Heterogeneity of radial spokes structural components and associated enzymes in Tetrahymena cilia

Reviewer #1 (Public Review):

Summary:
Radial spokes (RS) are made of >20 proteins and are believed to be a transducer to coordinate axonemal dyneins to enable the beating motion of motile cilia. While the atomic structure of RS from green algae Chlamydomonas and H. Sapience has been solved by single particle cryo-EM recently, this work by Bicka et al. provided the atomic structure of RS from ciliate Tetrahymena. They identified component proteins of Tetrahymena RS, which correspond to those in the atomic structure of Chlamydomonas and human RS. These proteins were likely already guessed as RS components, based on sequence similarity, but in this work experimentally identified for the first time. Furthermore, they discovered novel isoforms of RS proteins and characterized them structurally and functionally. RSP3 has three isoforms (A, B, and C). They are distributed specifically in the three radial spokes within the repeating unit as proved by mutant analysis, cryo-EM, and proteomics. By high-speed video microscopy, they proved the essential roles of RSP3B for ciliary beating. These isoforms have never been reported in past works and this demonstrates the novelty of this work.

Strength:
Their discovery of RSP3 isoforms is unexpected and, although it is still not clear why Tetrahymena needs to have these isoforms, will evoke future research. The authors characterized the multi-facet aspects of these proteins precisely, structurally by cryo-EM, functionally by waveform and velocity analysis, and in terms of protein networking by co-IP and bioID studies.

Weakness:
While the first half of this manuscript about RSP3 isoforms is very well organized and described (this reviewer still has some advice to make this story convincing and attractive), the later part has room for improvement. Some results are presented in the current manuscript without mentioning figures or tables, for example in "250: The components of the Tetrahymena radial spoke stalks" no figure/table is cited. There is also inconsistency - in 327 RSP9 is mentioned as a MORN protein, but in Fig.6 Sup.3 Table.1, it is mentioned as "unknown".

Reviewer #2 (Public Review):

Summary:
Radial spokes are evolutionarily conserved protein complexes that are important for cilia motility. So far, the composition of certain radial spokes was investigated in the algae Chlamydomonas, mice, and humans. This work by Bicka et al. investigated the composition of radial spokes in the ciliate Tetrahymena by analyzing knockouts and strains that express tagged radial spoke proteins, using mass spectrometry and cryo-electron tomography. While three specific types of radial spokes have been reported thus far, this study suggests that in Tetrahymena, there is another layer to the variability in radial spokes. Additionally, many proteins with predicted enzymatic folds have now been assigned to radial spokes. The comparison of ciliary complexes between species is important to define the basic principles that govern cilia motility, as well as to reveal the differences that enable cilia of various organisms to beat in diverse environments.

Strengths:
The manuscript includes a thorough bioinformatic analysis of radial spoke proteins in Tetrahymena and reveals the presence of multiple orthologs to certain algae and mammalian radial spoke proteins. The mass spectrometry analysis and cryo-electron tomography experiments are solid and informative. This work provides a lot of important data and thus, opens the door to resolve the exact composition and structures of radial spokes in Tetrahymena and perhaps other species.

Weaknesses:
The assignment of the three RSP3 orthologs to RS1, RS2, and RS3 is based only on missing structures in the knockouts. Although this method is informative, it is not sufficient to draw conclusions regarding the positions of the missing proteins. There are numerous examples where a structure was missing, but the absent protein was localized elsewhere (i.e., absence of central pair protrusions in patients with mutations in radial spoke proteins). To directly demonstrate the position of an RSP3 ortholog in a certain radial spoke, the protein can be labeled with a tag that is visualized in subtomogram averages (as was done in Oda et al., 2014 and other studies). Relying on the data from knockouts alone, the model for radial spoke composition in Tetrahymena (Fig. 6) may be incomplete.

The control for the bio-ID experiment was WT cells. Since there are many hits in the experiment, a better control would have been a strain with free BirA, or BirA fused to a protein that is distant from the radial spokes, such as one of the outer-dynein arm proteins, or a ciliary membrane protein.

Reviewer #3 (Public Review):

Summary:
The authors aim to study the role of axoneme radial spoke proteins in forming the three radial spokes that connect the central pair microtubules with the doublet microtubules of the ciliary axoneme. They combined existing and novel mutants to first study ciliary dynamics, followed by cryoET structure and proteomics to identify known and new radial spoke protein components, and assign those with radial spoke(s) to which they belong.

