The C-terminus of CFAP410 forms a tetrameric helical bundle that is essential for its localization to the basal body

  1. Alexander Stadler
  2. Laryssa De Liz
  3. Heloisa B. Gabriel
  4. Santiago Alonso-Gil
  5. Robbie Crickley
  6. Katharina Korbula
  7. Bojan Žagrović
  8. Sue Vaughan
  9. Jack D. Sunter
  10. Gang Dong

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Abstract

Cilia are antenna-like organelles protruding from the surface of many cell types in the human body. Defects in ciliary structure or function often lead to diseases that are collectively called ciliopathies. Cilia and flagella associated protein 410 (CFAP410) localizes at the basal body of cilia/flagella and plays essential roles in ciliogenesis, neuronal development, and DNA damage repair. It remains unknown how its specific basal body location is achieved. Multiple single amino acid mutations in CFAP410 have been identified in patients with various ciliopathies. One of the mutations, L224P, is located in the C-terminal domain (CTD) of human CFAP410 and causes severe s pondylo m etaphyseal d ysplasia, ax ial (SMDAX). However, the molecular mechanism for how the mutation causes the disorder remains unclear. Here, we report our structural studies on the CTD of CFAP410 from three distantly related organisms, Homo sapiens, Trypanosoma brucei , and Chlamydomonas reinhardtii. The crystal structures reveal that the three proteins all adopt the same conformation as a tetrameric helical bundle. Our work further demonstrates that the tetrameric assembly of the CTD is essential for the correct localization of CFAP410 in T. brucei , as the L224P mutation that disassembles the tetramer disrupts its basal body localization. Taken together, our studies reveal that the basal body localization of CFAP410 is controlled by the CTD and provide a mechanistic explanation for how the mutation L224P in CFAP410 causes ciliopathies in humans.

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    Reply to the reviewers

    We would like to thank both reviewers for their thorough and constructive evaluation and comments on our manuscript. Following their suggestions, we have edited our manuscript to address all criticisms and comments from them. We hope that, with these introduced changes, this manuscript will be suitable for publication in an appropriate journal. Detailed point-to-point responses are shown below.

    __Point-to-point response to reviewers’ critiques: __

    Reviewer 1

    Summary:* Stadler and colleagues characterized CFAP410, using molecular structural biology, biophysics, biochemistry, cellular imaging and genetic engineering, to address molecular mechanism of ciliopathies caused by defect of this protein. They crystalized the C-terminal domain of CFAP410 and its homologue, from Trypanosoma, human and Chlamydomonas. All of them take tetrameric complex formation with four bundle helices in the center. Then they mutated highly conserved residues, L219 and L224, which are located at the helix bundle, and characterized its biophysical properties, demonstrating oligomerization defect and, in case of L224 mutation, collapse of secondary structure as well. This was supported by molecular dynamics. Next they examined the effect of these mutation in vivo using Trypanosoma brucei. They visualized the localization of CFAP410 at the postrior cell tip and its transfer to the basal body. By mutation, transfer of CFAP410 is prohibited and cytokinesis defect occurs. The experiments are logically designed and the results are clearly and convincingly stated. Undoubtedly this work deserves publication after minor revision of the manuscript.*

    Strength:* This is a compact paper, clearly stating the biological aim and experiments designed for that.

    Limitation: While it is undoubtedly proved that the C-terminal domain of CFAP410 forms tetramer and L219 and L224 are key residues and that mutation of these residues causes severe defect at the localization of CFAP410, it is an overstatement to conclude the tetramer formation is essential for the localization. The authors experiments cannot exclude the possibility of another consequence of mutation (different from origomerization), which is the cause of abnormal localization.

    : Indeed, the reason for the disrupted localization of the disease-causing mutation L224P to the basal body could be caused by mis-folding of TbCFAP410 if that is the only mutation we had tested. However, as shown in our results, the point mutation A267E of TbCFAP410 that breaks the tetramer into two dimers (so as Hs/Cr-CFAP410-A219E) did not change its folding or structural stability. Nevertheless, TbCFAP410-A267E lost its ability to localize to the basal body effectively (Figure 6A). It suggests that even a folded dimer is insufficient to correctly localize to the basal body. Therefore, we can confidently conclude that the fully assembled tetramer of CFAP410 is required for its localization to the basal body.

