Molecular architecture of the C. elegans centriole

  1. Alexander Woglar
  2. Marie Pierron
  3. Fabian Zacharias Schneider
  4. Keshav Jha
  5. Coralie Busso
  6. Pierre Gönczy

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Abstract

Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. Caenorhabditis elegans was key in identifying evolutionary conserved components governing assembly of the centriole organelle. However, localizing these components with high precision has been hampered by the minute size of the worm centriole, thus impeding understanding of underlying assembly mechanisms. Here, we used Ultrastructure Expansion coupled with STimulated Emission Depletion (U-Ex-STED) microscopy, as well as electron microscopy (EM) and electron tomography (ET), to decipher the molecular architecture of the worm centriole. Achieving an effective lateral resolution of approximately 14 nm, we localize centriolar and PeriCentriolar Material (PCM) components in a comprehensive manner with utmost spatial precision. We found that all 12 components analysed exhibit a ring-like distribution with distinct diameters and often with a 9-fold radial symmetry. Moreover, we uncovered that the procentriole assembles at a location on the centriole margin where SPD-2 and ZYG-1 also accumulate. Moreover, SAS-6 and SAS-5 were found to be present in the nascent procentriole, with SAS-4 and microtubules recruited thereafter. We registered U-Ex-STED and EM data using the radial array of microtubules, thus allowing us to map each centriolar and PCM protein to a specific ultrastructural compartment. Importantly, we discovered that SAS-6 and SAS-4 exhibit a radial symmetry that is offset relative to microtubules, leading to a chiral centriole ensemble. Furthermore, we established that the centriole is surrounded by a region from which ribosomes are excluded and to which SAS-7 localizes. Overall, our work uncovers the molecular architecture of the C . elegans centriole in unprecedented detail and establishes a comprehensive framework for understanding mechanisms of organelle biogenesis and function.

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  1. Version published to 10.1371/journal.pbio.3001784
  2. Review Commons

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    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript Woglar et al. use several light and electron microscopy techniques combined with averaging/registration methodologies to produce a comprehensive molecular map of the centriole in the C. elegans gonad. The images produced are very impressive and potentially very informative, allowing the authors to draw several important conclusions (e.g. about the chirality of the structure, and the potential organisation of Sas-6 in the cartwheel, the latter of which has been controversial in this species). Thus, although the manuscript is largely descriptive, there is a lot here that will be of great interest to the centriole field. The manuscript is generally well written and well presented, and, although I am not a great expert in all of these techniques, the data seems to solidly support the main conclusions. I therefore have only a small number of relatively minor suggestions for improvements.

    Minor Comments:

    1. It should be clarified whether the centrioles being examined here are organising genuine PCM and MTs. I know that in the embryo SPD-2 and SPD-5 are considered the main organisers of the mitotic PCM, and these centrioles are in S-phase or G2 (so I'm not sure if they are organising any PCM). SPD-5 is located internally to SPD-2, perhaps suggesting that these centrioles are not organising a bona fide PCM? On the other hand, TBG-1 and MZT-1 are located at the periphery, so I assume these centrioles are organising MTs?
    2. I think the labels (A, B, C) in Figure S1 are probably in the wrong order and are not referred to correctly in the main text.
    3. In Figure S1A two centrioles are shown that seem to be touching at their proximal ends, which I initially interpreted as meaning the centrioles were engaged. If so, there seems to be a long tail of Sas-6 connecting the two centrioles that extends well below the centriole MTs. However, reading the legend, I think this interpretation is incorrect, and the images are showing two separate centrioles that just happen to be touching? Perhaps swap in another image that won't lead to this potential confusion?

    Significance

    Although several papers have reported high resolution molecular mapping of centrioles, this one is perhaps the most detailed and does a nice job of superimposing the molecular structures on high quality EM images. Not all of these C. elegans proteins are obviously conserved, but C. elegans is a 'poster-child' model organism for centriole research, and this broad architecture will be of great interest to the entire centriole/centrosome (and also cilia) fields. In addition, the observation of chirality that is intrinsic to the inner centriole structure, and that Sas-6 is likely organised into rings rather than a steep helix, are important conclusions.

