ALMS1 contributes to centriole proximal architecture and stability

  1. Sarah De Freitas
  2. Maria Giovanna Riparbelli
  3. Giuliano Callaini
  4. Marine H. Laporte
  5. Bénédicte Durand
  6. Véronique Morel

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Abstract

Centrioles are highly organised microtubular scaffolds which grow and mature progressively during successive cell cycles. Their molecular organisation is extensively characterized, yet the contribution of several components to centriole assembly, maturation or stability is incompletely understood. Here, using ultrastructure expansion microscopy and transmission electron microscopy, we show that ALMS1, the protein mutated in Alström syndrome, is required for proper centriole architecture. In absence of ALMS1, RPE1 cells exhibit shorter centrioles with defects in the microtubular wall, including broken or missing triplets or open B/C tubules. These structural defects arise after procentriole assembly. We show that ALMS1 loss selectively reduces the proximal region proteins CCDC77 and CEP44, leaving intact central and distal ones. ALMS1 is further required for the recruitment of the proximal CEP135 cap and the clearance of the γ-tubulin/GCP2 pool present at the procentriole base. Our findings thus identify ALMS1 as a key organiser of the centriole proximal domain and required for remodelling and stabilising the proximal end of centrioles during cell cycle progression.

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    Reply to the reviewers

    We thank the referees for their useful comments that we are able to fully address below.

    In this work we show for the first time a role of ALMS1 in the stabilization of the centriole architecture. We establish that centriole length reduction in absence of ALMS1 is supported by a specific decrease of the proximal region of the centriole. More, we identify for the first time a role for ALMS1 in the organization of the region proximal to the centriole and specifically in the formation or stabilization of CEP135 proximal cap.

    Although Ozaki et al. (2026) reported abnormal de novo centriole formation following an experimental ALMS1 forced depletion-and-add-back approach, they did not report alteration of proteins from the proximal region of the centriole nor destabilisation of CEP135 proximal cap or alteration of proximal y-TURC localisation.

    Therefore, our results identify for the first time ALMS1 as a key organizer of the proximal end of the centriole contributing to the remodeling and stability of the centriole.

    Considering the role of CEP135 as an anchor for rootlet fibres and regulator of cilia stability, this work thus provides a mechanistic basis for the alteration of centrosome cohesion and centrosome linker formation previously reported, as well as for cilia homeostasis alterations associated with ALMS1 mutations and Alström syndrome.

    1. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Referee 2

    Major comments

    The recent mammalian study by Ozaki et al. should also be cited and discussed in relation to the current work. That study reports ALMS1-dependent cartwheel-seed dynamics, impaired procentriole formation following acute ALMS1 depletion, and abnormal centriole architectures. The authors should discuss how these early functions relate to the later proximal maturation function proposed here and whether the differences could reflect acute depletion versus a stable knockout condition.

    Indeed, in 2026, Ozaki et al. revealed by experimentally slowing down G2 phase a role of ALMS1 in stabilizing Sas-6 cartwheel structure in late procentrioles. These observations are not incompatible with our observations as we do not look at the same time scale and parameters. The defects observable by Osaki et al. can only be observed if the G2 phase is slowed down leaving time to resolve defects during cartwheel shedding. In our study we reveal defects after SAS-6 removal and characterized by CEP135 loss from the centriole proximal end. It is important to take into account here that SAS-6 has also been shown by Lin et al. (2013) to directly interact with CEP135 and that this interaction is essential to stabilize the cartwheel structure. In Drosophila, CEP135 mutation is associated with a gradual loss of cartwheel structure over time (Roque et al. 2012). Given these close relationships between SAS-6 and CEP135 in the early stages of centriole development, we hypothesize that the defects we describe and the cartwheel instability reported by Ozaki et al. may occur simultaneously or represent two facets of ALMS1's role in stabilising centriolar proximal structures. Nevertheless, following Referee 2 suggestion, we are currently performing additional experiments to determine more precisely the timing of SAS-6 and CEP135 loss.

    We will include the results from the experiments asked by referee 2 to narrow the time window of ALMS1 associated defects in the final version of the results and discussion.

    The manuscript already compares its findings with the recent A-C linker study by Bournonville et al. and considers whether A-C-linker remodeling is a cause or consequence of proximal microtubule instability. This discussion should be used to define the remaining uncertainty rather than to imply that ALMS1 has been placed upstream of the A-C linker.

    Overall, the advance should be framed around the nanoscale analysis of proximal CEP135 and γ-tubulin/GCP2 organization rather than a general new function for ALMS1 in centriole biogenesis or architecture.

    In the proposed discussion, we indeed considered that ALMS1 might be upstream of CCDC77. This hypothesis relies on their relative timing of localisation at the procentriole and the correct length of the A-C linker region in procentrioles lacking ALMS1. We however do not exclude the possibility that A-C linker could act upstream of, or in parallel to, ALMS1.

    As suggested by referee 2, we are currently testing the relative hierarchical position of ALMS1 and A-C linker by performing CCDC77 siRNA depletion and investigating the consequences on ALMS1 recruitment. We will also measure the intensity of CCDC77 fluorescence at procentrioles and G1 centrioles to determine if CCDC77 recruitment could be impaired in term of protein quantity at both stages in ALMS1KO.

    We will also perform CEP135 y-Tub co-staining to investigate their relative timing of recruitment and removal. This should provide more information on the reorganisation of the proximal cap of the centriole.

    The discussion of the revised manuscript concerning the relationship between ALMS1 and A-C linker as well as the proximal cap organisation will be rethought in light of the results of these new experiments.

    Figure 3 analyzes procentrioles that are already present. Similar dimensions among these structures do not exclude failed or delayed initiation in a subset of cells. The authors should quantify the number of SAS-6- or STIL-positive procentrioles in clearly identified S-phase cells, using EdU or another cell-cycle marker.

