TTBK2 controls cilium stability through actin and the centrosomal compartment

  1. Abraham Nguyen
  2. Sarah C. Goetz

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Abstract

The serine-threonine kinase Tau Tubulin Kinase 2 (TTBK2) is a key regulator of the assembly of primary cilia, which are vital signaling organelles. TTBK2 is also implicated in the stability of the assembled cilium, through mechanisms that remain to be defined. Here, we use mouse embryonic fibroblasts (MEFs) derived from Ttbk2 fl/fl ; UBC-CreERT+ embryos (hereafter Ttbk2 cmut ) to dissect the role of TTBK2 in cilium stability. This system depletes TTBK2 levels after cilia formation, allowing us to assess the molecular changes to the assembled cilium over time. As a consequence of Ttbk2 deletion, the ciliary axoneme is destabilized and primary cilia are lost within 48-72 hours following recombination. Axoneme destabilization involves an increased frequency of cilia breaks and is partially driven by altered actin dynamics and a reduction in axonemal microtubule modifications. At the same time, we find that TTBK2 is required to regulate the composition of the centriolar satellites and to maintain the basal body pools of intraflagellar transport (IFT) proteins. Altogether, our results reveal parallel pathways by which TTBK2 maintains cilium stability.

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  1. Review Commons

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    Reply to the reviewers

    The current resubmission is our revision plan only. Therefore the authors do not wish to provide a response at this time. We will include our response to reviewers with our full resubmission.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Nguyen and Goetz here explore the roles of Tau tubulin kinase 2 (TTBK2) in ciliary stability. Using a murine conditional null MEF model, they ablate TTBK2 from cells with established cilia and monitor the impact on ciliary structures. They observe loss of cilia over time, acoompanied by changes in centriolar satellites, axonemal microtubule composition and intraflagellar transport protein levels at the basal body. They note changes in autophagy proteins in the absence of TTBK2 and find that pharmacological manipulation of actin dynamics impacts on the ciliary phenotypes seen in TTBK2 deficient cells.

    Major comments:

    1. The phrase 'centrosomal compartment' is potentially ambiguous. This is not a generally-used term and its use to mean the base of the cilium, PCM and satellites is spatially uninformative; there is not a 'compartment' meant here in the sense of a discrete structure. Especially in the context of the title, I suggest this phrase should be revised for greater clarity, but it may also be useful to rephrase it in the Discussion.
    2. As a question for discussion/ consideration: Is TTBK2 a satellite component, or is it envisaged that these functions are all derived from a CEP164-associated fraction? This point is related to the point raised above about a 'compartment', in that different populations of TTBK2 might be involved differently in cilium regulation. An experimental approach to this might be to use a CEP164-TTBK2 fusion (such as was described by Cajanek and Nigg in PNAS 2014) and test if this can rescue the TTBK2 deficiency.
    3. The statement on p.8 that 'Loss of TTBK2 results in increased actin activity' is not directly supported by the data, so should be revised. The actin analyses are indirect and show attenuated effects, so some caution is warranted in the interpretation of these findings (as is the case in the Discussion). With that point in mind, I am unsure if the title's inclusion of an actin-based mechanism for TTBK2 functions in cilium stability is optimal.
    4. A key technical issue is that the description of how intensity measurements were made should be improved. It is not clear what area or volume was used for this in the various experiments that measured axonemal/ centrosomal/ peri-centrosomal regions, particularly when the centrioles are further apart from one another. As the intensity measurements form a key part of the paper, this should be clarified throughout.
    5. From the images presented in Fig. 2A, the classification of the number of buds/ axonemal breaks is not clear. Improved images of the different outcomes of TTBK2 removal should be shown to make the basis for the proposed differentiation between these phenotypes more convincing. This is visible in the movies, but the still images are not ideal.
    6. A control should be presented for the loss of TTBK2 in the drug treatment experiments in Figure 4, to confirm that there is no impact on the recombination.
    7. A control for the relative expression level of the rescuing TTBK2-GFP protein should be provided in support of the data in Fig. S1D. This should also be included for the data in Fig. S4.