    I am an expert in centrioles and high resolution imaging, but not EM.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Woglar et al describe molecular features of the C. elegans centriole with unprecedented detail. By adapting U-ExM to extracted gonads and combining it with EM and TEM data, the authors precisely mapped the location of 12 components. They uncovered that these centrioles are shorter than in the embryo, have the same structural elements, and show an offset of centriolar proteins distribution relative to microtubules which results in chirality. Their detailed analysis also identified two novel electron-dense regions: the Inter Paddlewheel Density (IPD); and the SAS-6/4/1 Containing Density (SCD). This manuscript is a very nice description of C. elegans centrioles and we have mostly minor comments to improve it.

    1. Regarding the duplication and maturation section, the authors state in the abstract: "We uncovered that the procentriole assembles from a location on the centriole margin characterized by SPD-2 and ZYG-1 accumulation.". The data collected by the authors do not provide evidence of enrichment of ZYG-1 and SDP-2 prior to procentriole assembly (in the main text the authors clearly say they are speculating). This statement in the abstract should be corrected to more accurately match what is described in the main text and supported by the results.
    2. It is stated in the main text that the procentrioles can emanate from the middle of the centriole but no representative image is shown (only shown for off-centered procentrioles or very short templates). It is also referred that this may have implications on chirality- it would be important to explain better those implications, as well as offer an example of this configuration.
    3. The authors mention "core PCM" throughout the manuscript without explaining or referencing its definition. Would be useful to the reader if more information is provided.
    4. FigS1.A looks strange because procentrioles seem much longer than centrioles and their relative orientation does not seem to be orthogonal. If this image is representative, it would be helpful to have a diagram explaining the image.
    5. In the main text it is said: "Four components were found to localize to the paddlewheel: HYLS-1[N], SPD-2, SPD-5 and PCMD-1." and SDP-5 is represented in the final scheme (Fig. 7). However, an overlay of SPD5 and EM data is never shown. The authors may extrapolate that SPD-5 localizes there because it is interior to SPD-2 with no offset compared to α-tubulin, but if this is the case it should be clearer in the text.
    6. A statistics section is missing in which the program used is detailed and whether the {plus minus} values in the figures depict SD or SEM. The number of independent experiments should also be mentioned.
    7. Although symmetrization has been increasingly adopted by the field, it would still be useful to reference previous examples of its application in centriole structure analysis.
    8. S1B and S1C figure labels are swapped.
    9. The authors claim that "the procentriole likewise harbors little SAS-4 initially and that more protein is recruited at prometaphase, resulting in similar levels of SAS-4 in the centriole and the procentriole by then (Fig. 2D)". Can the authors provide some sort of semi-quantitative readout?
    10. In Figure 5A side view, the presence of an inner tube is not very clear. Given that diameter quantifications were done using the mostly side views, it would be beneficial if the authors could provide a clearer image.

    Significance

    Overall, these observations contribute toward a better understanding of centriole structure, molecular composition and diversity, with a particular focus on C. elegans. The precision of the approach developed by the authors (U-ExM and EM overlay) is a valuable tool and will be of interest to the centriole biology field and to cell biologists in general.

    Reviewer expertise: Cellular and molecular biologists working in the field of centrioles.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors analyze here the organization of centrioles in C. elegans, by combining the physical expansion of the specimens (by about 5-fold) with stimulated emission depletion (STED) microscopy. They analyze a large number of centriole components in different experiments, and they combine the data into a convincing model of the centriole, which is presented in conjunction with electron microscopy images of this structure. The work is solid, well-performed and technically sound. While this reviewer is not a centriole expert, the work also appears to be sufficiently novel, simply due to its precision, to warrant publication.

    Significance

    I only have one suggestion, which the authors may consider. Most of their work involves analyzing the symmetry of the structures, as presented, for example, in Fig. 4. However, symmetry problems, observable in individual structures, may also be informative. Are specific proteins more prone to variable localization, as, for example, SPD-2-C or SPD-5, while others are more stereotypically organized? Could an analysis of the variability of the stainings provide information on flexibility in the centriole organization?

  6. Version published to 10.1101/2022.05.09.491129v1 on bioRxiv