Myristoylated Neuronal Calcium Sensor-1 captures the preciliary vesicle at distal appendages
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eLife assessment
The identification of NCS1 as a distal appendage protein that captures preciliary vesicles has important implications for understanding the early steps of ciliary assembly. Furthermore, the work has important implications for the broader understanding of NCS1, which prior to this work was focused on roles in neurotransmission, but now must be considered in a broader context. The investigators used a variety of state-of-the-art methodologies, and the conclusions are convincingly supported by the experimental data. This work will be of interest to cell biologists studying ciliary assembly, human geneticists exploring the pathology of cilia as well as neurobiologists studying NCS1.
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Abstract
The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of preciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures preciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the preciliary vesicle recruitment, but not for other steps of cilium formation (Kanie et al., 2025). The lack of a membrane-binding motif in CEP89 suggests that it may indirectly recruit preciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and the centriole-associated vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similar to CEP89 knockouts, preciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the preciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the preciliary vesicles.
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Author response:
Reviewer #2 (Public Review):
Comment 1: In terms of the biological significance of this interaction, it would be good to examine (via co-immunoprecipitation) whether the CEP89/NCS-1/C3ORF14 interaction takes place upon serum starvation. Does the complex change?
NCS1 centriolar localization requires CEP89 as no NCS1 localization was observed in CEP89 knockout cells (Figure 2L; Figure 2-figure supplement 2B). Both CEP89 and NCS1 centriolar localization were observed (Figure 2C; Figure 1D of the PMID: 36711481) in cells grown in serum containing media, although their localization was further enhanced in serum starved cells. From these results, we predict that CEP89 and NCS1 can interact and colocalize in both serum-fed and serum-depleted condition. We think it may not be easy to assess the change in interaction with …
Author response:
Reviewer #2 (Public Review):
Comment 1: In terms of the biological significance of this interaction, it would be good to examine (via co-immunoprecipitation) whether the CEP89/NCS-1/C3ORF14 interaction takes place upon serum starvation. Does the complex change?
NCS1 centriolar localization requires CEP89 as no NCS1 localization was observed in CEP89 knockout cells (Figure 2L; Figure 2-figure supplement 2B). Both CEP89 and NCS1 centriolar localization were observed (Figure 2C; Figure 1D of the PMID: 36711481) in cells grown in serum containing media, although their localization was further enhanced in serum starved cells. From these results, we predict that CEP89 and NCS1 can interact and colocalize in both serum-fed and serum-depleted condition. We think it may not be easy to assess the change in interaction with the co-immunoprecipitation assay, as interactions occur in a test tube, which may not reflect the binding condition inside the cells.
Comment 2: Also, for the subdistal appendage localization of NCS-1 and C3ORF14, would this also change upon serum starvation?
We agree that it would be interesting to see whether the subdistal appendage localization changes upon serum starvation, as NCS1 may capture the ciliary vesicle at the subdistal appendages as we discussed. However, the loss of the subdistal appendage protein, CEP128, blocks subdistal appendage localization of CEP89 [PMID: 32242819] without affecting cilium formation [PMID: 27818179]. This suggests that the subdistal appendage localization of NCS1 or C3ORF14 is likely dispensable for cilium formation.
Comment 3: For the ciliation results and the recruitment of IFT88 in CEP89 knockout cell lines, this contradicts previous work from Tanos et al (PMID: 23348840), as well as Hou et al (PMID: 36669498). A parallel comparison using siRNA, a transient knockout system, or a degron system would help understand this. A similar point goes for Figure 4, where the effect on ciliogenesis is minimal in knockout cells, but acute siRNA has been shown to have a stronger phenotype.
Hou et al. [PMID: 36669498] investigated the role of distal appendage proteins, CEP164, CEP89, and FBF1 in the ciliated chordotonal organ of Drosophila melanogaster by generating knockout Drosophila strains. The results were markedly different from what was observed in mammalian cells. Notably, CEP164 is not required for cilium formation, and CEP89 is required for FBF1 localization in the animal. CEP89 was required for cilium formation in the cells in the ciliated chordotonal organ, of which cilium formation is dependent on IFT machinery. They did not show if IFT centriolar recruitment is affected in the CEP89 mutant cells. These differences likely reflect the divergence of the organization of distal appendage during evolution.
