Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates

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1. 

   eLife

   Apr 16, 2023

   Author Response

   Reviewer #2 (Public Review):

   Yamaguchi et al. studied the roles of two proteins, Calaxin and Armc4, in the assembly of the outer arm dynein (OAD) docking complex (DC). By combination of the improved cryo-ET analysis and gene knockout zebrafish lacking each of these proteins, they found that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction in the docking.They further showed an evidence that Calaxin changes the conformation of another compartment of DC comprising CCDC151/114. This new information provides an important basis for understanding how the DC is assembled and regulates docking of OAD. The authors' conclusion is well supported by the data but some data presentation and discussion need to be completed.

   Gui et al. (2021) already reported on a cryo-EM …

   Author Response

   Reviewer #2 (Public Review):

   Yamaguchi et al. studied the roles of two proteins, Calaxin and Armc4, in the assembly of the outer arm dynein (OAD) docking complex (DC). By combination of the improved cryo-ET analysis and gene knockout zebrafish lacking each of these proteins, they found that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction in the docking.They further showed an evidence that Calaxin changes the conformation of another compartment of DC comprising CCDC151/114. This new information provides an important basis for understanding how the DC is assembled and regulates docking of OAD. The authors' conclusion is well supported by the data but some data presentation and discussion need to be completed.

   Gui et al. (2021) already reported on a cryo-EM observation in bovine tracheal cilia, with the conclusion similar to this paper in the structure of OAD/DC on DMT. Using knockout zebrafish strain, the authors present detailed interaction of calaxin with other DC components. They show that the binding of calaxin induces the changes of conformation in N-terminal region of CCDC151/114. The conformation further changes in the presence of Ca2+; specific conformation of N-terminal region of CCDC151/114 becomes undetectable, instead additional structure appears in the vicinity of calaxin.

   1. The authors conclude that the Ca2+-dependent conformational change of DC is subtle and not dynamic. This result is eventually valuable information but may be somewhat unexpected from the point of view that calaxin plays an important role in the regulation of flagellar motility in Ciona sperm. The authors found that calaxin changes the conformation of N-terminal CCDC151/114 region but the core dynein structure shows no dynamic change. What about the changes in the interaction between calaxin, core dynein, and DMT? Is this beyond the resolution of cryo-ET analysis?

   Since Mizuno et al., 2009 reported that Ciona Calaxin switches its interactor depending on Ca2+ concentration, it is highly expected that zebrafish Calaxin also changes its interactor in 1 mM Ca2+ buffer conditions. However, the resolution of our cryo-ET data was insufficient to detect the change of Calaxin interactors. More detailed structural analyses are required to understand the OAD structures in the Ca2+ buffer conditions. We discussed this point as follows:

   (line 389-395)

   Regarding the Calaxin conformation, a previous biochemical analysis reported that Ciona Calaxin switches its interactor depending on Ca2+: β-tubulin at lower Ca2+ concentration and OAD γ-HC at higher Ca2+ concentration (Mizuno et al., 2009). Moreover, a crystal structure analysis revealed the conformational transition of Ciona Calaxin toward the closed state by Ca2+-binding (Shojima et al., 2018). In this study, however, such conformation change of Calaxin was not detected, probably due to insufficient resolution of our cryo-ET analysis. More detailed structural analyses in the Ca2+ condition are required to understand the mechanism of the Ca2+-dependent OAD regulation.

   1. It would be very helpful if the authors could add the cryo-ET images of calaxin-/- axoneme in the presence of 1 mM EGTA in Figure 7. Although these images are thought to be similar or identical to Figure 4F, it would help to confirm that the conformational changes in CCDC151/114 and additional part of DC are induced in a Ca2+-dependent manner.

   We added the cryo-ET images of calaxin-/- OAD-DC (1 mM EGTA) in Figure 7D.

   1. To clarify the molecular interaction of calaxin with other components, it would also be helpful if the authors add the images rotated 80 degree to Figure 4F and G, in similar way in Figure 7.

   We added the images of OADs rotated 80 degrees in Figure 4F and G.