    Planned experiment

    We thank the referee for this comment. We are currently performing experiments where we are assessing the cell cycle stage by EdU staining as suggested and measuring the % of cells in S-phase (Edu+) with SAS6 cartwheel and / or procentrioles in control and ALMS1KO cells.

    Likewise, the conclusion that the defect arises specifically between late G2 and G1 requires validated staging. A synchronized or pulse-labelled late-G2-to-early-G1 analysis would directly test this timing.

    Planned experiment

    We agree that we currently do not discriminate between G2 and next G1 for the appearance of centriolar defects.

    To fill this gap, we are pursuing our efforts to analyze centrioles from mitotic cells. These are however extremely difficult to image as there are embedded in the mitotic spindle poles. Following the referee’s suggestion, we will also perform EdU labelling to formerly identify G2 stages (Edu- cells with duplicated centrioles) and document potential centriole length defects between control and ALMS1KO cells.

    We hope that these two combined approaches will allow to answer the referee’s question regarding a more precise staging of the phenotype.

    The proximal CEP135-cap phenotype should be quantified for frequency, intensity, and position rather than supported only by representative images (Fig. 5A). The γ-tubulin/GCP2 contact phenotype is quantified (Fig. 5C), but the numbers of cells, centrioles, biological replicates, and statistical analyses should be provided.

    Planned experiment

    As suggested, the quantification of CEP135 cap phenotype, as done for the yTub phenotype, will be added in the revised version of the manuscript. The numbers of cells, centrioles, biological replicates will also be added.

    A co-localization of CEP135 and γ-tubulin or GCP2 at the same centrioles would be informative. This would establish whether loss of the CEP135 cap correlates with the ectopic proximal γ-TuRC signal. A synchronized late-G2-to-G1 analysis could further determine whether CEP135-cap loss precedes or accompanies altered γ-TuRC positioning.

    Planned experiment

    As suggested, we are currently co-staining cells for CEP135 and y-Tub to investigate their relative timing of recruitment and removal. This analysis will be performed in EdU labelled cells to formerly distinguish S from G2 stages. This should allow us to determine in the control condition whether CEP135-cap is first observed concomitantly to or before the reorganisation of y-TURC below the centriole.

    The proximal-domain data require additional quantification.

    Figure 4 quantifies the length and position of the CEP44- and CCDC77-positive domains. However, their shorter absolute length may reflect shortening of the proximal microtubule wall rather than reduced protein recruitment. In particular, CCDC77 covers a similar proportion of stage-matched control and ALMS1-deficient centrioles. The authors should distinguish changes in domain length and position from changes in protein abundance. Conclusions on reduced recruitment or protein levels would require fluorescence-intensity measurements and immunoblotting to determine their global levels.

    Modifications already included in the manuscript

    Indeed, with Figure 4 we aim to determine whether the reduction in centriole size in ALMS1KO reflects a homogeneous shortening of the centriole or is supported by the reduction of a specific subregion of the centriole. We used CEP44, CCDC77 and POC5 localisation regions as rulers of the pinhead, A-C linker and inner scaffold respectively. We conclude that the reduced length of CCDC77 and CEP44 regions reflects a decrease in the length of the proximal region of the centriole.

    lines 184-185 “the global shortening of ALMS1KO centrioles is sustained by a specific decrease in length of the proximal region.”

    To clarify this aim, we have modified the first sentence of the paragraph to:

    __“__Centriolar regions have been shown to differentially contribute to centriole structure and function, including stabilization of the centriole structure for A-C linkers and Inner Scaffold, control of centriole length and ciliogenesis for distal cap proteins and formation of the rootlet key for cilium stability for CEP135 proximal cap. We therefore next investigated whether the reduced centriole length observed upon ALMS1 loss was supported by the reduction in length of all or only some subregions of the centrioles when procentrioles have reached the G1 phase and become daughter centrioles.”

    We also replaced “size” by “length” in the text, as suggested by referee 3, which should remove the ambiguity between “protein region length” and “protein intensity”.

    We agree that in addition to an effect on the length of centriolar subregions, including A-C linker region, loss of ALMS1 could also impact the amount of A-C linker proteins recruited at the centriole.

    Planned experiment

    To investigate this point, we will thus quantify the amount of CCDC77 proteins at the centriole in the different conditions used in this work. Comparing protein abundancy between different conditions from U-ExM images however requires the use of a reference protein for normalisation. Tubulin is commonly used as centriolar protein ruler. However, since ALMS1 loss impacts centriole structure, we are concerned that the analysis might not be fully reliable. While a decrease in the ratio CCDC77/tub in ALMS1KO compared to control would validate a decrease in CCDC77 abundancy (adding to a decrease in the length of the subregion covered by A-C linker), any other result will be impossible to interpret with certainty.

    Positioning of ALMS1 within or parallel to the A-C-linker/pinhead pathway has not been resolved.

    The current experiments test only one direction: ALMS1 loss alters the organization of CEP44 and CCDC77. They do not determine whether ALMS1 directly regulates the pinhead/A-C-linker machinery or whether these changes arise secondarily from proximal microtubule-wall instability.

    The most focused experiment would be to acutely or partially deplete one A-C-linker/pinhead component, such as CCDC77 or CEP44, and quantify endogenous ALMS1 localization, domain size, and position by U-ExM. Testing whether the CEP135 and γ-TuRC phenotypes are reproduced under the same perturbation would further connect the molecular observations.

