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

Abstract

Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, C3ORF14) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assay revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.

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

“…Similar to the centriolar localization of CEP89, which was significantly reduced in CEP83 or SCLT1 knockouts, NCS1 localization was also greatly diminished in these knockouts (Figure 2-figure supplement 3D). This is consistent with the observation that CEP83-SCLT1 module serves as a structural component of the distal appendage (see (Tomoharu Kanie et al, 2023) for the detail). In contrast, the feedback complex CEP164-TTBK2 (see (Tomoharu Kanie et al, 2023) for the detail) was required for proper centriolar localization of NCS1 (Figure 2- figure supplement 3D) but not for CEP89 (Figure 2F of (Tomoharu Kanie et al, 2023)).…”

“…Like C3ORF14, NCS1 formed a slightly smaller ring than CEP164 (Figure 2I). Consistent with this, the ring diameter of NCS1 and C3ORF14 was 319.5±7.7 nm (n=13, average±SEM) and 348.8±8.0 nm (n=16, average±SEM), respectively (see Figure 1C of (Tomoharu Kanie et al, 2023)). It is notable that both C3ORF14 and NCS1 also localized to the region close to subdistal appendage in some but not all centrioles (Figure 2D and F), consistent with what was observed for CEP89 localization (Chong et al, 2020).…”

“…The raw data and detailed statistics are available in Figure 3E-Source data. This experiment was synchronized with the experiment shown in the Figure 4C of (Tomoharu Kanie et al, 2023), hence the values for sgGFP are exactly the same as the ones shown in Tomoharu Kanie et al, 2023).…”

“…We then sought to understand how NCS1 is involved in cilium formation. In an accompanying paper (Tomoharu Kanie et al, 2023), we showed that CEP89 participates in cilium formation by regulating ciliary vesicle recruitment without affecting IFT88::CEP19 recruitment, important steps that require distal appendage proteins (Schmidt et al, 2012) (Dateyama et al, 2019)(see Figure 5 of (Tomoharu Kanie et al, 2023)). We tested if NCS1 has similar roles to its binding partner, CEP89.…”

“…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 thef ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner.…”