   1. Despite the molecular phylogenetic difference, there are several similarities between calaxin and Chlamydomonas DC3, not only in the in situ structure and configuration but in the phenotype of mutants; Chlamydomonas mutant lacking DC3 shows OAD loss in the distal part of a flagellum (Casey et al, MBC, 2003). It may be a good reference if the authors add the position of DC3 in Figure 4. A', B', and C.

   To answer this comment, we created Figure 4—figure supplement 1, which shows the cryo-ET structures and models of OAD-DCs in vertebrates and Chlamydomonas.

   1. There is a significant difference in sperm motility between WT and calaxin-/- or WT and armc4-/- (Figure 2E). However, it is not clear whether immotile sperm were included in the data for VAP (Figure 2F) or BCF (Figure 2G). For example, WT and calaxin-/- show similar VAP, although both are significantly different in the percent of motile sperm.

   In our CASA study, spermatozoa with less than 20 μm/s velocities were considered immotile and excluded from the data for VAP (Figure 2F) and BCF (Figure 2G). To clarify this point, we revised the manuscript as follows:

   before

   Swimming velocity and beating frequency were calculated from the trajectories of the motile spermatozoa (Figure 2F-G; Figure 2—figure supplement 1; Video3).

   after (line 139-141)

   Swimming velocity (VAP) and beating frequency (BCF) were calculated from the trajectories of the motile spermatozoa, which have 20 μm/s or more velocities (Figure 2F-G; Figure 2—figure supplement 2; Video3).

   1. In calaxin-/- zebrafish, OAD was clearly detected from the base to two-thirds of a flagellum with unclear border (Figure 2A). Typical distribution of OAD+class and OAD-class are shown in Figure 5 in the ~3 micrometer tomograms. Were these taken from around this unclear border? Are proximal most region of a flagellum occupied with OAD+class only? The authors should clearly indicate the region of a flagellum where the tomograms in Figure 5C and D were selected.

   1. Line 229~: It is not clear what the authors meant by "probably reflecting the different distance from the sperm head". In relation to this and the comment 6, does the "proximal" in the sentence "OAD loss occurred even in the proximal part of the flagella" (line 232) indicate the region near the base of a flagellum?

   In general, axonemes are tangled on the cryo-TEM grids, which makes it difficult to identify the ends of all axonemes, especially for the long zebrafish sperm flagella. Thus, we could not clarify the region of a flagellum about the tomograms shown in Figure 5D.

   However, to answer comments (6) and (7), we created Figure 5—figure supplement 1. In this experiment, we newly generated cryo-TEM grids with sparse sperm axonemes and succeeded in finding two areas containing clear axonemal ends with suitable ice conditions for cryo-ET observations (Figure 5—figure supplement 1B). The polarity of the axonemes was judged from the 3D-reconstructed structures of the axonemes (Figure 5—figure supplement 1B, red dotted lines). By the structural classification of OAD+ class and OAD- class in the tomograms, we confirmed the OAD loss in calaxin-/- even in the proximal part of the flagella, which is near the base of a flagellum (Figure 5—figure supplement 1D, (a) and (c)). To clarify these points, we revised the manuscript as follows:

   before

   In calaxin-/-, the ratio of OAD+ class to OAD- class varied among tomograms (Figure 5D), probably reflecting the different distance from the sperm head. However, all calaxin-/- tomograms showed multiple clusters of OAD- class, indicating that the OAD loss occurred even in the proximal part of the flagella.

   after (line 236-239)

   In calaxin-/-, the ratio of OAD+ class to OAD- class varied among tomograms (Figure 5D), reflecting the different distances from the sperm head. Analysis of detailed OAD distributions along calaxin-/- axoneme revealed that OAD loss occurred even in the proximal part of the flagella (Figure 5—figure supplement 1D).

   1. In conjugation with comment 7, it would be appreciated to show an authors' idea on why distal region of flagella tends to lack calaxin, if they do not discuss anywhere in the text.