    Planned experiment

    A-C linker presence and microtubule triplet stability are interconnected and will thus be difficult to discriminate. We can however, as suggested, investigate if the altered localisation of CEP135 and yTURC are a consequence of A-C linker alteration and microtubule instability or directly relate to ALMS1 loss by depleting CCDC77 by RNAi (as performed in Bournonville et al. 2025) to trigger proximal microtubule wall instability. In this context we will investigate whether and how this impacts CEP135 and y-TURC phenotypes. Additionally, we will further document potential impact of ALMS1 localisation as suggested.

    2. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    Referee 1

    __Major comment __

    How were longitudinally aligned centrioles selected, and was the flatness of this selection verified in any way? Some degree of orientation-related error is always present. However, because centrioles in wild-type and ALMS1-knockout cells have different lengths, the selection procedure might result in a higher proportion of centrioles that are not perfectly parallel to the imaging plane in the knockout condition, thereby biasing the quantifications. Since the following quantification is at the core of Figure 4, I would suggest verifying that centriole orientation was selected with similar accuracy in the two conditions, for example by comparing the uniformity of the tubulin profiles between wild-type and knockout cells.

    Orientation of centrioles has been a major concern throughout this study for these exact reasons.

    Since we observed an impact on centriole architecture in our mutant conditions, we did not consider tubulin intensity profiles as an adequate read-out of centriole orientation in the plane of focus.

    Also, we often detect in control conditions a difference in tubulin intensity between the proximal and the distal ends of the centriole, likely reflecting the difference in the centriole structure with microtubule triplets being found in the proximal 2/3rd of the centriole while the distal 1/3rd is made of microtubule doublets.

    To avoid a measurement bias due to centriole orientation, we rather chose to only measure centrioles “aligned in the plane of focus”, i.e. which both distal and proximal ends are in focus either in the same z-plane or in two consecutive z-planes.

    In all experiments, acquisition was done with Spinning Disks microscope with a z-step of 0.2um, thus 5 times smaller than the centriole diameter as measured on expanded centrioles with the achieved 4-fold expansion.

    Details concerning centriole selection have been added in the material and method.

    In “Imaging” section:

    “Expanded gels were mounted onto 24 mm coverslips coated with poly-D-lysine (0.1 mg/ml). For centriole substructure analysis, gels were imaged using an IX 83 inverted microscope from Olympus, equipped with a Yokagawa CSU-X1 Spinning Disk Unit, Borealis technology for homogeneous illumination and Ixon3 888 EM-CCD camera from Andor. The oil immersion Plan Apochromat 60x/1.42 NA objective from Olympus was used for all acquisitions. For all acquisitions, the z-step was set at 0,2mm.”

    In “__Quantification and statistical analysis / Longitudinal measurements from side viewed centrioles” __section we added:

    “To avoid a measurement bias due to centriole orientation, we only measured centrioles “aligned in the plane of focus”, i.e. which both distal and proximal ends are in focus either in the same z-plane or in two consecutive z-planes.”

    Minor comments

    1. The authors state: "Thus, ALMS1 appears to be required for the preservation of centriole architecture and the integrity of centriolar microtubule triplets."

    The data supporting an effect on centriole architecture are very clear. However, it is not clear to me why the authors interpret this specifically as a requirement for the preservation of centriole architecture rather than for the correct formation of the centriolar wall.

    More generally, throughout the manuscript-except where this issue is explicitly discussed, as in the case of CEP135-the distinction between defective stabilisation and potentially incomplete formation is not always made clear. I would recommend making this distinction explicit throughout the manuscript.

    This interpretation relies on the absence of difference between control and ALMS1KO procentrioles while they are being formed. For technical reasons we however indeed lack data documenting procentriole structure as we have for the G1 Mother and Daughter centrioles. We thus agree that our interpretation is potentially too restrictive and we modified the text to suggest the two mechanisms.

    This possibility is already mentioned in the manuscript:

    *lines 207-208 “ our observations also suggest that centrioles could grow from their proximal end during maturation from daughter to mother and that ALMS1 is required for this growth. * “

    line 258-260: “ALMS1 contributes to the organisation of the centriole proximal end, and may either stabilise the centriole or promote its proximal growth.”

    we also modified the text as follows:

    Discussion, lines 289-292: “Together, these observations suggest that in absence of ALMS1, microtubules might be partially disassembled proximally, which could in turn lead to a remodelling of the A-C linker. Whether this reflects a role for ALMS1 in the formation of the microtubule triplets or in their stabilisation remains to be investigated.”

    lines 346-347: “We therefore propose that ALMS1 contributes to the organisation of the centriole proximal end, either by stabilising it or by promoting its formation or growth”

    1. I find the profile plots in Figure 4 rather confusing. Distance and intensity are not clearly labelled, and it is not evident why the plots show distances only up to 200 nm. It would also be useful to indicate A′, A", B′ and C′ on the corresponding profile plots. In addition, B" and C" are not shown in the image.

    We thank the referee for this comment. Figure 4 has been modified as follows. We modified the plots to show the full extent of the centriole rather than just the proximal half. We also added the suggested legends that were indeed missing. Regarding the B” and C” references to the quantifications, they were difficult to indicate on the image but have been added on all plot-profiles, as A’, A”, B’ and C’ to explicit the quantifications.

    We further added a section in the Material and Methods to better document how the profiles were generated:

    “Protein domain plot profiles from side viewed centrioles

    Protein domains plot profiles were performed with Fiji software. Centrioles representatives of the different measurements done were selected to appear on figures and for fluorescence intensity profiles. Briefly, a line was drawn from the proximal to the distal end of the centriole, extending outside of the centriole and with its thickness adjusted to match the centriole width. Plot profiles for both tubulin and the selected protein were generated and X-Y values extracted. X values were normalized to account for the expansion factor and to set the proximal end of the centriole (defined as 50% of the maximum tubulin intensity at proximal end) at zero. Fluorescence values were normalized for each protein, with the maximum set at 100. For centrioles co-labelled with CEP164 or CP110 for orientation purposes, the normalization was done to set the maximum fluorescence value of the protein of interest at 100. Hence, the peak corresponding to CP110 or CEP164 can be above 100 and is cut on the plots. Plots were drawn using Prism.”

