Ciliopathy-associated mutations of IFT122 impair ciliary protein trafficking but not ciliogenesis

Abstract

The intraflagellar transport (IFT) machinery containing the IFT-A and IFT-B complexes mediates ciliary protein trafficking. Mutations in the genes encoding the six subunits of the IFT-A complex (IFT43, IFT121, IFT122, IFT139, IFT140, and IFT144) are known to cause skeletal ciliopathies, including cranioectodermal dysplasia (CED). As the IFT122 subunit connects the core and peripheral subcomplexes of the IFT-A complex, it is expected to play a pivotal role in the complex. Indeed, we here showed that knockout (KO) of the IFT122 gene in hTERT-RPE1 cells using the CRISPR/Cas9 system led to a severe ciliogenesis defect, whereas KO of other IFT-A genes had minor effects on ciliogenesis but impaired ciliary protein trafficking. Exogenous expression of not only wild-type IFT122 but also its CED-associated missense mutants, which fail to interact with other IFT-A subunits, rescued the ciliogenesis defect of IFT122-KO cells. However, IFT122-KO cells expressing CED-type IFT122 mutants showed defects in ciliary protein trafficking, such as ciliary entry of Smoothened in response to Hedgehog signaling activation. The trafficking defects partially resembled those observed in IFT144-KO cells, which demonstrate failed assembly of the functional IFT-A complex at the base of cilia. These observations make it likely that, although IFT122 is essential for ciliogenesis, CED-type missense mutations underlie a skeletal ciliopathy phenotype by perturbing ciliary protein trafficking with minor effects on ciliogenesis per se.

Robust interaction of IFT70 with IFT52–IFT88 in the IFT-B complex is required for ciliogenesis

“…1 E–G) was different between the control and IFT22 -KO cells. As described previously ( Hirano et al, 2017 ), IFT88 was localized mainly around the base of cilia and faintly along cilia, and IFT140 was mainly found around the ciliary base; note that, as described previously ( Hirano et al, 2017 ; Takahara et al, 2018 ), the commercially available polyclonal antibody against IFT140 also stained undetermined structures in the nucleus of RPE1 cells; see the manufacturer's website ( ).…”

“…The strategy of CRISPR/Cas9-mediated gene disruption of hTERT-RPE1 cells using homology-independent DNA repair was previously reported in detail ( Katoh et al, 2017 ); also see ( Funabashi et al, 2017 ; Hirano et al, 2017 ; Nishijima et al, 2017 ; Nozaki et al, 2018 , 2017 ; Takahara et al, 2018 ). Single guide RNA (sgRNA) sequences targeting the IFT22 gene or those targeting both the IFT70A and IFT70B genes (see ) were designed using CRISPR design ( Hsu et al, 2013 ).…”

Interaction of WDR60 intermediate chain with TCTEX1D2 light chain of the dynein-2 complex is crucial for ciliary protein trafficking

“…In considerable contrast, in WDR60 -KO cells, IFT88 was observed throughout cilia and often found around the distal tips ( Figure 4B ; also see Figure 4I ); this phenotype is reminiscent of that of IFT139 -KO and IFT121 -KO cells ( Hirano et al. , 2017 ; Takahara et al. , 2018 ); both IFT139 and IFT121 are peripheral subunits of the IFT-A complex and are encoded by the causative genes of SRTD ( SRTD4 and SRTD7 , respectively).…”

Ciliary Genes in Renal Cystic Diseases

“…Functional analysis showed that ift122 knockdown in zebrafish embryos leads to reduced number of basal bodies and cilia in the pronephric duct and shorter primary cilia of Kupffer vesicles. In human HEK293T cells IFT122 knockout leads to cilia loss [129][130][131][132], while Wdr35 knockout in mouse fibroblasts reveals strongly reduced level of IFT43 [133]. Human and mouse fibroblasts lacking WDR35 fail to produce cilia [134].…”