Alstrom syndrome proteins regulate centriolar cartwheel assembly by enabling Plk4-Ana2 amplification loop in Drosophila

  1. Marine Brunet
  2. Joelle Thomas
  3. Jean-Andre Lapart
  4. Léo Krüttli
  5. Marine Laporte
  6. Maria Giovanna Riparbelli
  7. Giuliano Callaini
  8. Benedicte Durand
  9. Veronique Morel

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Abstract

Centrioles play a central role in cell division by recruiting pericentriolar material (PCM) to form the centrosome. Alterations in centriole number or function lead to various diseases including cancer or microcephaly. Centriole duplication is a highly conserved mechanism in eukaryotes. Here, we show that the two Drosophila orthologs of the Alstrom syndrome protein 1 (Alms1a and Alms1b) are unexpected novel players of centriole duplication in fly. Using Ultrastructure Expansion Microscopy, we reveal that Alms1a is a PCM protein that is loaded proximally on centrioles at the onset of procentriole formation whereas Alms1b caps the base of mature centrioles. We demonstrate that chronic loss of Alms1 proteins affects PCM maturation, whereas their acute loss completely disrupts procentriole formation before Sas-6 cartwheel assembly. We establish that Alms1 proteins are required for the amplification of the Plk4-Ana2 pool at the duplication site and the subsequent Sas-6 recruitment. Thus, Alms1 proteins are novel critical but highly buffered regulators of PCM and cartwheel assembly in flies.

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    Referee #3

    Evidence, reproducibility and clarity

    Brunet and colleagues utilized expansion microscopy to identify the distinct localizations of Alms1a and Alms1b at the centrosome in transgenic flies expressing Alms1a-Tomato or Alms1b-GFP-6Myc. Their imaging results are of exceptional quality and include insightful observations and discussion. However, these experiments were exclusively conducted in the transgenic flies, and no evidence has been provided for the expression of endogenous Alms1b proteins. Moreover, the defect in centriole duplication was observed only when Alms1 was depleted via RNA interference, not through gene knockout. Although some discussion is provided to address this discrepancy, the evidence remains unconvincing. More robust evidence is necessary to support their findings.

    Major comments

    1. One of the primary observations is the defect in centriole duplication in the absence of Alms1. This finding was only observed with RNAi and not with gene knockout. The authors performed the RNAi experiments in Alms1-deleted cells (Alms1del3) and did not observe the defects in centriole duplication. Therefore, authors concluded that acute loss of Alms1 is responsible for centriole duplication in germline stem cells. However, it is still difficult for me to reach that conclusion with only this evidence.

    A. Could the authors provide the western blot and staining results of Alms1a and Alms1b proteins after RNAi and gene knockout?

    B. The most reliable and widely accepted method to determine the specificity of RNAi (whether it is an off-target effect or not) is through genetic rescue experiments. Could the authors provide rescue data? It would be great if the authors could show the rescue of centriolar signals of Plk4, Sas-6, and Ana2, in addition to centriole duplication.

    C. The authors failed to discriminate which gene is crucial for centriole duplication in GSGs due to the sequence similarity. Linked to the above point, could the authors show the rescue results with either or both Alms1a and/or Alms1b genes? If only one gene can rescue the centriole duplication defects, it would clarify the specific functions of Alms1a and Alms1b in this process.

    D. (Optional) Considering the results in the manuscript, the authors appear to have good techniques in genetic manipulation in flies. If so, generating transgenic flies of Alms1a and Alms1b tagged with a degron (e.g., dTAG or destabilization domain [DD]) to observe centriole duplication defects upon rapid protein degradation might be feasible to support their observations.

    1. There were no results with endogenous Alms1a and Alms1b in the manuscript. Could the authors provide immunostaining results for endogenous Alms1a and Alms1b? If expansion microscopy is not available due to antibody specificity issues, standard confocal imaging would be sufficient.
    2. In figure 3C and D, the authors noted measurements from 7 to 8 testes per group. It would be helpful to also know how many centrosomes were measured, as done in Figure 4C.