    The conclusion that distal organization is unaffected is based on representative CEP290 images. This should either be quantified.

    An intensity profile plot for CEP290 localisation as well as the length of the overlap between CEP290 region and the distal end of the centriole, as shown in Figure 4, have been added together with U-ExM images in Figure S3. Numbers of events observed have been added in the figure legend.

    Referee 2 Major comments

    1. The findings should be positioned more clearly relative to recent ALMS1 studies. The authors' 2025 Drosophila study already established a role for Alms1 in centriole duplication and cartwheel assembly and described ALMS1 localization at the base of elongating procentrioles in RPE1 cells. Figure 3B should therefore be presented as a quantitative refinement of this localization rather than as a new finding.

    Indeed, we already published a time-line of ALMS1 recruitment to the procentriole in RPE1 cells (Brunet et al. 2025). The reference to the paper was added in the text:

    “ALMS1 precedes A-C linker recruitment as it is first detected when the procentriole reaches a length of approximately 140 nm, while the A-C linker protein CCDC77 arrives on 180 nm long procentrioles (Fig. 3B,F, (Brunet et al., 2025). “

    We thus modified the legend of Fig 3B to state it more clearly.

    “As previously shown in (Brunet et al. 2025), ALMS1 localises at the base of, and slightly overlaps with, the procentriole. In our current experimental conditions, ALMS1 is first detected as the procentriole reaches a length of 140 nm. “

    Cell-cycle staging and the conclusion that procentriole formation is unaffected require clarification.

    The Methods do not explain whether cells were synchronized or how S, G2, and G1 cells were identified. CEP164 distinguishes the mature mother centriole from the younger centriole but does not by itself establish that a cell is in G1. The criteria used to assign cell-cycle stage and centriole age should be described clearly, as several conclusions depend on this classification.

    We have modified the Method section to fill this gap.

    In this work, since we aimed to characterize ALMS1 contribution to centriole homeostasis at all centriole stages, cells were not synchronized.

    G1, S and G2 were identified based on

    • presence of only 2 centrioles/cell for G1

    • presence of procentrioles for S/G2 which we do not formally distinguish.

    CEP164 was only used as a marker of mother centrioles in opposition to daughter centrioles. Hence, we wrote lines 123-124 “the daughter and the mother stages (discriminated thanks to the presence of distal appendages labelled with CEP164 specific to the mother centriole,…)”

    We added__ in Material and Methods/Human cell culture section __

    “Cell cultures were not synchronized. Stages were identified based on centriole features: cells with one pair of centrioles were considered as G1, with duplicating centrioles as S/G2. “

    We added__ in Material and Methods/____Antibody staining__

    CEP164 was used to identify Mother centrioles, CP110 to identify the distal end on Daughter centrioles.

    The structural phenotypes, image-analysis methods, and statistical reporting need to be strengthened.

    The U-ExM and TEM observations are convincing, but the structural phenotype should be reported more systematically. For TEM, the authors should provide corresponding control frequencies and state the numbers of experiments, cells, unique centrioles, and sections analyzed, including whether multiple sections were obtained from the same centriole. The broken-centriole phenotype observed by U-ExM should also be quantified using defined criteria.

    We modified the legend of Figure 2 as follows to include quantification of broken-centriole observed by U-ExM (for top views: control broken centrioles 0/7; ALMS1KO broken centrioles 9/19) as well as TEM details asked by the referee.

    *“D–P- Transmission electron microscopy (TEM) images of centrioles shown in longitudinal (D-I) and transverse (J-O) views. D,J, control centrioles. E-I and K-O, ALMS1KO centrioles (F-I) **Longitudinal sections of **ALMS1KO centrioles reveal incomplete centriolar wall (G and H are two consecutive sections of the same centriole, I and F two different centrioles; n=10 centrioles from different cells) and shorter centrioles in ALMS1KO (E) compared to control (D) (ALMS1KO centriole in E is representative of 5 shorter centrioles from 5 different cells). Alterations of centriolar wall are also observed on cross-sections. (M-O) Examples of cross sections of ALMS1KO centrioles with incomplete wall (n=10 different cells) with K-L and M-O, sections of the same centriole (centriole1: K distal to L; centriole 2: M distal, N intermediate and O proximal). These defects *in the centriole wall are observed at both the proximal and distal ends (distal: arrowheads in H, I, K, N; proximal: L, O). In addition, the microtubule triplet architecture is altered, with some B- and C-tubules either missing (arrows in N and L) or incompletely closed (arrows in M). asterisk: Distal appendages. TEM control centrioles longitudinal views, n=8, cross sections, n=7. In all images (U-ExM and TEM) scale bar = 200 nm.”

    For all imaging experiments, the Methods and legends should report the numbers of biological replicates, independent gels, cells, and centrioles; image-selection and orientation criteria; acquisition and normalization procedures.

    Legends and methods have been modified accordingly. For experiments currently quantified, numbers will be added.

    Minor comments

    ALMS1-dependent ciliogenesis defects have already been reported in several models, and the ciliation time course is peripheral to the main structural study. I suggest moving it to the supplementary information or presenting it only briefly as context.

    This data is now presented in Figure S2.

    The Results refer to 24, 48, 72, and 96 hours, whereas the Methods and Figure 1 show 0, 24, 48, and 72 hours.