   We discussed this point as follows:

   (line 316-323)

   calaxin-/- spermatozoa exhibited a unique OAD distribution, with OAD-missing clusters at various regions of the flagella. Interestingly, OADs decreased gradually toward the distal end, by which the mechanism is unclear. The axoneme is elongated by adding flagellar components to its distal end during ciliogenesis (Johnson & Rosenbaum, 1992). IFT88, a component of the IFT machinery, disappears as the spermatozoa mature (San Agustin et al., 2015). Thus, we speculate that the OAD supply at the distal sperm axoneme is insufficient to compensate for the OAD dissociation in the calaxin-/-. Consistent with this idea, distal OAD loss is the sperm-specific phenotype, as olfactory epithelial cells in calaxin-/- have Dnah8 along the entire length of the cilia (Figure 6B).

   1. Immunofluorescence in twister-/- epithelial cilia showed that the localization of calaxin is independent of OAD (line 271-274). Based on the authors' finding, the localization of calaxin requires Armc4, which is preassembled with calaxin in the cytoplasm. If this is true and the localization of calaxin is NOT resulting from diffusion, Armc4 must be localized with calaxin along the entire length of cilia in twister-/- epithelial cilia (Figure 6D). Although Armc4 is shown localized in cryo-ET images (e.g. Figure 1, Figure 7), authors may provide the immunofluorescence of Armc4 along the entire length of sperm flagella and epithelial cilia.

   To answer this comment, we obtained a commercially available anti-ARMC4 (human) antibody and checked the cross-reactivity of the antibody against zebrafish Armc4, but no signal was detected in our western blot analysis. Thus, we could not assess the localization of zebrafish Armc4 in twister-/- epithelial cilia.

   In our study, we found an ectopic accumulation of Calaxin at the ciliary base in armc4-/- cells (Figure 6C, white arrowheads). The small molecular weight of Calaxin (~25 kDa) suggests the possible diffusional entry of Calaxin into the ciliary compartment. However, in armc4-/- cells, Calaxin accumulated at the ciliary base, strongly suggesting that Calaxin requires Armc4 to be localized to cilia.

   Reviewer #3 (Public Review):

   ODA-DC anchors ODA, the main force generator of ciliary beating, onto the doublet microtubules. Vertebrate ODA-DC contains 5 proteins, including Calaxin and Armc4, whose mutations are associated with defective ciliary motility in animals and human. By generating calaxin-/- and armc4-/- knockout zebrafish lines, this manuscript examined the Kupffer's vesicle cilia and spermatozoa. They showed that calaxin-/- and armc4-/- knockouts both affect ciliary motility but to different degrees. The authors conducted careful structural analyses using cryo-ET and subtomo averaging on both mutants, revealing a partial loss of ODA in calaxin-/- and a complete loss of ODA in armc4-/-. I really like the distribution analysis of calaxin-/- OADs (Figure 5), which emphasizes the strength of cryo-ET in uncovering the molecule distribution of distinct conformational states in situ. Fitting of the atomic models of ODA and ODA-DC into the cryo-ET density maps and Calaxin rescue experiments showed how Calaxin stabilizes ODA at a molecular detail. By using olfactory epithelium, the authors also presented the possible assembly mechanism of ODA-DC proteins, which is also a beautiful experiment. Finally, the authors also investigated how Ca2+ regulate the ODA-DC using cryo-ET.

   The thorough structural and functional analyses of Calaxin and Armc4 in WT and gene KO animals could serve as a reference for future study of the detailed function of other ciliary proteins. The experiments are overall well designed and conducted, but some aspects need to be clarified and improved.

   The authors interpret the vertebrate ODC-DC to include four linkers (line 193). However, the authors also said that loss of one linker (Calaxin) makes ODA to attach on the DMT through two linkers (line 199 and 246). These descriptions are confusing. It would make more sense to interpret the vertebrate ODC-DC as containing three linkers (CCDC151/114, Armc4/TTC25, Calaxin).