    Minor comments

    1. In figure S1, the orientation of the gene is not consistent between endogenous and transgenic Alms1. Could the authors make it consistent for readers to intuitively recognize it? Additionally, the promoter used for transgenic gene expression is not specified. Please provide this information as well.
    2. On page 5, the statement '~, whereas Alms1b is associated with post-duplication maturation of the centriole." needs revision. The authors observed strong Alms1b-GFP signals in the centrioles at the end of the SC stage or during meiotic division but did no observe any defects in centriole maturation at the current stages. Furthermore, Alms1b was not detected from rapidly duplicating centrioles in syncytial embryos and dividing neuroblasts (Page 6), making it making it hard to understand that that Alms1b has a role in post-duplication centriole maturation.
    3. In figure S2, the centrosome drawings are unclear (initially thought to be 'v' marks). Could a centrosome drawing be added to the legend for clarity?

    Significance

    ALMS1 is a well-known protein which is important for cilia formation in human cells. Recent research by Chen and Yamashita (eLife, 2020) highlighted its significance in centrosome duplication in Drosophila's germ line stem cells (GSGs). Brunet and colleagues' findings align with these previous studies but go further by elucidating the localization of Alms1a as a centriolar and pericentriolar material protein, and Alms1b solely as a centriolar protein, using expansion microscopy. Moreover, their research suggests that only acute, not chronic, loss of Alms1 leads to defects in centriole duplication, proposing that cells may develop compensatory mechanisms for Alms1 loss in GSGs.

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    Referee #2

    Evidence, reproducibility and clarity

    The study takes a look at the role of Alms1a and b in centriole formation in Drosophila tissues using genetics and microscopy approaches. Alsm1a RNAi was previously shown to lead to loss of centrioles on the male germ line, but the molecular mechanism of how Alsm1a provides this function is not known. This is what this manuscript addresses.

    The general proposal of this manuscript is that Alsm1a is a key regulator of centriole formation in Drosophila tissues (embryos, male germ line and neuroblasts analysed). Alms1a is an inner PCM protein and functions downstream of PLK4 to drive procentriole formation.

    This is an interesting advance in understanding of centriole formation in flies. The authors managed to pull of beautiful expansion microscopy and produced images of centrioles both by immunofluorescence and electron microscopy. The data quality is very high, the quantifications lack a little behind in places. The manuscript would benefit from thinking about structure and presentation. The proposal that chronic loss of Alms1a (mutants) is well tolerated while acute (RNAi) is not is a bit puzzling. It is not impossible, but RNAi is not that acute either. It would be important to clarify this. To make sure there is no glitch in the tools, the newly generated alms1 deletions should be better characterised to clarify whether a compensatory mechanism exists, that upon chronic depletion rescues all phenotypes described by RNAi. There are also ways to test whether the Alsm1a fluorescent transgene introduce confounding effects, which if tested would be an improvement (see below). The genetic and immunofluorescence analysis got the authors quite far, the manuscript lacks biochemical analysis to strengthen the proposed molecular mechanism and clarify whether key and easy to predict interactions of Alms1 actually do occur. This would be a big plus but is not limiting. There are also inconsistencies that need clarification, for instance the title of figure 6 is that Alms1 proteins act downstream of Plk4, yet the model in the discussion proposes that Alsm1 stabilises Plk4 which does not fit and hinges on quantification of Plk4 levels that is perhaps currently not robust enough. The statement that Alms1 is required for PLK4 activity is too strong based on the data provided or provide data on PLK4 activity. The attempt to check what is going on in other systems is appreciated, in RPE cells Alms1 comes in only after 120nm elongation. Some of the quantifications could be done more robustly, using ratiometric analsysis rather than directly comparing intensity levels. Referencing in the manuscript and discussion should be improved.

