ATP induced conformational change of axonemal outer dynein arms studied by cryo-electron tomography

  1. Noemi Zimmermann
  2. Akira Noga
  3. Jagan Mohan Obbineni
  4. Takashi Ishikawa

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Axonemal dyneins in the outer dynein arm (ODA) generate force for ciliary beating. We analyzed three states of ODA during the power stroke cycle using in situ cryo-electron tomography, subtomogram averaging and classification. These states of force generation depict the pre-power stroke, post-power stroke conformations and an intermediate state. Comparison of these conformations to published in vitro atomic structures of cytoplasmic dynein, ODA and Shulin-ODA complex showed that the orientation and position of the dynein head and linker differs. Our analysis shows that all dynein linkers in the in the absence of ATP interact with AAA3/AAA4, indicating interaction to the adjacent B-tubule direct dynein orientation. For the pre-power stroke conformation, we found changes of the tail anchored on the A-tubule. We built pseudo-atomic models from high-resolution structures to generate a best fitting atomic model to the in-situ pre- and post-power stroke ODA, thereby showing that the Shulin-ODA display similar conformation as the active pre-power stroke ODA conformation in the axoneme.

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

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

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #4

    Evidence, reproducibility and clarity

    In the manuscript "ATP induced conformational change of axonemal outer dynein arms studied by cryo-electron tomography", Noemi Zimmermann et al. built pseudo atomic models of outer dynein arms (ODA) from the native axonemes with and without ATP treatment using a combination of cryo-electron tomography (cryo-ET), sub-tomogram averaging and atomic model fitting analysis. The authors clearly distinguished several important conformations of ODA using their high-quality cryo-ET maps. The authors showed that in situ ODA conformation in post-power stroke state is different from in vitro ODA structures, either lacking B-tubule binding or A-tubule binding. In my opinion, this is a very important observation by taking the advantage of cryo-ET analysis on intact axonemes. Furthermore, by freezing the activated axoneme immediately after ATP treatment, the authors obtained the active pre-power stroke and an intriguing intermediate conformation of ODA. By generating pseudo atomic models, the authors were able to compare the structural changes in dynein heads and stalks among different states and highlighted geometrical constraints from neighboring MTDs on ODA. Overall, the findings by Noemi et al have provided really exciting insights into ODA conformational changes during the power stoke. I therefore highly recommend publication of the manuscript. However, before the official publication, I do have some comments and believe the authors can further improve their manuscript to make it more exciting to the field.