    We thank the reviewer for noticing this discrepancy. The Results section has been modified accordingly to:

    “To further characterise the ciliation phenotype associated with ALMS1 loss (Fig. 1A), we first aimed to quantify the percentage of ciliated cells over time following serum starvation-induced quiescence (0h, 24 h, 48 h and 72 h). In control cells, the percentage of ciliated cells progressively increases from 17% at 0 h (fed state) to 60% after 24 h serum starvation and reaches a plateau of approximately 70% at 48 h and 72 h (Fig. 1B).”

    Primary-antibody dilutions should be provided.

    Method section has been modified accordingly.

    Methods do not contain sufficient detail, it should be expanded significantly.

    In particular, we have added details concerning imaging procedure and image analysis as well as statistical analysis. Details asked by the referee in major point 2 were also added. Added information is in red in the modified manuscript.

    Referee 3

    Major comment

    1. Functional connection between the ciliation and centriole phenotypes The manuscript begins by showing altered ciliation following ALMS1 loss, providing a functional phenotype relevant to Alström syndrome. The remainder of the study then focuses on the structural organisation of ALMS1-deficient centrioles. Although the Discussion appropriately presents possible links through CEP135/C-NAP1 recruitment, centriole integrity, and cilium anchoring or maintenance as hypotheses, no experiment directly relates the identified proximal centriole defects to the ciliation phenotype.

    The authors should therefore consider providing additional evidence connecting these two observations.

    …

    A more direct analysis could determine whether loss of the CEP135 cap or other proximal architectural abnormalities is associated, at the single-cell level, with impaired cilium formation or maintenance. Given the large size of ALMS1 and the focused scope of the study, such experiments may be technically challenging. If further functional analysis is beyond the scope of the present work, the authors should make clear that Figure 1 provides functional context for the subsequent structural investigation and that the relationship between the two phenotypes remains unresolved.

    Ciliation impairment associated with ALMS1 mutations had already been described. Here we refined the analysis by investigating the ciliation defect over-time. Our aim however was to provide a general frame for the more specific study we are presenting here which is to investigate if and how ALMS1 impacts centriole homeostasis. We therefore moved the data on ciliation in Figure S2 and rephrased the first paragraph to clarify our scope:

    *“The percentage of ciliated cells subsequently declined in ALMS1KO cells at 48 h and 72 h, whereas it remained stable in control conditions (Fig. S2). These observations are in agreement with the reduction of ciliated cells previously described and further suggest that the decreased percentage of ciliated cells in ALMS1KO could result from both a decreased ability of ALMS1KO cells to initiate ciliation but also to maintain a cilium. *

    Ciliogenesis and cilium stability rely on centriole proximo-distal ultrastructure and the correct assembly of the associated molecular scaffold (Fig S1)(Yang et al., 2005; Graser et al., 2007; Pearson et al., 2009; Bayless et al., 2012; Tsai et al., 2019; Curinha et al., 2025)*. *We thus investigated to which extent cilium homeostasis alteration in absence of ALMS1 could reflect a role of ALMS1 in centriole organisation.”

    the discussion has also been modified as follows:

    “In absence of ALMS1, lack of CEP135/C-NAP1 recruitment at the base of the centriole could thus explain cilium instability. A contribution of altered structural integrity of ALMS1KO centrioles to defective cilia homeostasis however remains to be challenged.”

    __ __Minor comments

    Figure 4 lacks clearly labelled units and descriptions for both axes of the fluorescence profiles. It is not immediately apparent that values such as −100, 0 and 100 refer to positions along the proximo-distal centriole axis, or that the reference position at 0 corresponds to the proximal tubulin boundary defined at 50% of maximum tubulin intensity. A simple measurement schematic and clearer axis labels would substantially improve interpretation. Figure 5 legend also contains typographical errors (thus).

    The figure 4 has been modified to clarify the plot-profiles and legends have been clarified. It now states:

    “The y-axis represents the proximo-distal axis of the centriole with 0 corresponding to the proximal end of the centriole, set to 50% of maximum tubulin intensity value.”

    The Method section has also been implemented as follows:

    “Protein domain plot profiles* from side viewed centrioles*

    Protein domains plot profiles were performed with Fiji software. Centrioles representatives of the different measurements done were selected to appear on figures and for fluorescence intensity profiles. Briefly, a line was drawn from the proximal to the distal end of the centriole, extending outside of the centriole and with its thickness adjusted to match the centriole width. Plot profiles for both tubulin and the selected protein were generated and X-Y values extracted. X values were normalized to account for the expansion factor and to set the proximal end of the centriole (defined as 50% of the maximum tubulin intensity at proximal end) at zero. Fluorescence values were normalized for each protein, with the maximum set at 100. For centrioles co-labelled with CEP164 or CP110 for orientation purposes, the normalization was done to set the maximum fluorescence value of the protein of interest at 100. Hence, the peak corresponding to CP110 or CEP164 can be above 100 and is cut on the plots. Plots were drawn using Prism.“

    The typo in Figure 5 legend has been corrected.

    Where the manuscript refers to "tubulin size," "tubulin length" would be more appropriate, as the measurement represents the longitudinal extent of the tubulin signal. More generally, the terms "size" and "length" are used interchangeably between the text, figures and legends and should be harmonised. "Length" would be preferable for measurements along the proximo-distal axis.

    We thank the referee for this comment and changed the text accordingly.

    In the description of Figure 2, the statement that longitudinal sections identified shorter centrioles cites "Fig. 2D,E." Please clarify whether the intended comparison is between the control centriole in D and the ALMS1KO centriole in E, or revise the figure reference accordingly.

    The figure legend has been modified to clarify the comparison and the text has been changed to:

    *“Longitudinal section analysis identified shorter centrioles in *ALMS1KO compared to control (respectively Fig 2E and D)”

    The use of "domain" throughout the manuscript should be defined clearly. In most instances, the authors appear to mean a spatial or structural region occupied by a protein within the centriole rather than a molecular domain within the protein itself.