    Specific points

    1. Provide a thorough characterisation of the alsm1 deletions generated using qPCR to measure RNA levels in the flies. Provide whether the alleles are viable and fertile and clarify all genotypes in the manuscript. Can the mutants suppress the Plk4-ND overexpression effect?
    2. BamGal4 alms1a RNAi, SCs loose centrioles, those that keep centrioles, have Alms1a, but fail to initiate procentriole formation. To strengthen this view please provide nos-Gal4 alms1a RNAi, Alms1a-Tomatoe data showing that now Alms1a-Tomato is not present accept on the mother centrosomes in GCS, ruling out anything unpredicted happens by introducing the Alsm1a-Tomatoe.
    3. Quantifications, measure fluorescence ratios (e.g. Figure 3 C,D quantify the ratio of Asl/PlP to Bld10, similarly for PLK4-ND in the relevant figure).
    4. A model for Alms1a in the style of Figur5A would be great.
    5. What happens to Alsm1a upon Plk4 inhibition? Experiments like this could strengthen the validity of the hierarchy of events proposed.

    Other comments

    The organisation and presentation should be improved. The figures reorganised to group what belongs together, I would suggest moving human RPE cell analysis to supplementary data and bring the beautiful EM of BamGal4-Alms1 RNAi, Alsm1a-Tomatoe into the main manuscript.

    In the deletions asterless levels are reduced in SC but not in the testis tip, why is that?

    Conclusion: "while Alms1b is only detected on mature centrioles as it is absent from rapidly duplicating centrioles of syncytial embryos or from duplicating centrioles of dividing neuroblasts." Perhaps add, Alms1b when expressed.

    BamGal4 alms1a RNAi, SCs loose centrioles, those that keep centrioles, have Alms1a, but fail to initiate procentriole formation. To strengthen this view please provide nos-Gal4 alms1a RNAi, Alms1a-Tomatoe data showing that now Alms1a-Tomato is not present accept on the mother centrosomes in GCS.

    The idea to compare nos-gal4 and bam-gal4 driving Alms1a RNAi is good. Providing a scheme of when these are expressed in the male germ line will help illustrate the experimental strategy. BamGal4 Alsm1a RNAi leads to loss of centrioles in SCs

    Alms1b (CG12184) is according to published RNAseq data not expressed in neuroblasts, so it makes sense they do not observe (Knoblich lab data), which could be cited here: "in agreement with RNA-seq data showing low expression of alms1b in neurons and glial cells (Li et al., 2022)."

    Fig S5 what cells were analysed by TEM?

    Fig S6 monochrome images confirm what is what.

    Figure 4 A for clarity it would be helpful to provide landmarks, marker for stem cells (Vasa) or the Hub to be able to understand what we are looking at. Same for Fig 4E, Mira or Dpn? The DAPI staining does not allow in the images provided to identify NBs.

    Figure 5 For the fluorescence profile plots it would be nice to see the average plus the standard deviation of the signal in the quantifications.

    Figure 6 D is not convincing, how were the dots visible chosen for the quantification?

    Figure 1C. Quantification of protein levels is not provided (is this because the expanded ultrastructural approach is not linear precluding quantification?)

    Figure 2 B radial dimensions, it would be great to show the sample average and standard deviation.

    I don't find figure S2 helpful

    Significance

    Understanding the process of centriole duplication is an important topic that should be relevant to a broader cell biological community especially since the proteins of interest have disease relevance.. This study provides new insights into how Plk4 dependent centriole duplication takes place in Drosophila.

    The manuscript is strong on the microscopic images both immune fluorescence and TEM. The function of Alms proteins is dissected genetically no biochemical analysis is provided, which is a limitation. Another limitation currently is the uncertainty about the discrepancy between the RNAi and the mutant results. If the mutants are confirmed and technical issues can be ruled out the finding that a compensatory mechanism exists that suppresses chronic loss of Alms1 proteins could bar very interesting.