    1. The authors only showed the maps from sub-tomogram averages (Supply Fig 1). I suggest the authors also show a representative reconstruction of the whole tomogram as a supplementary figure so that we have a better overview of the reconstruction.
    2. Since this is a typical piece of structural work, I highly suggest the authors summarize their cryo-ET data collection and processing parameters as a supplementary table, such as standard microscopy parameters, image pixel sizes, number of tomograms, number of particles etc.
    3. On page 5 and Supplementary Figure 2H, I, the authors fitted Lis1 model to the additional density at the interface between AAA2 and AAA3. This is really intriguing. However, according the currently published Lis1-dynein structures (PMID: 28886386, PMID: 34994688), it seems that Lis1 interacts with dynein on AAA4 and AAA5. Can the authors discuss anything about the evolutionary conservation of Lis1 binding? In addition, the authors did not fit LC5 model into the density map. I am a bit worried that there might be some bias on Lis1. With the fast development of protein prediction tools like Alphafold and Rosetta fold, the authors would be able to have a nice prediction of the LC5 structure to fit the additional density. I therefore suggest the authors try to do so if it is technically feasible, and then discuss a bit more on this point.
    4. On Page 6, the authors mentioned that "neither of the two structures (MTBS1, MTBS2) represented our conformation of ODA". This is an interesting finding since in the reconstituted ODA array on MTD by Rao et al., 2021 paper, they observed both MTBS1 (MTBD: 0 nm; MTBD:0nm; MTBD:8nm) and MTBS2 (MTBD: 0 nm; MTBD:8nm; MTBD:8 nm) conformations (Here, 0nm and 8nm represent the relative longitudinal positions along the tubulin lattice among the three MTBDs). According to the post-ODA structure from this manuscript, the authors found all three heavy chains are in the post-2 states, or equivalently with MTBDs at the 8-nm position (MTBD: 8nm; MTBD:8nm; MTBD:8nm, Fig3G). The authors also mentioned that the conformations of minimum energy of ODA are different in vivo and in vitro in the discussion. On the other hand, many structures previously determined by X-ray and EM in vitro show that Post-1 were overwhelmingly preferred before Rao et al reported the Post-2 state. This raises a very interesting question, how many MTBS states can ODA actually adopt in vivo? In theory, the three MTBDs can be arranged in at least a certain subset of the eight states (000,001,010,100,011,101,110,111) if the distance between any two MTBDs is restricted to 8nm, and the movement of each MTBD is restricted along one direction. There might be more states if the movement is more than one step. Therefore, from the results of both this manuscript and Rao et al., 2021 paper, probably not all states could have been observed. I wonder if the authors can perform more 3D classification on their STA particles in the post-PS state to demonstrate and see if there is any chance to see more states in vivo. I was a bit surprised because I felt there might be more states in vivo than in vitro reconstitution. The idea that the two neighboring MTDs can restrict the ODA conformation is great. I suggest the authors discuss more about the possible effects from two neighboring instead of just a general concept of energy minimization (probably it is impossible to estimate the total energy of such a complex system under physiological conditions using any kind of currently available techniques).
    5. In Figure 4, the authors observe structural changes of ODA among different states. The figures clearly show the differences among post-PS, intermediate state, and pre-PS state. For the pre-PS and intermediate state, I wonder if the authors can map the two conformations back onto the raw tomograms and show how they look like in a relatively large region with more repeating units.
    6. In Figure 4, I really appreciate the authors pointing out the distortion (changes in distances and the rotation angles) between adjacent MTDs. To my knowledge, the distortion of neighboring MTDs during ODA power stroke cycle has not been well analyzed in many previous publications. To gain more insights on this part, I wonder if the authors can perform more quantitative analysis on all adjacent MTDs with and without ATP from their current data sets. There are some nice publications on filament distortion analysis using single particle approaches, including one from the Sindelar lab (PMID: 32636254, Fig 4 and 6). More specifically, since the authors already have the position and Euler angle information of each particle from the subtomogram averaging, it is possible to extract the distortion information from two adjacent MTDs. After extracting distortion information from all MTD pairs and plotting the data points in different ways, the authors may be able to correlate the ODA conformation, MTD bending and see whether they could find some intriguing patterns. The authors do not have to incorporate all their results from this analysis into the current manuscript since there are already many interesting things, but briefly showing some curvature distribution would be highly appreciated, and the authors can still publish other interesting results in their future publications.
    7. It seems the authors have not deposited their maps and PDBs (as they are XXXX's in the current manuscript). It would be nice to if they can do so at their earliest convenience.
    8. On page 5, the authors found an additional density next to the  dynein which could be Lis1 or LC5 (see also minor comment #1). Again, this is an advantage using cryo-ET. This observation is also missing from ODA SPA papers, and I appreciate the authors for the careful examination. Since there are several 96-nm MTD maps from previously studies from Chlamydomonas and Tetrahymena, I wonder if this additional density is also present from previous cryo-ET maps.
    9. On page 5, the sentence "one unit of the dimeric Homo sapiens Lis1 (PDB-5VLJ (Htet et al., 2020, p. 1)) and fitting it into our density allowed us to assess its likeability." The Lis1 model in PDB-5VLJ is from Saccharomyces cerevisiae, not from Home sapiens. In addition, the reference paper doesn't match the PDB-5VLJ. The authors should cite the correct paper.
    10. On page 6 Figure 2 legend D, B HC should be  HC.
    11. On page 8 Figure 3 legend "A and B) Rigid body fit of the whole MTBS1 map (Walton et al., 2021).". The citation here should be Rao et al., 2021.
    12. In Figure 5, the authors generated models for the pre-PS conformation of ODA. From the cryo-ET density map, the authors suggested that -MTBD was in a bent conformation, which was similar to the conformation in shulin-ODA. This is a novel observation. Since the authors have atomic models, I suggest the authors directly use the PDB models for better visualization of structural changes among post-PS ODA, intermediate ODA, and pre-PS ODA. A supplementary figure or movie will be very nice.
    13. On page 16 "EM grids" session, I suggest the authors provide slightly more details on their sample preparation, such as the concentration of the axoneme, blotting time, temperature, humidity etc.

    Significance

    Significance: This is a very nice manuscript for better understanding of the motile cilia system. It is a significant progress in the field with lots of interesting findings.

    Audience: People in the field of dynein, motile cilia, cytoskeleton and in cryo-ET technique as well.

    My expertise: I am very confident in reviewing this paper, both biologically and technically, and I have recently published in this field as well.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The study attempts to reconcile cryo-EM SPA structures of ODAs with in situ tomographic reconstructions.

    Several key discrepancies between SPA structures and the native in situ structures (here) are highlighted in the study with a particular focus on the positions of various ODA motor head components (linker, tails etc.) during the powerstroke cycle.