   This comment is reasonable because vertebrate OAD is tethered to DMT through three linker structures (the distal CCDC151/114, Armc4/TTC25, and Calaxin). However, vertebrate DC is composed of four parts (a) Calaxin, (b) the Armc4-TTC25 complex, (c) the proximal CCDC151/114, and (d) the distal CCDC151/114 (Figure 4E). The (c) part is embedded in the cleft between protofilaments A07 and A08. To clarify this point, we revised the manuscript as follows:

   before

   The bovine DC model shows that vertebrate DC is composed of four linker structures: (a) Calaxin, (b) the Armc4-TTC25 complex, (c) the proximal CCDC151/114, and (d) the distal CCDC151/114 (Figure 4E).

   after (line 196-200)

   The bovine DC model shows that vertebrate DC is composed of four parts: (a) Calaxin, (b) the Armc4-TTC25 complex, (c) the proximal CCDC151/114, and (d) the distal CCDC151/114 (Figure 4E). Among the four parts, three (a, b, and d) work as linkers between OAD and DMT, while (c) the proximal CCDC151/114 is embedded in the cleft between protofilaments of the DMT.

   To confirm whether Calaxin directly interacts with β-tubulin (line 213), a control experiment could be needed by incubating WT axoneme with mEGFP-Calaxin followed by IF imaging.

   In our manuscript, we wrote as follows:

   (line 218-224)

   To assess the specificity of Calaxin binding, we also performed a rescue experiment with mEGFP-Calaxin (Figure 4H-I; Figure 4—figure supplement 2). Ciona Calaxin was reported to interact with β-tubulin (Mizuno et al., 2009), suggesting the possible binding of Calaxin along the entire length of the axoneme. However, the rescued axonemes showed partial loss of EGFP signal (Figure 4H, white arrowheads). This pattern resembled the OAD localization of calaxin-/- in immunofluorescence microscopy, suggesting the preferential binding of Calaxin to the remaining OAD-DC. mEGFP alone showed no interaction with the axoneme (Figure 4H, asterisk).

   Therefore, our manuscript is NOT intended to support or deny the interaction between Calaxin and β-tubulin, which was reported by Mizuno et al., 2009. Instead, we focused on the interaction between Calaxin and OAD-DC, revealing that Calaxin binds to Calaxin-deficient OAD-DC (Figure 4G, H, and I). Thus, we assume this comment refers to the interaction between Calaxin and OAD-DC.

   To further discuss the interaction between Calaxin and OAD-DC, we created Figure 4—figure supplement 2. We tested Calaxin’s interaction by incubating recombinant mEGFP-Calaxin with sperm axonemes of calaxin-/-, armc4-/- (representing OAD-missing DMT), and WT (representing DMT with Calaxin and OAD). The localization of mEGFP-Calaxin was assessed by fluorescence microscopy of mEGFP signals. In calaxin-/-, mEGFP-Calaxin was bound to the limited region of the axoneme, with the partial loss of EGFP signals (Figure 4—figure supplement 2A, white arrowheads), consistent with Figure 4H. On the other hand, mEGFP-Calaxin showed no significant interaction with armc4-/- axoneme (Figure 4—figure supplement 2B) or WT axoneme (Figure 4—figure supplement 2C). These data show the preferential binding of Calaxin to the Calaxin-deficient OAD-DC than OAD-missing DMT or WT OAD. Although Mizuno et al., 2009 reported the interaction between Calaxin and β-tubulin, our analysis could not detect the signals for such interaction, probably due to the different binding affinity of Calaxin against OAD-DC and β-tubulin.

   The Immunoblotting experiment should be improved in Figure 5E. Could the authors get the same results in repeating experiments? Why is the Dnah8 signal higher in 50 mM NaCl of the (+)Calaxin group compared to that in 0 NaCl? This makes me doubt if the difference between (-)Calaxin and (+)Calaxin groups are significant.

   This comment is reasonable because NaCl concentration-dependent detachment of OAD-DMT suggests the highest Dnah8 signal in 0 mM NaCl of the (+)Calaxin group. To discuss this point, we created Figure 5—figure supplement 2, which shows the experimental replication of the immunoblot analysis in Figure 5E. In this experiment, we used calaxin-/- sperm axonemes collected independently of the Figure 5E data.

