisms, the authors identify Msps/Xmap215 as an important nucleation factor in this scenario.

    Major points:

    1. The authors use fly embryos with mutant Grip71, Grip75 and Grip163 alleles, which are central to the study. Most conclusions are based on the assumption that some mutants contain only gamma-TuSC, whereas wildtype cells contain a mix of gamma-TuSC and gamma-TuRC. It would be important to show sucrose gradient analyses of extracts to confirm the expected presence/absence of gamma-TuSC/gamma-TuRC.
    2. Given the advantage of the CnnΔCM1 separation of function mutant, I do not understand why it is not used throughout the study. Instead, full Cnn loss is used, which results in strongly reduced Spd-2 levels (Figure 2C,D). Are the observed differences between wild-type and mutants in Figure 2-5 dependent on defective PCM or do they also occur in a CnnΔCM1 background?
    3. Statistical tests should support the conclusions in the text. If the authors claim differences between different genetic backgrounds (e.g. that spd2-mutants only have ~77% of gamma-tubulin at mitotic centrosomes compared to wild-type), statistical tests must compare mutant mitosis vs. wild-type mitosis.
    4. While Cnn, grip71, grip163 mutants do not accumulate gamma-tubulin at centrosomes in mitosis, they still have low levels of centrosomal gamma-tubulin. It is therefore misleading to refer to "gamma-tubulin negative centrosomes".

    Minor points:

    1. The abstract states that gamma-TuRC is a catalyst of microtubule nucleation. By definition, a catalyst takes part in a reaction but is not part of the final product. Although our knowledge of the nucleation mechanism is still incomplete, mechanistic studies suggest a non-catalytical mechanism since gamma-TuRC was found to stay attached to the microtubule end after nucleation (Consolati et al. 2020, Wieczorek et al. 2020).
    2. CnnΔCM1 flies: genotyping data should be provided besides describing gRNAs.
    3. Is it important to combine spd-2 with all four mutants, grip75 grip128 grip163 and grip71? What about spd-2 grip71 cells and spd-2 grip75 grip128 grip163 cells? Should that not have the same effect?
    4. CM1-containing factors are the only known factors able to directly bind and activate gamma-TuRC. How do the authors envision activation of gamma-TuRC in the absence of Cnn?
    5. Do the authors think that each identified pathway to microtubule nucleation (i.e. Spd-2/gamma-TuRC, Cnn/gamma-TuSC, Msps/mei38) as revealed by mutant genetic backgrounds contributes to a similar extent to overall nucleation capacity also in an unperturbed genetic background?
    6. How does CM1 mediate binding to gamma-TuRC? Using recombinant Cnn fragments, the authors find that a Cnn triple mutant (R101Q, E102A and F115A) no longer binds gamma-tubulin, suggesting these residues together mediate binding to gamma-tubulin complexes. However, it is not tested to what extent R101, E102 and F115 individually contribute to gamma-tubulin binding. Does the binding mode in Drosophila resemble more the one in humans or in budding yeast? Also, was this done with extracts from Grip71, Grip75, Grip128RNAi, Grip163 embryos or normal embryos?
    7. Figure 2C: Should the green channel not correspond to Spd-2?
    8. I suggest to reconsider the color-coding of graphs. While the colored background of the dot plots in Figure 1 and 2 are a matter of taste, the coloring of graphs in Figure 4F-H is confusing. Here, genetic backgrounds of fly lines are colored in the same way as the microscopy channels in Figure 4A-E, but they do not belong together.
    9. A tacc mutant allele is used in experiments, but is not further described. Please provide the necessary background information.
    10. The authors assess spindle quality in Cnn, grip71, grip163 cells and show that spindle quality worsens with ectopic msps. For comparison it would be good to compare spindle quality side by side with a wild-type situation.
    11. Introduction: "[...], however, as they depend upon each other for their proper localisation within the PCM and act redundantly." - Sentence is incomplete.
    12. Introduction: "Cnn contains the highly conserved CM1 domain (Sawin et al., 2004), which binds directly to γ-tubulin complexes in yeast and humans (Brilot et al., 2021; Wieczorek et al., 2019)". - Choi et al 2010 should also be cited here.
    13. Results: "Typically, interphase centrosomes have only ~5-20% of the γ-tubulin levels found at mitotic centrosomes, [...]". - Citation is needed
    14. The authors should discuss that Msps was found to act non-redundantly with gamma-tubulin in interphase nucleation (Rogers, MBC, 2008), contrary to the conclusions in the current manuscript.

    Referees cross-commenting

    This is a good paper in my opinion, they need to add some controls though, to determine the expected presence/absence of gTuSC/gTuRC in the different mutants. An important advance is the finding that gTuSC can function as nucleator in parallel to gTuRC, depending on the recruitment mechanism. Different recruitment mechanisms, nucleation templates, and regulatory strategies co-exist and provide complex regulation and robustness to nucleation/spindle assembly.

    Significance

    This is a very well-executed study and the data is presented clearly. However, some findings would benefit from additional experiments to substantiate the main interpretations. If these points are addressed, the study would provide an important conceptual advance in the field, namely that animal cells may rely on two different gamma-tubulin complexes for nucleation at mitotic centrosomes, gamma-TuSC and gamma-TuRC, which differ not only in their composition of GCP proteins but also the mode of recruitment to the centrosome. The findings will be of interest to all cell biologists.

  5. Version published to 10.1101/2022.09.23.509043 on bioRxiv