Cep164 triggers ciliogenesis by recruiting Tau tubulin kinase 2 to the mother centriole

Abstract

Primary cilia play critical roles in development and disease. Their assembly is triggered by mature centrioles (basal bodies) and requires centrosomal protein 164kDa (Cep164), a component of distal appendages. Here we show that loss of Cep164 leads to early defects in ciliogenesis, reminiscent of the phenotypic consequences of mutations in TTBK2 (Tau tubulin kinase 2). We identify Cep164 as a likely physiological substrate of TTBK2 and demonstrate that Cep164 and TTBK2 form a complex. We map the interaction domains and demonstrate that complex formation is crucial for the recruitment of TTBK2 to basal bodies. Remarkably, ciliogenesis can be restored in Cep164-depleted cells by expression of chimeric proteins in which TTBK2 is fused to the C-terminal centrioletargeting domain of Cep164. These findings indicate that one of the major functions of Cep164 in ciliogenesis is to recruit active TTBK2 to centrioles. Once positioned, TTBK2 then triggers key events required for ciliogenesis, including removal of CP110 and recruitment of intraflagellar transport proteins. In addition, our data suggest that TTBK2 also acts upstream of Cep164, contributing to the assembly of distal appendages.primary cilium | centrosome

CEP164-null cells generated by genome editing show a ciliation defect with intact DNA repair capacity

“…3E). Thus, our disruption of the CEP164 locus confirms the findings made on the roles of CEP164 in primary ciliogenesis with siRNA experiments (Č ajanek and Nigg, 2014;Graser et al, 2007;Schmidt et al, 2012). We next tested whether CEP164 deficiency impacted on the ability of cells to withstand UV-induced DNA damage.…”

“…Previous data have indicated a role for CEP164 in primary ciliogenesis (Č ajanek and Nigg, 2014;Graser et al, 2007;Schmidt et al, 2012;Chaki et al, 2012). Despite the feasibility of inducing ciliogenesis in DT40 cells (Prosser and Morrison, 2015), we prefer to examine the roles of CEP164 in a cell line with high levels of primary ciliation.…”

Eupatilin rescues ciliary transition zone defects to ameliorate ciliopathy-related phenotypes

“…TTBK2 recruitment to the centrosomal area and CP110 cap removal from the mother centriole are known to occur at the beginning of ciliogenesis (11,17). Both TTBK2 recruitment and CP110 removal appeared normal in CEP290 null cells (Supplemental Figure 5).…”

“…The rescue effect of the flavonoids was compared by a dose-response analysis, and eupatilin was selected for further analyses ( Figure 1D). To determine whether eupatilin could exert a general rescue effect on the loss of ciliopathy proteins, we treated CEP290 WT RPE1 cells with eupatilin after siRNA-mediated knock down of several ciliopathy genes (4,11,12). Serum starvation-induced ciliogenesis was not rescued by eupatilin in cells depleted of the selected ciliopathy genes, whereas eupatilin facilitated ciliogenesis in cells transfected with negative control siRNA ( Figure 1E).…”

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry

“…Nek2B, encoded by a splice variant of nek2, has been shown to be required for normal γ-tubulin recruitment to centrioles in Xenopus zygotic extracts (Fry et al, 2000); however, this has not been followed up with in vivo work until now. A number of components of the distal and sub-distal appendages of the mother centriole, which branch off the γ-tubulin core (and in many cases, bind directly to γ-tubulin), are essential for cilium biogenesis (Brito et al, 2012;Cajanek and Nigg, 2014;Ishikawa et al, 2005). Thus, it follows that a loss of γ-tubulin from the mother centriole would preclude cilium biogenesis.…”