    Minor points: p.7: It would be interesting, if the authors, beside MD, attempt to predict conformation of the mutants using Alphafold2. *

    : We tried to predict conformational changes of the point mutations of CFAP410 via AlphaFold2, but did not observe significant changes in the generated models. It was not so surprising to us though, because, as shown previously, although experimentally point mutations induced complete unfolding of some proteins, the AlphaFold2 models of the same point mutants folded similarly to the wild-type crystal structures (Buel & Walters, 2022, Nat. Struct. Mol. Biol. 29:1-2.)

    p.7-8: A cartoon to describe what proteins exist between the posterior tip and the basal body will help readers to understand. Do the authors have any thought how CFAP410 is transported to the basal body? *

    : We thank the reviewer for the suggestion and have revised Figure 7 to illustrate the two distinct localization sites of CFAP410 in T. brucei.

    We do not know the mechanism through which CFAP410 localizes to the posterior cell tip and the basal body. Both structures are microtubule based, with the basal body consisting of a barrel of microtubule triplets and the posterior cell tip is the site at which the ends of the microtubules which form the sub-pellicular array in T. brucei are located. This suggests that CFAP410 could interact with microtubules or microtubule binding proteins in these locations. Additionally, in human cells CFAP410 appears to interact with NEK1 and the equivalent interaction in trypanosomes may be important for CFAP410 localization. We have refined our ideas on this in the Discussion section (p.11): “It was shown previously that the L224P mutant of CFAP410 abolishes its interaction with NEK1 [4]. Given that the mutant L224P disassembles the tetramer of CFAP410-CTD (Figure 3), the tetrameric assembly of CFAP410 seems to play an essential role in its interaction with NEK1. Therefore, the disrupted location of TbCFAP410-L272P to the basal body we observed here could be attributed to its abolished interaction with the trypanosome equivalent of NEK1 as occurs in human cells. However, we cannot exclude another possibility that CFAP410 localizes to the basal body by interacting with an unidentified anchoring target there and NEK1 is subsequently recruited via its binding to CFAP410.”

    It is worth mentioning though that the localization of TbCFAP410 to the posterior cell tip has only been reported in T. brucei and no other cellular localization sites have been reported for CFAP410 orthologs in other organisms including human. Moreover, in the genome-wide protein tagging project TrypTag many other proteins were found to localize to both the posterior tip and another site in the cell, including the basal body (Billington et al, 2023, Nat. Microbiol. 8: 533-547). The following paragraph discussing about this has been added to the Discussion section (p. 10): “Notably, recent genome-wide protein localizations revealed that the posterior cell tip in T. brucei has unexpectedly high complexity and contains many proteins that also localize to other organelles ____[16]____. The tip may thus serve as a “moonlighting” site for those proteins. However, the extra localization site of CFAP410 at the cell tip has only been reported in T. brucei and no other cellular localization sites have been observed for CFAP410 in any other organisms.”

    p.10: Can the authors define "NTD linker" precisely (from which to which residues)? *

    : We have defined the sequence range of both the NTD (aa1-160) and NTD-linker (aa1-254) of TbCFAP410 both in the text (p. 9 & p. 10) and legend of Figure 6.

    p.12: Alphafold-multimer may help to have information, which part of CFAP410 is likely interface to NEK1 and SPATA7. *

    : We attempted to predict how CFAP410 interacts with NEK1 or SPATA7 by Alphafold-multimer. Results of the former prediction, which are consistent with previous studies (Gregorczyk et al, 2023, Life Sci. Alliance), have been added as a new figure (Figure S4). However, no convincing results were obtained for the latter pair.

    p.16: The paragraph starting with "DSF measurements were ..." is probably not necessary. Figure 4 caption: "HsCFP0-CTD" should be defined precisely. Or is it a typo of HsCFAP410-CTD? *

    : We thank the reviewer for pointing out this mistake. This paragraph has been removed.

    Figure 5ad, Figure 6: blue is not defined.

    : We have now defined blue in the figure legends for 5A, D and 6A. These cells have been counterstained with the DNA stain Hoechst 33342 to highlight the nucleus and kinetoplast (mitochondrial DNA) and this is the blue element in the images.

    Advances: This interdisciplenary work nicely characterized CFAP410 at atomic, molecular and cellular levels and acquired insight of its functional mechanism. *

    : We appreciate the reviewer’s constructive comments and positive feedback.

    Reviewer 2

    *In this paper, the authors described 3 crystal structures of the CTD of CFAP410 from 3 different species. They explored the phenotype mutation L224P in humans which causes ciliopathies using in vitro and in vivo analysis. They were able to explain the oligomerization role of the L224P mutations and its importance for correct localization. In addition, using their structure, they also found A219 as an important residue for tetramerization as well. The paper is well-written and easier to read. *

    __There are some minor concerns: __

    1. Fig. 5C: Why would the line reduce to lower value at 24hr, 48hr for both non-induced and induced one. *

    : During routine culture of T. brucei, we had to split the cells to ensure they do not overgrow and are maintained in log phase growth. The reduction in value at each time point represents the cell splitting event and gives these characteristic “sawtooth” graphs for cell growth. At each time point the cell density is measured and then the cells (both non-induced and induced) are diluted to the same cell density, in this case 2x106 cells/ml, and grown for a further 24 hours before the next measurement.

