Fig. 2

Mutations found in ICK affect ciliogenesis and ciliary localization. a, b The cellular localization of ICK was analyzed in HEK293T cells overexpressing wild-type or two mutated forms of mRFP-ICK: p.R272Q (positive control, mutation previously studied in ECO infants) and p.G120C (new mutation detected in the current study). Nuclei were stained with DAPI (blue) and mRFP-ICK proteins are shown in red. mRFP-ICK with the p.G120C mutation showed the same nuclear localization as wild-type mRFP-ICK transfected cells, while nuclear import was completely lost when mRFP-ICK with the p.R272Q mutation was expressed (Fisher’s exact test two-tailed p < 0.0001, >35 cells counted per condition). c, d The ciliary localization of ICK was studied in mIMCD3 cells transiently transfected with wild-type or two mutant forms of mRFP-ICK: p.R272Q or p.G120C. Wild-type mRFP-ICK mostly localized to the ciliary axoneme and was often enriched at the ciliary base, while both mRFP-ICK mutants enriched at ciliary tips. Ciliary axonemes were visualized with anti-ARL13B (green), ciliary transition zones that are present at the ciliary base were marked with anti-RPGRIP1L (pink), mRFP-ICK constructs were shown in red, and nuclei were stained with DAPI (blue). Per construct >50 transfected, ciliated cells were counted. e, f Cilium presence was studied in serum-starved fibroblasts derived from an ECO patient with a homozygous missense mutation in ICK (c.815G > A; p.R272Q) and compared to fibroblasts derived from two healthy unrelated controls. Control I represents a non-Amish individual, while control II is from the Amish community. Ciliogenesis was significantly reduced in fibroblasts from the ECO patient compared to the controls (Fisher’s exact test two-tailed p < 0.0001 for both). At least 80 cells were counted per condition. Ciliary axonemes were visualized with anti-ARL13B (green), ciliary transition zones were marked with anti-RPGRIP1L (red), and nuclei were stained with DAPI (blue)