    Minor comments:

    1. Fig. 1B- 'lambda tubulin' should be corrected. Fig 7A should also correct the tubulin designation.
    2. It would be helpful to indicate in individual figures throughout that the bar graphs show means +/- SEM (it is stated in the Methods, but it would be desirable to have the Figures be entirely self-contained).
    3. Fig S1E does not show individual experiments as data points; this should be corrected for consistency.
    4. The phospho-Aurora A staining in Fig. S2C should be quantitated; there appears to be an increased level of this signal in the absence of TTBK2.
    5. The Ac-Tub staining shown in Fig. 3A is confusing, given the intensity measurements presented. More representative images should be shown.
    6. It is unclear whether there is a decline in PCM1 intensity levels over the timecourse of the (vehicle only) experiment in Fig. 5B. This should be tested for. It should also be specified in the Figure Legend that these cells remain serum starved for the duration of the timecourse (assuming the experiment follows the design outlined in Fig. 1A).
    7. The images in Figure 7A and 7C are not at sufficient resolution to distinguish IFT88 or IFT140 signals; blow-up panels should be included.
    8. Scale bars should be included in Figs. 2, S2, 3, 5, S3, S4, 6, 7.
    9. Details of the serum starvation regime should be provided in the Methods (% serum).

    Referees cross-commenting

    The comments from Reviewers 1 and 2 are detailed and constructive. There is good convergence between all three reviewers on a requirement for additional data on the involvement of actin and on the analysis of the centriolar satellites. As these are the main themes of the study, such information seems essential to support the principal conclusions drawn.

    I question Reviewer 1's stipulation that a quantitative proteomic analysis of PCM1 interactors is needed to sustain the conclusion that the satellites are substantively altered upon TTBK2 depletion. This would be a very strong experiment, but I feel that the analysis of a selection of individual satellite components, as done here, is sufficient to support the conclusion that the satellites are impacted by TTBK loss. Obviously, the more detailed the analysis (i.e., the more proteins examined), the better, but I am not convinced that this will bring us so much closer to the mechanism of TTBK2. I concur with the point raised for revision by Reviewer 2, that the actual role of the satellites in cilium stability should be addressed more robustly, however.

    Significance

    The importance of the primary cilium as a signalling organelle makes TTBK2 function a theme of general interest. A potential role for TTBK2 in the maintenance of cilium stability through a link to the centriolar satellites is new. Readership would include people working on centrosomes/ cilia and related themes.

    My expertise: cell biology of centrosomes/ cilia.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    This study by Nguyen and Goetz, explores the role of Tau tubulin kinase 2 (TTBK2) in cilium integrity in cultured cells. TTBK2 is a serine-threonine kinase that plays a key role in cilium initiation and has been implicated in maintaining assembled cilia in adult mice. The authors developed an inducible system to deplete TTBK2 once cilia are assembled to investigate the role of this kinase in cilium stability in a cell culture system. Using this system and live- and fluorescent- imaging approaches, the authors find that TTBK2 promotes cilium stability.

    The authors suggest three parallel pathways by which TTBK2 maintains cilia: 1) through tubulin polyglutamylation and actin dynamics, 2) through regulation of IFT pools at the centrosome, and 3) through regulation of centriolar satellite composition. Some of these conclusions (especially 1 and 3) need to be revisited and need some additional experiments (detailed below).

    Major comments:

    1. In the abstract and discussion, the authors suggest that TTBK2 functions via centriolar satellites to promote cilium stability. While the data support a role for TTBK2 in centriolar satellite homeostasis, it is unclear whether satellites contribute to cilium stability. Given that restoration of centriolar satellite composition using rapamycin does not rescue cilia loss upon TTBK2 depletion, these sections need to be revised.
    2. The authors show that TTBK2 regulates centriolar satellite composition via its kinase activity. Is TTBK2's kinase activity important for cilium stability, actin dynamics and IFT localization?
    3. Do changes in tubulin poly-glutamylation precede the loss of cilia phenotype observed upon depletion of TTBK2? Currently changes in tubulin post-translational modifications are assessed at 72h post-tamoxifen treatment. In order to support the hypothesis that changes in tubulin poly-glutamylation drives cilia loss upon TTBK2 depletion (as suggested in the abstract), these experiments must be repeated at an earlier time points, such as 24-48 h post tamoxifin treatment).
    4. By using small molecule inhibitors that alter actin dynamics, the authors suggest that loss of TTBK2 regulates actin polymerization leading to cilium instability. Are there any observable changes in cellular F-actin upon loss of TTBK2? Phalloidin staining and/or a biochemical assay to directly assess soluble vs. filamentous actin would be helpful to bolster the claim that TTBK2 regulates actin dynamics.