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    Referee #1

    Evidence, reproducibility and clarity

    Much is known about the centriole duplication machinery and the centriole duplication cycle thanks to key screens performed in C.elegans in the past 20-25 years and subsequent work in Drosophila or human cells. It has been proposed that the core duplication machinery consists of 5 proteins implicating Cep192-PLK4-SAS6-SAS5 and SAS4, with specific variations according to the model system or cell line studied. Recently, a role for ALM proteins in the duplication of centrioles in the Drosophila male germ line has been reported, but in this study the mechanism involved has not been identified or described.

    In the Brunet study, the authors show a role for Alm proteins in centriole duplication in different tissues in flies- showing that these proteins have more a ubiquitous role in centriole duplication rather than a restricted role in the male germ line as initially described. This is already accounting for the novelty of this paper. However, this study goes well beyond previous studies as it proposes two very novel concepts. The first one is that Alm proteins can stabilize PLK4, the sole kinase implicated in centriole duplication, and so be responsible for maintaining kinase activity during a time window where daughter centrioles are generated. This is extremely interesting and makes a lot of sense and the data is very convincing. The second novel concept is that loss of Alm can have different consequences according to the- I do not even know how to call it- loss condition- leading to Alm deficiency. While I think this is quite novel, interesting and maybe even real, I think the authors conclude very strongly on the differences between a gene knock out or gene knock down conditions. I think that they may want to tune down their conclusions related to this part, as many more data would be required to conclude in this way.

    In other words, I think the paper is of high interest to a broad field of cell biology with interests in centrosome, cilia and the regulation of centriole duplication.

    Major points:

    1. I did not find a rescue experiment of the Alm deletions with the Alm transgenes.
    2. If I understood the authors correctly, Alm def compensate for centrosome duplication by a yet unknown process, while Gal4-induced depletions do not. First, calling Gal4- induce RNAi- acute depletion is not correct. This is certainly not acute and so another designation has to be found. Acute is something like a degron such as Auxin or dTag where the protein is degraded in a very acute manner. RNAi targets the mRNA, so if the protein is already made, it will not suffer from the RNAi treatment. Second, are the authors sure that either their crispr strategy did not generate any other knock out- hence the essentiality of rescuing these mutations. Or alternatively, are they sure about their RNAi conditions? So can they add the RNAi conditions on the background of their deficiency and see that nothing changes? Third if depletion through RNAi indeed leads to a more evident role of Alm proteins, one is expected to see this over time. Can they do a clone-using an FRT site recombined with their Alm mutation, so that the initial cell divisions does not contain Alm, and so it is expected to fail duplication, which may be overcome with time? So a large clone might have progeny with cells with centrosomes (the young ones) and without (the older ones). Can they show that RNAi depletion in cells that will generate sensory cilia- these are not assembled? Because I am assuming that the mutants are not uncoordinated...
    3. If they think that Almdef flies compensate for Alm loss - can they analyse levels of PLK4, SAS6, Ana2 and SAS4 in the mother centrioles?

    Minor points

    1. Some figures (the majority) lack scale bars.
    2. I do not think that one can consider centrioles that are not in rosettes to be made de novo. They might just have disengaged. The "novo" centriole should be removed. Actually, PLK4 ND generates extra centrosomes, this is sufficient.

    Significance

    The article by Brunet and colleagues investigates the role of ALMs proteins in centriole duplication. Centrioles are the core constituents of centrosomes and as such contribute to microtubule nucleation. Centrioles can behave as basal bodies, providing essential function sin cilia assembly and function.

    Here the authors have use Drosophila to characterize the role of Alm proteins. They show that 2 isoforms with distinct behaviour are expressed with different localizations. Further, through the assessment of different tissues and loss-of-function conditions, they propose a role for Alm proteins in centriole duplication.

    Overall the paper is very well written and easy to follow.

  5. Version published to 10.1101/2023.09.13.557518v2 on bioRxiv
  6. Version published to 10.1101/2023.09.13.557518v1 on bioRxiv