    The word domain has been replaced by region when to discriminate with protein domain.

    The phrase "the removal of γ-TuRC, as timely described in Laporte et al." is unclear. If the authors mean that the sequence was resolved over time, "temporally described," "temporally resolved," or "described in the time-resolved analysis" would be more precise.

    “as timely described in Laporte et al." was replaced by “*as temporally staged in Laporte et al.” * to clarify the sentence.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Referee 2

    4…Rescue experiments have not been performed for any of the phenotypes. If possible, rescue of the ALMS1-deficient phenotype with wild-type ALMS1, and ideally with centriole proteins from the relevant pathway, would provide stronger causal evidence.

    Referee 3

    Major comment

    1. Functional connection between the ciliation and centriole phenotypes Rescue or validation using an independent ALMS1 perturbation would strengthen attribution of the ciliation phenotype to ALMS1 loss, although this would not by itself establish that the proximal structural alterations are causative.

    We have already attempted to rescue ALMS1KO cells by re-expressing ALMS1 with no success so far. It seems that cells are very sensitive to copy number for ALMS1 and do not survive to transfection with ALMS1 expression plasmid. We are thus unable to perform the suggested rescue by ALMS1.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    De Freitas and colleagues investigate the contribution of ALMS1, the protein mutated in Alström syndrome, to centriole maturation and structural maintenance using a previously generated ALMS1-deficient hTERT-RPE1 cell line. Following an initial analysis of ciliation after serum starvation, the authors use ultrastructure expansion microscopy and transmission electron microscopy to characterise centriole architecture in the absence of ALMS1. The authors show that ALMS1-deficient centrioles are shorter and exhibit defects in the centriolar wall and microtubule-triplet architecture. Overall, this is a focused and careful descriptive study. Its principal strength is that it places the ALMS1-associated phenotype spatially and temporally within the centriole life cycle, rather than simply documenting shorter or structurally abnormal centrioles. The central claims are generally appropriately restrained and consistent with the presented data. I have no major objection to the main conclusions. The comments below principally concern the presentation and integration of the findings rather than any substantial expansion of the experimental scope.

    Major comment

    1. Functional connection between the ciliation and centriole phenotypes The manuscript begins by showing altered ciliation following ALMS1 loss, providing a functional phenotype relevant to Alström syndrome. The remainder of the study then focuses on the structural organisation of ALMS1-deficient centrioles. Although the Discussion appropriately presents possible links through CEP135/C-NAP1 recruitment, centriole integrity, and cilium anchoring or maintenance as hypotheses, no experiment directly relates the identified proximal centriole defects to the ciliation phenotype. The authors should therefore consider providing additional evidence connecting these two observations. As centriole duplication appears to be preserved in this model, the manuscript implicitly attributes the altered ciliation phenotype to qualitative defects in centriole maturation or architecture rather than to reduced centriole number. Rescue or validation using an independent ALMS1 perturbation would strengthen attribution of the ciliation phenotype to ALMS1 loss, although this would not by itself establish that the proximal structural alterations are causative. A more direct analysis could determine whether loss of the CEP135 cap or other proximal architectural abnormalities is associated, at the single-cell level, with impaired cilium formation or maintenance. Given the large size of ALMS1 and the focused scope of the study, such experiments may be technically challenging. If further functional analysis is beyond the scope of the present work, the authors should make clear that Figure 1 provides functional context for the subsequent structural investigation and that the relationship between the two phenotypes remains unresolved.

    Minor comments

    1. Figure 4 lacks clearly labelled units and descriptions for both axes of the fluorescence profiles. It is not immediately apparent that values such as −100, 0 and 100 refer to positions along the proximo-distal centriole axis, or that the reference position at 0 corresponds to the proximal tubulin boundary defined at 50% of maximum tubulin intensity. A simple measurement schematic and clearer axis labels would substantially improve interpretation. Figure 5 legend also contains typographical errors (thus).
    2. Where the manuscript refers to "tubulin size," "tubulin length" would be more appropriate, as the measurement represents the longitudinal extent of the tubulin signal. More generally, the terms "size" and "length" are used interchangeably between the text, figures and legends and should be harmonised. "Length" would be preferable for measurements along the proximo-distal axis.
    3. In the description of Figure 2, the statement that longitudinal sections identified shorter centrioles cites "Fig. 2D,E." Please clarify whether the intended comparison is between the control centriole in D and the ALMS1KO centriole in E, or revise the figure reference accordingly.
    4. The use of "domain" throughout the manuscript should be defined clearly. In most instances, the authors appear to mean a spatial or structural region occupied by a protein within the centriole rather than a molecular domain within the protein itself.
    5. The phrase "the removal of γ-TuRC, as timely described in Laporte et al." is unclear. If the authors mean that the sequence was resolved over time, "temporally described," "temporally resolved," or "described in the time-resolved analysis" would be more precise.

    Significance

    General assessment

    This study represents a focused, careful and predominantly descriptive advance. Its principal strength is the detailed spatial and temporal positioning of the ALMS1-associated phenotype within the centriole life cycle. Rather than simply showing that ALMS1-deficient centrioles are short or structurally abnormal, the authors provide evidence that initial procentriole assembly is broadly preserved and that the most prominent alterations arise later, during maturation into the G1 daughter centriole, with selective involvement of the proximal region. The study is technically well suited to this question, combining U-ExM, TEM and localisation analysis of several sub-centriolar components. The conclusions are generally appropriately calibrated, and the manuscript does not substantially overstate the mechanistic or disease implications of the findings.