    Minor comments:

    The manuscript is well written, and easy to follow. Prior studies are referenced appropriately. I have some minor points that would improve the presentation and clarity of the manuscript:

    1. I would recommend the authors improve the presentation of figures by making the size of graphs more consistent across figures. For example, graphs in Figure 4 should be increased in size as they are currently very hard to read. Importantly, scale bars are missing from most immunofluorescence images.
    2. Statistical tests are missing in Figure S1 A and S1 E.
    3. Are the additional puncta observed around the centrosome in the TTBK2 immunofluorescence images (Figure 1B) non-specific signals? Also, gamma-tubulin is mislabeled as lambda-tubulin in the figure.
    4. Insets would be helpful for images Figure 1C, S1C, 2A, 4B, 7A, 7C.
    5. Quantification of fluorescence intensity is missing for Figure S2 C.
    6. The title for Figure S2 is misleading as it suggests there is a change in cilia disassembly factors upon loss of TTBK2, while the data show no changes in any of the factors assessed.

    Significance

    Although we now have some understanding of how cilium assembly is initiated, how the cilium is maintained at steady state remains poorly understood. Therefore, this study exploring the role of TTBK2 in ciliary structure maintenance is timely and will be of interest to cilia biologists. TTBK2 has previously been implicated in cilium maintenance, and the link between TTBK2 and IFT recruitment, and tubulin post-translational modifications has been previously described using hypomorphic mutants of TTBK2. Though this study specifically looks TTBK2's role in cilium maintenance at steady state, these previous studies (referenced in the manuscript) do diminish the novelty of the manuscript. Mechanistic details of how TTBK2 regulates actin, IFT dynamics and tubulin post-translational modifications to control cilium stability remain unknown but are important avenues for future research.

    Reviewers' expertise: cilia and centrosome biology, microscopy.

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    Referee #1

    Evidence, reproducibility and clarity

    TTBK2 is kinase mutated in spinocerebellar ataxia type 11 (SCA11). It is well characterized for its functions and molecular mechanism of action during cilium assembly and ciliary signaling. In this manuscript, Nguyen et al. investigated the role of TTBK2 during cilium stability using mouse embryonic fibroblasts derived from Ttbk2cmut embryos. This system allowed them to inducibly deplete TTBK2 in ciliated cells and thus, address the specific functions of TTBK2 during cilium maintenance and stability in ciliated cells. Upon TTBK2 loss, the ciliary axoneme was destabilized in part via an increased frequency of cilia breaks and primary cilia was lost, which was shown by live imaging experiments. To dissect the mechanism of axoneme destabilization, they performed rescue experiments with drug treatments as well as quantified of basal body, satellite and ciliary abundance of key ciliogenesis factors and tubulin modifications. Based on results from these experiments, the authors concluded that altered actin dynamics, reduction in axonemal polyglutamylation as well as changes in satellite-associated PCM1 and OFD1 and IFT levels at the basal body together underlies the axonemal destabilization phenotypes associated with TTBK2. Overall, the manuscript is well-written and the presented data is robust.

    I list below three major concerns I have on the manuscript along with detailed explanation. Although questions addressed in the manuscript and the tools the authors generated will be of general interest, the presented data falls short in supporting the major conclusions of the manuscript that pertain to the mechanisms by which TTBK2 regulate cilium stability and maintenance.

    1. Previous papers from the Goetz lab showed that TTBK2 is important for the structure and stability of the ciliary axoneme (i.e. reduced polyglutamylation of cilia in Ttbk2 hypomorphic mutants and null cells, disorganized axonemal microtubules). Although authors study the roles of TTBK2 in cilium stability with temporal control and identify altered centriolar satellite composition and actin dynamics as potential mechanisms, TTBK2's function in this process is not unexpected.
    2. The authors suggest that TTBK2 regulate cilium stability through parallel pathways that operate via actin, centriolar satellites, autophagy and IFT machinery. The presented data is not sufficient to assess whether these are direct or indirect consequences of TTBK2 depletion, which results in lack of a coherent model for how TTBK2 operates in ciliated cells. For example, what is the regulatory/functional link between TTBK2, actin and myosin VI during cilium maintenance? The authors discuss that TTBK2 BioID data includes actin-binding proteins and suggest actin polymerization as one potential mechanism. To gain insight into how TTBK2 alters actin dynamics, they can follow-up on the BioID hits to explain how TTBK2 depletion alters actin dynamics. Alternatively, they can treat cells with specific inhibitors of actin polymerization to determine whether axonemal destabilization phenotypes are rescued.
    3. The authors define changes in centriolar satellite composition as a consequence of TTBK2 depletion based on reduced PCM1 and elevated OFD1 and CEP290 intensity at the pericentrosomal region in TTBK2-depleted cells. Centriolar satellites are composed of about 200 proteins and a significant number of these proteins are implicated in ciliogenesis including the previously characterized interactors of TTBK2. Changes in PCM1, OFD1 and CEP290 levels in the 1 uM ROI authors defined around the basal body is not sufficient to conclude that satellite composition is altered and that this change underlies the axoneme destabilization and disassembly. Proteomic pulldown of PCM1 before and after tamoxifen addition will reveal how the satellite proteome is affected by TTBK2 depletion and will strengthen authors' conclusions.