The ciliation phenotype of our CEP89 knockout cells are milder than what was shown in Tanos et al [PMID: 23348840], but largely consistent with the results from Bornens group, which used siRNA to deplete CEP89 [PMID: 23789104]. Besides, NCS1 knockout cells showed very similar phenotype to the CEP89 knockout cells, and relatively acute deletion of NCS1 (14 days after infection of the lenti-virus containing sgNCS1 without single-cell cloning) displayed an almost identical ciliation defect (Figure 4B-C). Thus, we believe CEP89 is only partially required for cilium formation in RPE-hTERT cells and that the differences are more technical than definitive.
Comment 4: An elegant phenotype rescue is shown in Figure 5. An interesting question would be, how does this mutant and/or the myristoylation affect the recruitment of C3ORF14?
NCS1 is not required for the localization of C3ORF14 (Figure 2M; Figure 2- figure supplement 2C), so we can assume that the myristoylation defective mutant does not affect C3ORF14 recruitment.
Comment 5: For the EF-hand mutants, it would be good to use control mutants, from known Ca2+ binding proteins as a control for the experiment shown.
In the Figure 5-figure supplement 1A-C, we generated a series of EF-hand mutant of NCS1 to see if the calcium binding affects the CEP89 interaction, NCS1 localization, and cilium formation. NCS1 is only protein among the calcium binding NCS family proteins that was found as a positive hit in the mass spec data of CEP89 tandem affinity purification. Therefore, we cannot use other NCS1 family proteins as a control for CEP89 binding, NCS1 localization, and cilium formation.
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eLife assessment
The identification of NCS1 as a distal appendage protein that captures preciliary vesicles has important implications for understanding the early steps of ciliary assembly. Furthermore, the work has important implications for the broader understanding of NCS1, which prior to this work was focused on roles in neurotransmission, but now must be considered in a broader context. The investigators used a variety of state-of-the-art methodologies, and the conclusions are convincingly supported by the experimental data. This work will be of interest to cell biologists studying ciliary assembly, human geneticists exploring the pathology of cilia as well as neurobiologists studying NCS1.
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Reviewer #1 (Public Review):
In this work, Kanie and colleagues explored the role of NCS1 in capturing the ciliary vesicle. The microscopy was well executed and appropriately quantified. The authors convincingly show that while NCS1 is important for capturing the ciliary vesicle, another unknown distal appendage component is partially redundant in that ciliary vesicle capture and ciliary assembly are not fully dependent on NCS1. Overall, I am convinced by the data, and my only concern is that the discussion of the mouse phenotypes does not do a good job of putting this gene into the greater context of the complexity of mouse mutations.
Interestingly NCS1 has been previously studied in the context of neurotransmission and the new findings raise questions about whether prior findings are actually due to neuronal cilia defects.
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Reviewer #2 (Public Review):
Kanie et al have recently characterized DAP protein CEP89 as important for the recruitment of the ciliary vesicle. Here, they describe a novel interacting partner for CEP89 that can bind membranes and therefore mediates its role in ciliary vesicle recruitment. An initial LAP tag pull-down and mass spectrometry experiment finds NCS-1 and C3ORF14 as CEP89 interactors. This interaction is mapped in the context of the ciliary vesicle formation. From the data presented, it is clear that, upon knockout, the function of these proteins might be compensated by others, as the phenotype can eventually recover over time.