    *2. The authors wrote "Although we observed only little change in the average distance from the posterior cell tip to kinetoplast in 1K1N cytoskeletons after induction, there was a substantial increase in the range of these measurements, with cytoskeletons observed having a more reduced or increased distance from the kinetoplast to the posterior cell tip (Figure 5F)." *

    *Why not back the wide range with a standard deviation calculation in the figure caption of 5F or display the std dev directly in the figure if it looks good? Also, the author can include legends for color dots in the figure as Replicate 1,2,3 for easy reading/comprehension. *

    : We have included the standard deviations for each of the replicates in 5F in the figure legend. The spread of data is shown in the figure already with the individual points and the overlay of the standard deviation was not clear when we tried it. We have now included a legend for the dots in the figure as suggested.

    *3.The information about the construct of Trypanosoma used for Figure 6 is not described at all. What exactly is the region of the construct of the NTD and NTD-linker? *

    : The following sentence has been added to the legend of Figure 6: “Except for NTD (aa1-168) and NTD-linker (aa1-254), all other constructs are full-length proteins.”

    *4. The author wrote "In the mutant A267E, the mNG::CFAP410 signal was exclusively found at the posterior of most cytoskeletons (63.5%), while full-length TbCFAP410-L272P and the two CTD-lacking constructs, NTD and NTD-linker ... This suggests that both the presence of the CTD and the integrity of its oligomerization are essential for the interaction of TbCFAP410 with the basal body and posterior cell tip." *

    *This statement needs to be revised a bit. First, seems like the tetramerization is important for the localization, not dimerization. Second, is there any evidence that full-length TbCFAP410-L272P folds properly? Without the evidence that the NTD and NTD-linker region can fold properly in both full-length TbCFAP410-L272P and CTD truncation, it is not possible to exclude that the N-terminal is essential for the localization as well. *

    : Thanks for the comments. We have managed to express and purify TbCFAP410-NTD, which shows that it folds properly on its own. We further checked whether NTD directly interacts with CTD, but ended up with negative results. The following part has been added to the last paragraph of the Results section to address the reviewer’s question. “We found that TbCFAP410-NTD folded properly on its own when expressed in bacteria, and no direct interaction between NTD and CTD was detected (data now shown). It suggests that the two structural modules of TbCFAP410 that are connected by a long disordered linker are folded independently. Therefore, we conclude that the localization of TbCFAP410 to the basal body and the posterior cell tip requires the CTD and its ability to oligomerize.”

    Small things:

    1. It is worth define 1K1N, 2K1N and 2kK2N stages in the text for reader not in Trypanosoma field.

    *: We have explained the KN nomenclature, and how during the cell cycle the kinetoplast and nucleus are duplicated and segregated in a defined order.

    *2.Fig. 5C: label Non-induced black & induced orange as legends directly in the figure so readers don't have to read the Figure captions.

    *: This has been done as suggested.

    *3. In Methods, there is the part about DSF measurement in the Molecular Dynamic (MD) simulations *

    : This paragraph is unnecessary and has been removed. We thank the reviewer for pointing it out.

    4.* Abbreviation not defined: DSF (in Methods/MD simulations). Also, the molecular dynamics phrase appears well before the abbreviation MD in the Methods section. *

    : As stated above, this paragraph has been removed.

    Reviewer #2 (Significance (Required)):

    Overall I found the methodology and results of the paper solid, and the interpretation and conclusion are sound. *

    *The paper addresses the molecular mechanism of a mutation in CFAP410 resulting in severe spondylometaphyseal dysplasia, axial. *

    The audience of the paper should be the cilia field but also the paper is also good for other researchers as the paper is easy to read.*

    : We appreciate the reviewer’s constructive feedback and positive evaluation.

  2. Review Commons

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

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper, the authors described 3 crystal structures of the CTD of CFAP410 from 3 different species. They explored the phenotype mutation L224P in humans which causes ciliopathies using in vitro and in vivo analysis. They were able to explain the oligomerization role of the L224P mutations and its importance for correct localization. In addition, using their structure, they also found A219 as an important residue for tetramerization as well. The paper is well-written and easier to read.

    There are some minor concerns:

    1. Fig. 5C: Why would the line reduce to lower value at 24hr, 48hr for both non-induced and induced one.
    2. The authors wrote "Although we observed only little change in the average distance from the posterior cell tip to kinetoplast in 1K1N cytoskeletons after induction, there was a substantial increase in the range of these measurements, with cytoskeletons observed having a more reduced or increased distance from the kinetoplast to the posterior cell tip (Figure 5F)."