   However, again, the Dnah8 signal was higher in 50 mM NaCl of the (+)Calaxin group than that in 0 mM NaCl, confirming the result in Figure 5E. One possible explanation for this result is that the NaCl concentration affects the rescue efficiency of the Calaxin protein. We speculate that the Calaxin protein requires NaCl for efficient binding to OAD-DC, which caused the lower amount of OAD in 0 mM NaCl of the (+)Calaxin group compared to that in 50 mM NaCl.

   The authors have covered several important points in the Discussion section. Now that the function of Calaxin in both mouse and zebrafish have been reported, the authors could discuss the similarity and difference of Calaxin function in different species and tissues.

   To discuss this point, we inserted the following paragraph:

   (line 324-333)

   In mouse Calaxin-/- mutant, motile cilia in various organs (sperm flagella, tracheal cilia, and brain cilia) showed abnormal motilities, although OADs in the mutant cilia/flagella seemed mostly intact when observed by conventional transmission electron microscopy (Sasaki et al., 2019). In our study, however, we revealed that mutation of zebrafish calaxin caused OAD-missing clusters at various regions of the flagella, by using detailed cryo-ET analysis and immunofluorescence microscopy. Thus, we speculate that the same OAD defects to zebrafish calaxin-/- caused abnormal ciliary motilities in mouse Calaxin-/- mutant. One exception is the mouse nodal cilia. In mouse Calaxin-/- mutant, the formation of nodal cilia was significantly disrupted (Sasaki et al., 2019). On the other hand, zebrafish calaxin-/- mutant showed the normal formation of Kupffer’s vesicle cilia (orthologous to the mouse nodal cilia), suggesting the tissue-specific function of Calaxin on the ciliary formation.

   Because of the limited resolution, the authors should be more careful when interpreting the small densities in the difference map, for example, in Figure 4F-G black arrows. Considering that the CCDC151/114 coiled coil is overall poorly resolved both in the WT and mutant cryo-ET maps, the different densities could be due to different map quality or data processing. This makes the following statement suspicious "This structure corresponds to the N-terminus region of CCDC151/114, suggesting that Calaxin affects the conformation of neighboring DC components".

   This comment is reasonable because the resolution of our cryo-ET data was insufficient to identify each molecule in the cryo-ET map. To be more careful about the interpretation of our cryo-ET structures, we revised the manuscript as follows:

   before

   However, the difference map also showed an additional missing structure adjacent to Calaxin (Figure 4F’, black arrowhead). This structure corresponds to the N-terminus region of CCDC151/114, suggesting that Calaxin affects the conformation of neighboring DC components.

   after (line 207-210)

   However, the difference map also showed an additional missing structure adjacent to Calaxin (Figure 4F’, black arrowhead). When fitting the bovine DC model, this structure overlapped the N-terminus region of CCDC151/114, indicating that Calaxin can affect the conformation of neighboring DC components.

   To discuss the map quality and data processing of our cryo-ET analysis, we summarized the following points that can support the confidence of our data:

   (1) Two independent experiments showed the same results of OAD-DC structures, suggesting that the small changes in DC conformations were not due to different map quality or data processing:

   (a) For OAD structures in 1 mM EGTA condition, we analyzed the WT OAD (Figure 4D) and the calaxin-/- OAD rescued with recombinant Calaxin (Figure 4G). These samples were prepared in completely independent processes. However, in both cases, the small densities overlapping the N-terminus region of CCDC151/114 were visualized adjacent to Calaxin (Figure 4D and G, black arrowhead).

   (b) For OAD structures in 1 mM Ca2+ condition, we analyzed the WT OAD (Figure 7B) and the calaxin-/- OAD rescued with recombinant Calaxin (Figure 7C). These samples were prepared in completely independent processes. However, in both cases, the small densities overlapping the N-terminus region of CCDC151/114 were not observed. Instead, the additional densities appeared around DC (Figure 7B and C, white arrowheads).