    Advance

    The advance is modest but clear. The study maps the contribution of ALMS1 onto a specific stage and region of centriole maturation and identifies a coherent set of proximal architectural alterations. The principal limitations are the reliance on one immortalised cell line and one constitutive knockout model, the absence of biochemical insight into how ALMS1 influences CEP135 or γ-TuRC organisation, and the lack of a direct functional connection between the identified centriole defects and the diverse manifestations of Alström syndrome. These limitations restrict the mechanistic and disease-level reach of the study but do not undermine its central descriptive conclusions. The advance is therefore primarily structural and descriptive rather than biochemical, mechanistic, functional or clinical. Nevertheless, the work provides a useful framework for subsequent studies of proximal centriole maturation and ALMS1-associated disease.

    Audience

    The manuscript will be of greatest interest to a specialised basic-research audience working on centrioles, centrosomes, primary cilia, centriole ultrastructure and ciliopathies.

    Field of expertise

    My relevant areas of expertise are centrosome biology, mitosis, and cellular and mechanistic cell biology.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    De Freitas et al. use U-ExM and TEM to investigate the role of ALMS1 in centriole architecture and maturation in a previously characterized ALMS1-deficient RPE1 cell line. They show that ALMS1-deficient centrioles are shorter and exhibit defects in the microtubule wall and triplet organization, together with altered organization of proximal centriole components. The proximal CEP135 and γ-tubulin/GCP2 observations are potentially the most distinctive findings. However, the timing and molecular hierarchy of these defects are not yet established, and the methods for cell-cycle staging, image analysis, replication, and statistics require clarification. The data support a role for ALMS1 in proximal centriole organization and stability but do not yet establish all of the proposed recruitment, clearance, and proximal-growth mechanisms.

    Major comments

    1. The findings should be positioned more clearly relative to recent ALMS1 studies. The authors' 2025 Drosophila study already established a role for Alms1 in centriole duplication and cartwheel assembly and described ALMS1 localization at the base of elongating procentrioles in RPE1 cells. Figure 3B should therefore be presented as a quantitative refinement of this localization rather than as a new finding. The recent mammalian study by Ozaki et al. should also be cited and discussed in relation to the current work. That study reports ALMS1-dependent cartwheel-seed dynamics, impaired procentriole formation following acute ALMS1 depletion, and abnormal centriole architectures. The authors should discuss how these early functions relate to the later proximal maturation function proposed here and whether the differences could reflect acute depletion versus a stable knockout condition. The manuscript already compares its findings with the recent A-C linker study by Bournonville et al. and considers whether A-C-linker remodeling is a cause or consequence of proximal microtubule instability. This discussion should be used to define the remaining uncertainty rather than to imply that ALMS1 has been placed upstream of the A-C linker. Overall, the advance should be framed around the nanoscale analysis of proximal CEP135 and γ-tubulin/GCP2 organization rather than a general new function for ALMS1 in centriole biogenesis or architecture.
    2. Cell-cycle staging and the conclusion that procentriole formation is unaffected require clarification. The Methods do not explain whether cells were synchronized or how S, G2, and G1 cells were identified. CEP164 distinguishes the mature mother centriole from the younger centriole but does not by itself establish that a cell is in G1. The criteria used to assign cell-cycle stage and centriole age should be described clearly, as several conclusions depend on this classification. Figure 3 analyzes procentrioles that are already present. Similar dimensions among these structures do not exclude failed or delayed initiation in a subset of cells. The authors should quantify the number of SAS-6- or STIL-positive procentrioles in clearly identified S-phase cells, using EdU or another cell-cycle marker. Likewise, the conclusion that the defect arises specifically between late G2 and G1 requires validated staging. A synchronized or pulse-labelled late-G2-to-early-G1 analysis would directly test this timing.
    3. The proximal-domain data require additional quantification. Figure 4 quantifies the length and position of the CEP44- and CCDC77-positive domains. However, their shorter absolute length may reflect shortening of the proximal microtubule wall rather than reduced protein recruitment. In particular, CCDC77 covers a similar proportion of stage-matched control and ALMS1-deficient centrioles. The authors should distinguish changes in domain length and position from changes in protein abundance. Conclusions on reduced recruitment or protein levels would require fluorescence-intensity measurements and immunoblotting to determine their global levels. The proximal CEP135-cap phenotype should be quantified for frequency, intensity, and position rather than supported only by representative images (Fig. 5A). The γ-tubulin/GCP2 contact phenotype is quantified (Fig. 5C), but the numbers of cells, centrioles, biological replicates, and statistical analyses should be provided. A co-LOCALIZATION of CEP135 and γ-tubulin or GCP2 at the same centrioles would be informative. This would establish whether loss of the CEP135 cap correlates with the ectopic proximal γ-TuRC signal. A synchronized late-G2-to-G1 analysis could further determine whether CEP135-cap loss precedes or accompanies altered γ-TuRC positioning.
    4. Positioning of ALMS1 within or parallel to the A-C-linker/pinhead pathway has not been resovled. The current experiments test only one direction: ALMS1 loss alters the organization of CEP44 and CCDC77. They do not determine whether ALMS1 directly regulates the pinhead/A-C-linker machinery or whether these changes arise secondarily from proximal microtubule-wall instability. The most focused experiment would be to acutely or partially deplete one A-C-linker/pinhead component, such as CCDC77 or CEP44, and quantify endogenous ALMS1 localization, domain size, and position by U-ExM. Testing whether the CEP135 and γ-TuRC phenotypes are reproduced under the same perturbation would further connect the molecular observations. Rescue experiments have not been performed for any of the phenotypes. If possible, rescue of the ALMS1-deficient phenotype with wild-type ALMS1, and ideally with centriole proteins from the relevant pathway, would provide stronger causal evidence.
    5. The structural phenotypes, image-analysis methods, and statistical reporting need to be strengthened. The U-ExM and TEM observations are convincing, but the structural phenotype should be reported more systematically. For TEM, the authors should provide corresponding control frequencies and state the numbers of experiments, cells, unique centrioles, and sections analyzed, including whether multiple sections were obtained from the same centriole. The broken-centriole phenotype observed by U-ExM should also be quantified using defined criteria. For all imaging experiments, the Methods and legends should report the numbers of biological replicates, independent gels, cells, and centrioles; image-selection and orientation criteria; acquisition and normalization procedures.

