Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

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 ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation. The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary 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 a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing proper localization to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.

A hierarchical pathway for assembly of the distal appendages that organize primary cilia

“…This localization pattern is similar to what was observed for ARL13B (see Figure 4A of (Yang et al, 2018)). Localization of the novel distal appendage proteins, NCS1 and C3ORF14, are described in an accompanying paper (Tomoharu Kanie et al, 2023), but the predicted location and their diameter are shown here for convenience (Figure 1B; Figure 1C).…”

“…NCS1 then captures ciliary vesicles via its myristoylation motif. This story will be described in an accompanying paper (Tomoharu Kanie et al, 2023). Importantly, CEP89 or NCS1 depletion only partially inhibits ciliary vesicle recruitment, suggesting a compensation mechanism for the recruitment.…”

“…In this study, we sought to comprehensively characterize previously known and newly identified distal appendage proteins (KIZ, NCS1, and C3ORF14) to deepen our understanding of the distal appendages. Among the three proteins that we identified, the latter two will be described in detail in an accompanying paper (Tomoharu Kanie et al, 2023).…”

“…¯, weakly reduced; ¯¯, moderately decreased; ¯¯¯, greatly decreased or absent; , weakly increased; ®, unaffected. The detailed relationship between CEP89-NCS1-C3ORF14 as well as localization of each distal appendage protein in NCS1 knockout cells are available in an accompanying paper (Tomoharu Kanie, Ng, Abbott, Pongs, & Jackson, 2023). N. The updated hierarchy of the distal appendage proteins.…”

“…Here, we identify three more distal appendage proteins (KIZ, NCS1, and C3ORF14). The latter two will be described in an accompanying paper (Tomoharu Kanie et al, 2023). With this new set of distal appendage proteins, we sought to provide a comprehensive view of the structure, including precise localization, order of recruitment, and functional role of each distal appendage protein.…”