   (2) We assessed the statistical significance of the changes in DC conformations. We applied Student’s t-test for WT and calaxin-/- OAD-DC structures and created Figure 7—figure supplement 1. p-values of each voxel were calculated as described in Oda & Kikkawa, 2013. The isosurface threshold of p-values corresponds to 0.05% probability in one-tailed test. p-value maps indicate not only Calaxin structures but also the adjacent small density (Figure 7—figure supplement 1A, black arrowhead) and the additional density around DC (Figure 7—figure supplement 1B, white arrowheads) as the statistically significant difference between WT and calaxin-/- OAD-DC.

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2. 

   eLife

   Apr 15, 2023

   eLife assessment

   In vertebrates, ciliary motility is powered by axonemal dyneins, known as OADs, tethered to doublet microtubules by a pentameric docking complex including the Armc4 and Calaxin subunits. This valuable study combines zebrafish genetics with cryo-electron tomography to convincingly show that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction. The work will be of interest to those studying the structure and function of the axoneme, and motile cilia in general.

   Read the original source
3. 

   eLife

   Apr 15, 2023

   Reviewer #1 (Public Review):

   In vertebrates, ciliary motility is important for left-right body patterning, airway clearance, cerebrospinal fluid flow, and the locomotion of spermatozoa. The movement of cilia is powered by the action of dyneins tethered to axonemal doublet microtubules. The largest and most powerful axonemal dynein, OAD, is tethered by a pentameric docking complex (the OAD-DC). Here, Yamaguchi, Morikawa and Kikkawa show convincingly that the Calaxin and Armc4 subunits of the OAD-DC have discrete roles in docking OADs. Using zebrafish mutants, they show that loss of Armc4 causes complete loss of the OAD, whereas mutation of Calaxin causes only partial OAD loss. They demonstrate that Calaxin localization is dependent on Armc4 but independent of the OAD or calcium conditions. Using cryo-ET, they report a higher resolution …

   Reviewer #1 (Public Review):

   In vertebrates, ciliary motility is important for left-right body patterning, airway clearance, cerebrospinal fluid flow, and the locomotion of spermatozoa. The movement of cilia is powered by the action of dyneins tethered to axonemal doublet microtubules. The largest and most powerful axonemal dynein, OAD, is tethered by a pentameric docking complex (the OAD-DC). Here, Yamaguchi, Morikawa and Kikkawa show convincingly that the Calaxin and Armc4 subunits of the OAD-DC have discrete roles in docking OADs. Using zebrafish mutants, they show that loss of Armc4 causes complete loss of the OAD, whereas mutation of Calaxin causes only partial OAD loss. They demonstrate that Calaxin localization is dependent on Armc4 but independent of the OAD or calcium conditions. Using cryo-ET, they report a higher resolution structure of the wild-type zebrafish sperm axoneme than previously determined (Yamaguchi et al., 2018) and show that the OAD and OAD-DC structures resemble the cryo-EM structures of other organisms. Cryo-ET analysis of calaxin-/- axonemes reveals that without Calaxin, OADs have mostly normal conformations but make fewer connections with the OAD-DC and are less stably bound. The paper is well-written with appropriate methods and conclusions.

   Read the original source
4. 

   eLife

   Apr 15, 2023

   Reviewer #2 (Public Review):

   Yamaguchi et al. studied the roles of two proteins, Calaxin and Armc4, in the assembly of the outer arm dynein (OAD) docking complex (DC). By combination of the improved cryo-ET analysis and gene knockout zebrafish lacking each of these proteins, they found that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction in the docking.They further showed an evidence that Calaxin changes the conformation of another compartment of DC comprising CCDC151/114. This new information provides an important basis for understanding how the DC is assembled and regulates docking of OAD. The authors' conclusion is well supported by the data but some data presentation and discussion need to be completed.