In terms of the biological significance of this interaction, it would be good to examine (via co-immunoprecipitation) whether the CEP89/NCS-1/C3ORF14 interaction takes place upon serum starvation. Does …
Reviewer #2 (Public Review):
Kanie et al have recently characterized DAP protein CEP89 as important for the recruitment of the ciliary vesicle. Here, they describe a novel interacting partner for CEP89 that can bind membranes and therefore mediates its role in ciliary vesicle recruitment. An initial LAP tag pull-down and mass spectrometry experiment finds NCS-1 and C3ORF14 as CEP89 interactors. This interaction is mapped in the context of the ciliary vesicle formation. From the data presented, it is clear that, upon knockout, the function of these proteins might be compensated by others, as the phenotype can eventually recover over time.
In terms of the biological significance of this interaction, it would be good to examine (via co-immunoprecipitation) whether the CEP89/NCS-1/C3ORF14 interaction takes place upon serum starvation. Does the complex change?
Also, for the subdistal appendage localization of NCS-1 and C3ORF14, would this also change upon serum starvation?
For the ciliation results and the recruitment of IFT88 in CEP89 knockout cell lines, this contradicts previous work from Tanos et al (PMID: 23348840), as well as Hou et al (PMID: 36669498). A parallel comparison using siRNA, a transient knockout system, or a degron system would help understand this. A similar point goes for Figure 4, where the effect on ciliogenesis is minimal in knockout cells, but acute siRNA has been shown to have a stronger phenotype.
An elegant phenotype rescue is shown in Figure 5. An interesting question would be, how does this mutant and/or the myristoylation affect the recruitment of C3ORF14?
For the EF-hand mutants, it would be good to use control mutants, from known Ca2+ binding proteins as a control for the experiment shown.
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Reviewer #3 (Public Review):
This work addresses an important question aimed at understanding how membrane docking to the distal appendages is regulated during ciliogenesis. In this study, Tomoharu and colleagues identified interactions between CEP89 (important for RAB34-positive membrane localization to the mother centriole) and NCS1 and C3ORF14. Both these CEP89 interacting proteins were characterized as distal appendage localized proteins between CEP89 and RAB34 based on super-resolution microscopy. Ciliogenesis investigations using knockout cells indicated that NCS1 and CEP89 have similar impaired ciliation due to disruption in vesicle recruitment/RAB34 to the mother centriole, while C3ORF14 had less effect on ciliogenesis. The authors refer to the ciliogenesis requirement for CEP89/NCS1 as ciliary vesicles, which has been …
Reviewer #3 (Public Review):
This work addresses an important question aimed at understanding how membrane docking to the distal appendages is regulated during ciliogenesis. In this study, Tomoharu and colleagues identified interactions between CEP89 (important for RAB34-positive membrane localization to the mother centriole) and NCS1 and C3ORF14. Both these CEP89 interacting proteins were characterized as distal appendage localized proteins between CEP89 and RAB34 based on super-resolution microscopy. Ciliogenesis investigations using knockout cells indicated that NCS1 and CEP89 have similar impaired ciliation due to disruption in vesicle recruitment/RAB34 to the mother centriole, while C3ORF14 had less effect on ciliogenesis. The authors refer to the ciliogenesis requirement for CEP89/NCS1 as ciliary vesicles, which has been previously referred to as preciliary vesicle or distal appendage vesicles. NCS1 distal appendage localization was dependent on CEP89 and TTBK2, but it is not clear how TTBK2 affects NCS1. The authors subsequently performed double knockouts with NCS1 and other distal appendage proteins and showed stronger effects on mother centriole RAB34 levels, suggesting efficient membrane docking during ciliogenesis requires several distal appendage proteins. This is consistent with NCS1 knockout mice which do not display typical ciliopathy phenotypes. These mice do display obesity, which is associated with cilia dysfunction, and show reduced ciliary protein levels. As noted by the authors, the in vivo results for NCS1 knockouts could be affected by the mouse background which was not evaluated. The authors demonstrate the NCS1 myristoylation motif is required for RAB34 localization to the mother centrioles, providing a mechanistic explanation for how distal appendage proteins could interact with membranes during ciliogenesis. Overall the authors' findings support an important role for NCS1 in regulating ciliogenesis via myristoylation-dependent interaction with RAB34-positive membranes docked at the mother centriole.
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