    The study also highlights largely concordant inter and intra-ODA connections between previous SPA structures and the tomographic reconstructions.

    Major comments:

    Overall, the key conclusions are convincing. No additional experiments are suggested. The manuscript is acceptable provided minor comments below are addressed.

    Minor comments:

    The text could be improved throughout for improved clarity. Overall, the figures are good, but some panels are over-annotated which is confusing. Simplification or cartoon illustrations could add clarity to the figures.

    CROSS-CONSULTATION COMMENTS

    The paper still represents a significant and sufficient advance. Correction of factual errors flagged up by other reviewers (use of correct references and citations, correct species for Lis1 models used etc.) is required and essential prior to acceptance. Addition of more details in the sample preparation methods section would also be useful. Depositing PDBs and maps is recommended.

    Agree on the overall point of improving accessibility and readability of the text. Figures can be much improved to highlight the biological insights for the reader.

    The point of contention between extra density corresponding to either Lis1 or LC5 is valid. Tempering the assertion and removing bias towards Lis1 in the text would resolve this issue. The authors are putting forth a speculative model which is valid; this model can be tested in future work.

    Several minor comments highlighted by other reviewers are fair and should be addressed as best as possible.

    Several major comments highlighted by other reviewers (specifically: use of structure prediction and modeling, filament distortion analysis etc.) are well beyond the scope of the present work and do not advance the specific and main conclusions of the current study.

    Significance

    The study presents structures of ODAs during their powerstroke cycles in situ in their native context and integrates previous structural models of ODAs to provide novel insights.

    The identification of a Lis1 or LC5 like density adjacent to the alpha-HC and observation of a curved position of the beta-HC stalk in the native state adds further novelty to the study.

    The study will be of interest to researchers like myself working on cilia motility and dynein motors.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    • The authors report cryo-EM tomography of the axoneme of motile cilia in the presence and absence of ATP, providing new insight into the mechanism of action of the motors. They use crystal structures and information from single particle cryo-EM to fit these fragments into their new density obtained in situ, and show that distortions to these smaller structures are required for them to be accommodated in the complex and crowded environment of the axoneme. The movies provided show the relevant fits in 3D, which is important because the complexity of the structures makes 2D visualisations limited. Are the authors sufficiently confident in their atomistic models that they would be useful for other researchers, and if so are they planning to release them (e.g. as pdb files) with the paper, or on request?

    • There are potentially a few editorial additions and changes that the authors might consider making to improve the readability of the paper for non-specialists in the axoneme. For example, could they insert a sentence explaining what Shulin is and its biological significance? There are numerous abbreviations and acronyms throughout the manuscript - would it be helpful to maybe write some of those out in full where appropriate? In the very helpful Supplementary table containing the pdb IDs used to fit into the current structure, would it be useful to have a small picture of each system as one of the columns in this table? Would it also potentially be helpful to include a figure summarising the different types of dynein observed in this and other relevant studies - e.g the pre and post-powerstroke states, Shulin bound etc? This would help the reader to understand the magnitudes of the conformational changes between these various states that are under discussion. Could a schematic diagram representing the "winch" and "rotation" models be included potentially? In the Discussion section, I was not able to understand whether the winch or rotation models are most supported by the data in this paper, or whether a mixture of the two might be needed to understand axoneme mechanics, so further clarification of this would be helpful.

    • Please note that all of these comments are suggestions to improve accessibility and readability, and are not essential additions for the paper to be publishable.

    CROSS-CONSULTATION COMMENTS

    I was very interested to read the detailed and informative comments from the other referees. While I agree with referee 1 point 4 that the use of alpha-fold to predict how atomistic structures from different organisms may differ, and subsequent flexible fitting would be desirable, this in my opinion would be an enormous amount of work, and would be best reserved for subsequent publications. Sharing of the pdb files of the fitted structures obtained so far would open this mammoth task up to the rest of the community.

    Given the complexity of the axoneme, and the huge amount of expertise needed to obtain and process these tomograms, I did wonder if this community would consider forming a collaborative consortium where researchers worked together to construct a common model.

    Significance

    • The paper reports more complete and detailed structural information on the axoneme than (to my knowledge) has been obtained before. The fitting of atomistic level structures into the density to create a pseudo-atomic model is highly instructive.

    • To me, it was not in the least bit surprising that distortions from the structures obtained in isolation using single particle analysis are required for an optimal fit. In fact, theoretical work reported by Richardson et al, QRB 2020 showed for inner dynein arms that the crowded environment provided solely by the microtubule tracks within the axoneme modified the conformations of the dynein stalk that were accessible compared to a simple isolated dynein motor. While this study considers outer dynein arms, the conceptual physical rationale is equivalent to the findings here. In my opinion, the finding reported in this paper that considering fragments of biological ultrastructures is not necessarily equivalent to the whole functioning entity is both important and profound, and has implications beyond motile cilia, particularly as cryo-electron tomography enables us to visualise ever larger and more complex functional biological assemblies.