    Why not back the wide range with a standard deviation calculation in the figure caption of 5F or display the std dev directly in the figure if it looks good? Also, the author can include legends for color dots in the figure as Replicate 1,2,3 for easy reading/comprehension.

    1. The information about the construct of Trypanosoma used for Figure 6 is not described at all. What exactly is the region of the construct of the NTD and NTD-linker?
    2. The author wrote "In the mutant A267E, the mNG::CFAP410 signal was exclusively found at the posterior of most cytoskeletons (63.5%), while full-length TbCFAP410-L272P and the two CTD-lacking constructs, NTD and NTD-linker ... This suggests that both the presence of the CTD and the integrity of its oligomerization are essential for the interaction of TbCFAP410 with the basal body and posterior cell tip."

    This statement needs to be revised a bit. First, seems like the tetramerization is important for the localization, not dimerization. Second, is there any evidence that full-length TbCFAP410-L272P folds properly? Without the evidence that the NTD and NTD-linker region can fold properly in both full-length TbCFAP410-L272P and CTD truncation, it is not possible to exclude that the N-terminal is essential for the localization as well.

    Small things:

    1. It is worth define 1K1N, 2K1N and 2kK2N stages in the text for reader not in Trypanosoma field.
    2. Fig. 5C: label Non-induced black & induced orange as legends directly in the figure so readers don't have to read the Figure captions.
    3. In Methods, there is the part about DSF measurement in the Molecular Dynamic (MD) simulations
    4. Abbreviation not defined: DSF (in Methods/MD simulations). Also, the molecular dynamics phrase appears well before the abbreviation MD in the Methods section.

    Significance

    Overall I found the methodology and results of the paper solid, and the interpretation and conclusion are sound.

    The paper addresses the molecular mechanism of a mutation in CFAP410 resulting in severe spondylometaphyseal dysplasia, axial.

    The audience of the paper should be the cilia field but also the paper is also good for other researchers as the paper is easy to read.

  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 #1

    Evidence, reproducibility and clarity

    Summary: Stadler and colleagues characterized CFAP410, using molecular structural biology, biophysics, biochemistry, cellular imaging and genetic engineering, to address molecular mechanism of ciliopathies caused by defect of this protein. They crystalized the C-terminal domain of CFAP410 and its homologue, from Trypanosoma, human and Chlamydomonas. All of them take tetrameric complex formation with four bundle helices in the center. Then they mutated highly conserved residues, L219 and L224, which are located at the helix bundle, and characterized its biophysical properties, demonstrating oligomerization defect and, in case of L224 mutation, collapse of secondary structure as well. This was supported by molecular dynamics. Next they examined the effect of these mutation in vivo using Trypanosoma brucei. They visualized the localization of CFAP410 at the postrior cell tip and its transfer to the basal body. By mutation, transfer of CFAP410 is prohibited and cytokinesis defect occurs.

    The experiments are logically designed and the results are clearly and convincingly stated. Undoubtedly this work deserves publication after minor revision of the manuscript.

    Significance

    strength: This is a compact paper, clearly stating the biological aim and experiments designed for that.

    Limitation: While it is undoubtedly proved that the C-terminal domain of CFAP410 forms tetramer and L219 and L224 are key residues and that mutation of these residues causes severe defect at the localization of CFAP410, it is an overstatement to conclude the tetramer formation is essential for the localization. The authors experiments cannot exclude the possibility of another consequence of mutation (different from origomerization), which is the cause of abnormal localization.

    Minor points:

    p.7: It would be interesting, if the authors, beside MD, attempt to predict conformation of the mutants using Alphafold2.

    p.7-8: A cartoon to describe what proteins exist between the posterior tip and the basal body will help readers to understand. Do the authors have any thought how CFAP410 is transported to the basal body?

    p.10: Can the authors define "NTD linker" precisely (from which to which residues)?

    p.12: Alphafold-multimer may help to have information, which part of CFAP410 is likely interface to NEK1 and SPATA7.

    p.16: The paragraph starting with "DSF measurements were ..." is probably not necessary.

    Figure 4 caption: "HsCFP0-CTD" should be defined precisely. Or is it a typo of HsCFAP410-CTD?

    Figure 5ad, Figure 6: blue is not defined.

    advances: This interdisciplenary work nicely characterized CFAP410 at atomic, molecular and cellular levels and acquired insight of its functional mechanism.

    audience: cilia community

  4. Version published to 10.1101/2023.11.30.569397v1 on bioRxiv