A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling

Abstract

Summary CEP104 is an evolutionarily conserved centrosomal and ciliary tip protein. CEP104 loss-of-function mutations are reported in patients with Joubert syndrome, but their function in the etiology of ciliopathies is poorly understood. Here, we show that cep104 silencing in zebrafish causes cilia-related manifestations: shortened cilia in Kupffer’s vesicle, heart laterality, and cranial nerve development defects. We show that another Joubert syndrome-associated cilia tip protein, CSPP1, interacts with CEP104 at microtubules for the regulation of axoneme length. We demonstrate in human telomerase reverse transcriptase-immortalized retinal pigmented epithelium (hTERT-RPE1) cells that ciliary translocation of Smoothened in response to Hedgehog pathway stimulation is both CEP104 and CSPP1 dependent. However, CEP104 is not required for the ciliary recruitment of CSPP1, indicating that an intra-ciliary CEP104-CSPP1 complex controls axoneme length and Hedgehog signaling competence. Our in vivo and in vitro analyses of CEP104 define its interaction with CSPP1 as a requirement for the formation of Hedgehog signaling-competent cilia, defects that underlie Joubert syndrome.

Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome

“…Recent work has reported Hedgehog (Hh) signaling defects in cell lines with dysfunction of the JBTS genes CEP104, CSPP1, and Armc9 (33,39). Therefore, we tested whether TOGARAM1 dysfunction leads to attenuated ciliary SMO accumulation in response to Hh stimulation in the engineered TOGARAM1-mutant hTERT-RPE1 cells described above (Supplemental Figure 5).…”

“…To identify direct binding partners, we employed full-length ARMC9 and four fragments ( Figure 1A) as bait in a GAL4-based yeast two-hybrid (Y2H) interaction trap screen of two validated prey retinal cDNA libraries that were generated via random or oligo-dT primers (37). Using full-length ARMC9 as a bait, we identified four proteins previously implicated in ciliary function as binary interactors, including ARMC9 itself (suggesting a propensity to multimerize), TOGARAM1 (29,34), CCDC66 (36,38), and the JBTS-associated protein CSPP1 (39). Validation of these interactions and evaluation of the interacting domains was performed by Y2H co-expression.…”

“…CEP104 localizes both to the daughter centriole as well as to the apical tip of a growing cilium (30) and, similar to TOGARAM1, interacts with tubulin through its TOG domain (61). Moreover, CEP104 has been shown to interact with NEK1 (61), which is associated with the ciliopathy "short-rib polydactyly syndrome Majewski type" (62), and with the JBTS-associated protein CSPP1 (39). This interaction, that we confirmed in our co-IP experiments, is required for the formation of Hedgehog signaling-competent cilia, as mutations in CSPP1 and CEP104 significantly decreased ciliary SMO translocation (39), similar to what we observed with RPE1 TOGARAM1 mutant lines.…”

“…Moreover, CEP104 has been shown to interact with NEK1 (61), which is associated with the ciliopathy "short-rib polydactyly syndrome Majewski type" (62), and with the JBTS-associated protein CSPP1 (39). This interaction, that we confirmed in our co-IP experiments, is required for the formation of Hedgehog signaling-competent cilia, as mutations in CSPP1 and CEP104 significantly decreased ciliary SMO translocation (39), similar to what we observed with RPE1 TOGARAM1 mutant lines. Following the "guilt by association" paradigm, we next found biallelic TOGARAM1 variants in multiple individuals with JBTS, reiterating the relevance of the 20 complex to JBTS, and moving us closer to identifying all genetic causes of this disorder.…”

Genetic tests aid in counseling of fetuses with cerebellar vermis defects

“…CSPP1 interacts with centrosomes and microtubules and plays a role in cell‐cycle progression and spindle organization 28 . In vivo and in vitro analyses showed that CSPP1 directly interacts with CEP104 , which is required for the formation of Hedgehog signaling‐competent cilia, defects in which underlie Joubert syndrome 29 . Mutations in this gene were identified in patients with Joubert syndrome 21, which is an AR disorder characterized by CVH, elongated superior cerebellar peduncles, and deepened interpeduncular fossa, which together are recognized as the “molar tooth sign” on brain MRI 30 .…”