   Gui et al. (2021) already reported on a cryo-EM observation in bovine …

   Reviewer #2 (Public Review):

   Yamaguchi et al. studied the roles of two proteins, Calaxin and Armc4, in the assembly of the outer arm dynein (OAD) docking complex (DC). By combination of the improved cryo-ET analysis and gene knockout zebrafish lacking each of these proteins, they found that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction in the docking.They further showed an evidence that Calaxin changes the conformation of another compartment of DC comprising CCDC151/114. This new information provides an important basis for understanding how the DC is assembled and regulates docking of OAD. The authors' conclusion is well supported by the data but some data presentation and discussion need to be completed.

   Gui et al. (2021) already reported on a cryo-EM observation in bovine tracheal cilia, with the conclusion similar to this paper in the structure of OAD/DC on DMT. Using knockout zebrafish strain, the authors present detailed interaction of calaxin with other DC components. They show that the binding of calaxin induces the changes of conformation in N-terminal region of CCDC151/114. The conformation further changes in the presence of Ca2+; specific conformation of N-terminal region of CCDC151/114 becomes undetectable, instead additional structure appears in the vicinity of calaxin.

   1. The authors conclude that the Ca2+-dependent conformational change of DC is subtle and not dynamic. This result is eventually valuable information but may be somewhat unexpected from the point of view that calaxin plays an important role in the regulation of flagellar motility in Ciona sperm. The authors found that calaxin changes the conformation of N-terminal CCDC151/114 region but the core dynein structure shows no dynamic change. What about the changes in the interaction between calaxin, core dynein, and DMT? Is this beyond the resolution of cryo-ET analysis?

   2. It would be very helpful if the authors could add the cryo-ET images of calaxin-/- axoneme in the presence of 1 mM EGTA in Figure 7. Although these images are thought to be similar or identical to Figure 4F, it would help to confirm that the conformational changes in CCDC151/114 and additional part of DC are induced in a Ca2+-dependent manner.

   3. To clarify the molecular interaction of calaxin with other components, it would also be helpful if the authors add the images rotated 80 degree to Figure 4F and G, in similar way in Figure 7,

   4. Despite the molecular phylogenetic difference, there are several similarities between calaxin and Chlamydomonas DC3, not only in the in situ structure and configuration but in the phenotype of mutants; Chlamydomonas mutant lacking DC3 shows OAD loss in the distal part of a flagellum (Casey et al, MBC, 2003). It may be a good reference if the authors add the position of DC3 in Figure 4. A', B', and C.

   5. There is a significant difference in sperm motility between WT and calaxin-/- or WT and armc4-/- (Figure 2E). However, it is not clear whether immotile sperm were included in the data for VAP (Figure 2F) or BCF (Figure 2G). For example, WT and calaxin-/- show similar VAP, although both are significantly different in the percent of motile sperm.

   6. In calaxin-/- mouse, OAD was clearly detected from the base to two-thirds of a flagellum with unclear border (Figure 2A). Typical distribution of OAD+class and OAD-class are shown in Figure 5 in the ~3 micrometer tomograms. Were these taken from around this unclear border? Are proximal most region of a flagellum occupied with OAD+class only? The authors should clearly indicate the region of a flagellum where the tomograms in Figure 5C and D were selected.

   7. Line 229~: It is not clear what the authors meant by "probably reflecting the different distance from the sperm head". In relation to this and the comment 6, does the "proximal" in the sentence "OAD loss occurred even in the proximal part of the flagella" (line 232) indicate the region near the base of a flagellum?

   8. In conjugation with comment 7, it would be appreciated to show an authors' idea on why distal region of flagella tends to lack calaxin, if they do not discuss anywhere in the text,

   9. Immunofluorescence in twister-/- epithelial cilia showed that the localization of calaxin is independent of OAD (line 271-274). Based on the authors' finding, the localization of calaxin requires Armc4, which is preassembled with calaxin in the cytoplasm. If this is true and the localization of calaxin is NOT resulting from diffusion, Armc4 must be localized with calaxin along the entire length of cilia in twister-/- epithelial cilia (Figure 6D). Although Armc4 is shown localized in cryo-ET images (e.g. Figure 1, Figure 7), authors may provide the immunofluorescence of Armc4 along the entire length of sperm flagella and epithelial cilia.