Strengths: / Weaknesses:
The strengths of this study are in the genetic mutants combined with the cryoET to study the unique structural impacts of each mutant on the three radial spokes. The proteomics to study protein loss and interactions also enabled a comprehensive comparison of proteins at the radial spoke under normal and mutant conditions. This allowed the authors to predict that there are several classes of each type of radial spoke. While there are some limitations with overlapping phenotypes between the mutants, this tactic allows the authors to predict known and new proteins that are predicted to localize to each of the three radial spokes. However, in some places, the conclusions are overstated and the list of molecules without functional insight simply identifies new components that will need to be the target of future studies. Two examples of this are that the authors claim to have "solved the composition of individual radial spokes" and that "adenylate kinases [that] dock to specific RSs". Neither of these statements should be made based on the results in this manuscript. Moreover, the authors state that Rsp3Bp does not change in rsp3C knockouts and conclude that Rsp3B from the A-C heterodimer is still attached to the axoneme without maintaining the RS2 structure. To me, this makes a series of strongly stated conclusions without the results to justify the statement. The authors also report on unique features of ciliary dynamics resulting from the loss of each of the three Tetrahymena RSP3 genes. This showed a strong phenotype for rsp3b knockout. However, a quantitative measure of ciliary dynamics to understand how much the presented data represent the ciliary dynamics was not clear. Furthermore, the authors argue that metachrony or coordination between cilia was affected but the presented data are not interpretable or quantified. Furthermore, the authors state that all three Rsp3 paralogs localize along the entire length of the cilium. However, Rsp3A and B do not localize to ciliary tips, while Rsp3C does. This may inform the differences found in the ciliary waveform for rsp3C mutants compared to rsp3A and rsp3B. The authors state that they have defined a "large part of the protein composition of individual RSs...". It is not clear to me that they know how much of the total RS proteome they have identified.

This manuscript identifies new candidate proteins that may function with radial spokes, future work will be required to 1) confirm their localization to the radial spoke and 2) to study their function within radial spokes.

Author Response

We thank Editors and Reviewers for their positive evaluation of our work and appreciation of new findings and applied interdisciplinary approaches. We also thank for pointing out manuscript weaknesses as well as for all suggestions and advices that can strengthen this manuscript. We apologise for mistakes, overstatements or discrepancies in citing figures as well as omitted references.

The first part of the manuscript focuses on the Tetrahymena RSP3 genes mutants. Tetrahymena genome encodes three RSP3 paralogs that are the components of different radial spokes and likely form homo- and heterodimers. Thus, the proteomic analyses of Tetrahymena radial spokes are more complicated compared to the similar analyses in organisms having a single RSP3 protein.

Next, we attempted to identify proteins specific for each RS type. Conducting this research, we took advantage of six different radial spoke knockout mutants (RSP3A-KO, RSP3B-KO, RSP3C-KO, CFAP206-KO, CFAP61-KO, and CFAP91-KO) and compared wild-type and mutants’ ciliomes using two methods, LFQ and TMT (for each mutant the experiment was repeated three times). Comparative analyses of the wild-type and mutants ciliomes allowed us to identify Tetrahymena radial spoke proteins, in the case of RS1 (WT versus RSP3A-KO), RS2 (WT versus RSP3B-KO, RSP3C-KO, and CFAP206-KO), and RS3 (wild-type versus CFAP61-KO and CFAP91-KO). The extensive proteomic analyses were combined with detailed bioinformatics studies and co-immunoprecipitation and BioID assays to verify the presence of identified proteins in RS complexes.

Importantly, in the case of RS1 and RS2 spokes, our findings are in agreement with data obtained for Chlamydomonas and mammalian radial spokes. Thus, it is very likely, that newly discovered RS1 and RS2 proteins as well as identified Tetrahymena RS3 proteins are also true RS subunits.

As an outcome of this part, we propose a model of the RS protein composition in a ciliate Tetrahymena. We agree that this model requires further experimental verification (for example by pull-down experiments). However, considering the number of identified proteins, this is a considerable amount of additional work that we would like to publish as separate papers. We would like to add that our current analyses of additional RS3 mutant (that will be published separately) support findings regarding RS3 proteomic composition.

Reviewer 2:

The control for the bio-ID experiment was WT cells. Since there are many hits in the experiment, a better control would have been a strain with free BirA, or BirA fused to a protein that is distant from the radial spokes, such as one of the outer-dynein arm proteins, or a ciliary membrane protein.

The BirA* tag is approximately 30 kDa protein and thus it can be transported to cilia by diffusion. BirA* ligase present throughout the cilia could randomly biotinylate proteins including radial spoke proteins. Thus, expression of the BirA* alone is not the best control. We have performed numerous BioID experiments in which BirA* tag was fused with T/TH subunits (CFAP43, CFAP44, Urbanska et al., 2018), subunits of the small complex positioned parallel to N-DRC (CCDC113, CCDC96, Bazan et al., 2021), CFAP69, SPEF2A (C1b central apparatus complex, Joachimiak et al., 2021), N-DRC proteins (Ghanaeian et al., Biorxiv, 2023) and subunits of other ciliary complexes (our unpublished data). The comparison of the earlier obtained BioID data with RSP BioID data, prove that identified proteins are specifically associated with radial spokes. Therefore, in our model, wild-type cells are a good control for BioID experiments.