    Below are other comments I have on the data and its analysis and presentation:

    1. Fig. 1B: TTBK2 at the basal body was assessed as "positive" or "negative". Instead of classification into two groups, quantification of the basal body levels of TTBK2 in a time course manner will be more informative in correlating phenotype with TTBK2 depletion.
    2. Fig. 1C: In addition to percentage of ciliated cells, the cilium length should also be quantified in a time course manner to determine how TTBK2 depletion leads to cilium disassembly by 48-72 h. For representative images presented, insets are required.
    3. Fig. S1A: Western blot quantification of TTBK2 levels in addition to mRNA levels will be informative in assessing changes in protein levels upon tamoxifen addition.
    4. Statistical analysis for Fig. 2B is required. How many cilia / experimental replicates were quantified in Fig. 2B?
    5. Fig. S2: Bowie et al. 2018 paper reported increased Kif2a levels at the basal body as one possible mechanism for cilium instability in TTBK2 mutant cells. However, TTBK2 depletion in ciliated cells does not have a similar effect. How do the authors explain this difference on effects of TTBK2 in basal body Kif2a levels?
    6. How did the authors quantify between budding and axonemal events in Fig. 2? In general, the methods section for analysis of the microscopy data should be written in a more detailed way to be able to assess the presented data.
    7. Acetylated tubulin, but not glut tubulin, ciliary levels are decreased upon TTBK2 depletion. Can such change directly affect cilium stability? What triggers changes in glutamylation upon TTBK2 depletion?
    8. "Loss of TTBK2 results in an increase in actin activity" (pg.8) is an overconclusion based on the presented data. What is the effect of TTBK2 depletion on actin levels and organization? Specifically, the authors can also stain for actin in the cilia to determine whether it underlies the increased excision events reported by live imaging of cilia. Given that Nager et al. 2017 paper identified actin-myosin activity as a mechanism for ciliary ectocytosis, this will be interesting to test using similar assays to quantify actin dynamics in this paper. Moreover, Yeyati et al. 2018 paper on dissecting KDM3A's function during actin dynamics and cilium stability used quantitative approaches that can be adapted by the authors.
    9. Quantification of centriolar satellite levels of PCM1, OFD1 and CEP290 can be done in a different way to more specifically identify the satellite pools of these proteins. The quantification of their pericentrosomal levels was done by drawing 1 uM ROI around the centrosome. Therefore, the levels might represent changes in the centrosomal pool of these proteins, but not the satellite pool, which would then change the author's conclusions. A method that the authors can adapt is the Gupta et al. Cell 2015 paper. Such quantification will ensure that the authors are not drawing their conclusions based on changes in basal body levels of the proteins. Moreover, the representative images presented for centriolar satellite pools of these proteins in Fig. 5, Fig. 6 and Fig. S4 should be modified to include not only the basal body pool but the whole cell including an inset. Since satellites are distributed throughout the cell, presenting whole cell images is important to assess the remaining pool beyond the basal body.
    10. Does TTBK2 depletion alter the cellular abundance of CEP290, OFD1 and PCM1? This might underlie the changes in the basal body abundance of these proteins.
    11. Fig. 4C-E: Graphs are too small compared to figures.
    12. In multiple parts of the manuscript, the authors stated that the mechanisms of TTBK2 during cilium initiation is not known. Including major papers from the Goetz lab, there are many studies in literature that defines how TTBK2 regulates cilium initiation. The major unknowns relates to its functions during cilium maintenance and disassembly.

    Significance

    Although cilium assembly has been studied with extensive detail, relatively less is known about them mechanisms of cilium maintenance. This is in part due to lack of tools to manipulate protein expression in ciliated cells. The MEF cells used in this study is an elegant tool to address questions related to cilium maintenance and stability. Moreover, the live imaging experiments performed in TTBK2 depleted ciliated cells are excellent experiments in showing the spatiotemporal events that lead to axoneme destabilization. By identifying TTBK2 as a critical regulator of these processes, the results of the manuscript advances our understanding of how cilium is maintained as well as to the molecular defects that underlie SCA11. The topic is also of general interest to cell and developmental biologists.

    As a reviewer, my expertise is on questions that pertain to the biogenesis of centrosome and cilium and we extensively use cell biology, proteomics and biochemistry approaches.

  5. Version published to 10.1101/2022.06.02.494590v1 on bioRxiv