    Minor comments

    1. ALMS1-dependent ciliogenesis defects have already been reported in several models, and the ciliation time course is peripheral to the main structural study. I suggest moving it to the supplementary information or presenting it only briefly as context.
    2. The Results refer to 24, 48, 72, and 96 hours, whereas the Methods and Figure 1 show 0, 24, 48, and 72 hours.
    3. The conclusion that distal organization is unaffected is based on representative CEP290 images. This should either be quantified.
    4. Primary-antibody dilutions should be provided.
    5. Methods do not contain sufficient detail, it should be expanded significaintly.

    Significance

    General assessment and advance

    The study provides a useful but relatively modest advance. Its main strength is the combined U-ExM and TEM evidence that ALMS1-deficient centrioles are shorter and have defects in microtubule-wall and triplet organization. The proximal CEP135-cap phenotype and nanoscale redistribution of γ-tubulin/GCP2 are potentially the most important new observations. However, ALMS1 localization and its functions in ciliogenesis, centrosome cohesion, centriole duplication, cartwheel assembly, and centriole architecture have already been reported. Recent work has also established the importance of the A-C-linker and pinhead machinery for proximal triplet cohesion and centriole structural integrity. The present manuscript therefore represents primarily a structural and spatial extension of this work rather than a major conceptual advance. The study suggests an additional role for ALMS1 in proximal-domain remodeling during centriole maturation, but the direction of the pathway among ALMS1, CEP135, the A-C linker, and γ-TuRC remains unresolved. A focused temporal or reciprocal dependency experiment would increase the mechanistic significance of the study.

    Audience

    The study will primarily interest a specialized basic-research audience working on centrosomes, centrioles, cilia, ciliopathies, and nanoscale organelle architecture. It will also be relevant to researchers using U-ExM and electron microscopy. The work provides additional cellular context for Alström syndrome but is unlikely in its current form to have broad relevance beyond the centrosome and cilia fields.

    Field of expertise

    My expertise includes centrosomes, centrioles, primary and motile cilia, ciliogenesis, cytoskeletal organization, quantitative microscopy, super-resolution microscopy, U-ExM and proteomics. I do not have specialist clinical expertise in Alström syndrome.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by De Freitas et al. investigates the role of ALMS1 in centriole architecture and maturation. Using Ultrastructure Expansion Microscopy and Transmission Electron Microscopy, the authors show that loss of ALMS1 results in shorter centrioles with defects in the microtubule wall, including broken or missing triplets and open B- or C-tubules. The study further identifies a selective reduction in proximal-region components and implicates ALMS1 in the recruitment of the proximal CEP135 cap and the rearrangement of the γ-tubulin/GCP2 pool at the procentriole base.

    Overall, the manuscript is clearly written, the techniques adopted the most apt and complementary to investigate ALMS1 role in centriole ultrastructure and its conclusions are important to the community. Overall, I consider the manuscript to be of high quality and deserving of publication. I have only a few points that I believe should be addressed before publication.

    Major comment

    How were longitudinally aligned centrioles selected, and was the flatness of this selection verified in any way? Some degree of orientation-related error is always present. However, because centrioles in wild-type and ALMS1-knockout cells have different lengths, the selection procedure might result in a higher proportion of centrioles that are not perfectly parallel to the imaging plane in the knockout condition, thereby biasing the quantifications. Since the following quantification is at the core of Figure 4, I would suggest verifying that centriole orientation was selected with similar accuracy in the two conditions, for example by comparing the uniformity of the tubulin profiles between wild-type and knockout cells.

    Minor comments

    1. The authors state: "Thus, ALMS1 appears to be required for the preservation of centriole architecture and the integrity of centriolar microtubule triplets." The data supporting an effect on centriole architecture are very clear. However, it is not clear to me why the authors interpret this specifically as a requirement for the preservation of centriole architecture rather than for the correct formation of the centriolar wall. More generally, throughout the manuscript-except where this issue is explicitly discussed, as in the case of CEP135-the distinction between defective stabilisation and potentially incomplete formation is not always made clear. I would recommend making this distinction explicit throughout the manuscript.
    2. I find the profile plots in Figure 4 rather confusing. Distance and intensity are not clearly labelled, and it is not evident why the plots show distances only up to 200 nm. It would also be useful to indicate A′, A", B′ and C′ on the corresponding profile plots. In addition, B" and C" are not shown in the image.

    Significance

    While the requirement of Drosophila orthologs of ALMS1 in centriole biogenesis had been demonstrated, their role in centriole homeostasis was unknown. As such the findings presented in the manuscript are and significant since they identify a previously insufficiently understood role for ALMS1 in organising the proximal centriole domain and connect this function to specific ultrastructural defects in the centriolar microtubule wall. Some findings, such as the specific role of ALMS1 in γ-tubulin rearrangement, are clearly reported, although the underlying mechanism is not yet entirely understood. However, the authors are very transparent about this, and the distinction between what is directly demonstrated by the data and what remains to be unravelled is clearly stated. Overall the manuscript is going to be significant and important for the centriole community. More broadly, the work provides a valuable framework for investigating how defects in ALMS1-dependent centriole organisation may contribute to the cellular basis of Alström syndrome.

  5. Version published to 10.64898/2026.07.15.738620 on bioRxiv