   Read the original source
5. 

   eLife

   Apr 15, 2023

   Reviewer #3 (Public Review):

   ODA-DC anchors ODA, the main force generator of ciliary beating, onto the doublet microtubules. Vertebrate ODA-DC contains 5 proteins, including Calaxin and Armc4, whose mutations are associated with defective ciliary motility in animals and human. By generating calaxin-/- and armc4-/- knockout zebrafish lines, this manuscript examined the Kupffer's vesicle cilia and spermatozoa. They showed that calaxin-/- and armc4-/- knockouts both affect ciliary motility but to different degrees. The authors conducted careful structural analyses using cryo-ET and subtomo averaging on both mutants, revealing a partial loss of ODA in calaxin-/- and a complete loss of ODA in armc4-/-. I really like the distribution analysis of calaxin-/- OADs (Figure 5), which emphasizes the strength of cryo-ET in uncovering the molecule …

   Reviewer #3 (Public Review):

   ODA-DC anchors ODA, the main force generator of ciliary beating, onto the doublet microtubules. Vertebrate ODA-DC contains 5 proteins, including Calaxin and Armc4, whose mutations are associated with defective ciliary motility in animals and human. By generating calaxin-/- and armc4-/- knockout zebrafish lines, this manuscript examined the Kupffer's vesicle cilia and spermatozoa. They showed that calaxin-/- and armc4-/- knockouts both affect ciliary motility but to different degrees. The authors conducted careful structural analyses using cryo-ET and subtomo averaging on both mutants, revealing a partial loss of ODA in calaxin-/- and a complete loss of ODA in armc4-/-. I really like the distribution analysis of calaxin-/- OADs (Figure 5), which emphasizes the strength of cryo-ET in uncovering the molecule distribution of distinct conformational states in situ. Fitting of the atomic models of ODA and ODA-DC into the cryo-ET density maps and Calaxin rescue experiments showed how Calaxin stabilizes ODA at a molecular detail. By using olfactory epithelium, the authors also presented the possible assembly mechanism of ODA-DC proteins, which is also a beautiful experiment. Finally, the authors also investigated how Ca2+ regulate the ODA-DC using cryo-ET.

   The thorough structural and functional analyses of Calaxin and Armc4 in WT and gene KO animals could serve as a reference for future study of the detailed function of other ciliary proteins. The experiments are overall well designed and conducted, but some aspects need to be clarified and improved.

   The authors interpret the vertebrate ODC-DC to include four linkers (line 193). However, the authors also said that loss of one linker (Calaxin) makes ODA to attach on the DMT through two linkers (line 199 and 246). These descriptions are confusing. It would make more sense to interpret the vertebrate ODC-DC as containing three linkers (CCDC151/114, Armc4/TTC25, Calaxin).

   To confirm whether Calaxin directly interacts with β-tubulin (line 213), a control experiment could be needed by incubating WT axoneme with mEGFP-Calaxin followed by IF imaging.

   The Immunoblotting experiment should be improved in Figure 5E. Could the authors get the same results in repeating experiments? Why is the Dnah8 signal higher in 50 mM NaCl of the (+)Calaxin group compared to that in 0 NaCl? This makes me doubt if the difference between (-)Calaxin and (+)Calaxin groups are significant.

   The authors have covered several important points in the Discussion section. Now that the function of Calaxin in both mouse and zebrafish have been reported, the authors could discuss the similarity and difference of Calaxin function in different species and tissues.

   Because of the limited resolution, the authors should be more careful when interpreting the small densities in the difference map, for example, in Figure 4F-G black arrows. Considering that the CCDC151/114 coiled coil is overall poorly resolved both in the WT and mutant cryo-ET maps, the different densities could be due to different map quality or data processing. This makes the following statement suspicious "This structure corresponds to the N-terminus region of CCDC151/114, suggesting that Calaxin affects the conformation of neighboring DC components".

   Read the original source
6. 

   Version published to 10.7554/elife.84860 on eLife

   Apr 14, 2023
7. 

   Version published to 10.1101/2022.11.10.516068v1 on bioRxiv

   Nov 12, 2022