    • Please note that my area of expertise does not enable me to comment on the experimental procedures used to obtain the tomograms, as I am a computer modeller with an interest in dynein.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Zimmermann et al. provide a comparison between recent atomic models of the ODA determined by single particle cryo-EM and their conformation within intact axonemes by cryo-ET subtomogram averaging. They observed slight changes in the position of the motors for the structures of Kubo et al. and Walton et al., but the structure of Rao et al. required more changes, indicating that within the axoneme, the conformation of the ODA is influenced by the MTD on which it is docked, and the neighboring MTD to which its motors bind. They then use the information from their newly fit models to interpret cryo-ET maps of axonemes in the presence of ATP, which activates the ODA and other axonemal dyneins. They observe two states of the ODA, and describe how the position of the motor, linker, MTBD and LC tower change during the powerstroke cycle. A revised model of the ODA and the ability to describe conformational changes at the subunit level provides an advance on previous work and will be of interest to the dynein and cilia fields. However, the comments below must be addressed prior to publication, and additional work is needed to make the paper accessible.

    Major comments

    1. Greater clarity is needed in the introduction to explain the differences between the recent atomic models of the ODA. This is essential to understanding the paper, including Fig. 3. Arguably, the top half of Fig. S2 provides a stronger case for the study than any of the current main figures.

    2. In the manuscript, potential differences between Chlamydomonas and Tetrahymena ODAs are not considered but need to be explored. Comparison of Tetrahymena models within Chlamydomonas maps could result in misinterpretations.

    3. Systematic quantification of the fit-to-map should be provided for the models before and after refitting (together with evidence - see the point below - that the model has not been inappropriately distorted to fit the map). This information could be inserted into an expanded Supplementary Table.

    4. Because the revised pseudo-atomic model of the ODA is a chimera of PDBs from different organisms, it does not accurately represent the Chlamydomonas ODA. The modeling method also has the potential to introduce clashes between rigid-body fitted chains. Validation of the model is necessary, and alternative approaches to generate a more accurate model (e.g. AlphaFold and molecular dynamics flexible fitting) should be considered.

    5. Additional evidence needs to be provided to demonstrate that the intermediate state observed in Figure 4 is robustly detected and does not simply represent the data that doesn't fall into the "good" classes. In Fig. S1, the map looks very noisy and requires denoising. Are there other changes observed in the IDAs that would support the existence of an intermediate state?

    6. The speculation that the additional density bound to a-HC is Lis1 is not well-supported. Lis1 binds AAA4/5 (PDB: 5VH9), not AAA2/3. The fit of the Lis1 homolog into the cryo-ET density does not appear consistent with Lis1 binding the motor. The authors should consider other possibilities that could explain the additional density.

    Minor comments

    1. The results section "Post-PS structure and Fitting of the atomic models" is very dense. It should be split into subsections to help guide the reader through specific models or regions of the ODA.

    2. ODA numbering should be made consistent with previous papers (i.e. ODA1-4 as in Bui et al., 2012)

    3. The ODA-shulin model (PDB: 6ZYW) is inaccurately described as the state transported during IFT, but experimental confirmation of this hypothesis is lacking.

    4. The term TTH for tail-to-head contacts is too similar to T/TH for the tether/tetherhead complex and should be changed. An abbreviation may not be necessary.

    5. Please check to make sure that all figures and figure legends clearly specify which map/model/motor is being shown. This will make the figures easier to follow.

    6. The structures in Fig. 3 are from Rao et al., not Walton et al.

    7. Fig 5M-O is very difficult to interpret. Could the authors consider coloring by region, for one of the maps, or at least put the maps in a similar orientation to the ODA cores as in Fig 2?

    8. The final processing step in panel Fig S1B is confusing. Additional information is needed to explain the supervised classification step and how the final particle set was derived.

    9. Atomic resolution should not be used to describe structures determined to 4.3 Å resolution (e.g. EMD-11579).

    10. Supervised classification is not a method of validation

    11. Please check for grammatical and spelling errors throughout the manuscript.

    Significance

    While previous literature has interpreted ODA conformation in broad regions, this study goes farther by using recent atomic models to identify specific subunits that change conformations and interactions during the powerstroke. From my perspective as a structural biologist in the cilia field, I think this paper provides a conceptual advance to the study and interpretation of axonemes.

  6. Version published to 10.1101/2022.05.02.490280v1 on bioRxiv