Understanding the multifunctional domains of controller protein Centrin2
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Centrin2 is a centrosomal calcium-binding protein that plays a critical role in centriole dynamics and ciliogenesis. Structurally, it has four calcium-binding EF- hand motifs. Most of the cellular centrin2 is not associated with centrosomes, suggesting that it has other roles in the cell. Centrin2 is required for efficient DNA damage recognition in nucleotide excision repair (NER). To explore the functions of each of the EF-hands in NER and ciliogenesis, we used site-directed mutagenesis to disrupt the calcium-binding sites and analyzed the effects in a CETN2 null hTERT-RPE1 cell line that was previously generated in our lab. We generated stable rescue cell lines with wild-type centrin2 and the EF-hand domain mutants in CETN2 nulls. None of the mutants could rescue the ciliation defect. Ciliation capacity declined from 70% in cells that expressed wild-type centrin2 to 5% in cells with all 4 EF-hands mutated, suggesting that calcium-binding domains are essential for cilium formation. We also observed that centrosomal localization of centrin2 was almost completely lost in mutants with the C-terminal EF-hand mutations and when all the calcium-binding EF-hand motifs were disrupted. These results show that calcium-binding properties of centrin2 are important for its distribution within the cell. We observed a difference in ciliation frequency determined using Arl13B and acetylated tubulin, ciliary membrane and axonemal markers, respectively. This suggests a potential role for centrin2 in axonemal tubulin stabilization. We observed that the loss of centrin2 or centrin2 EF- hand domains reduced ciliary length, while overexpression of centrin2 in hTERT- RPE1 cells increased ciliary length, suggesting that centrin2 and its EF-hand domains play a role in ciliary length. Using STORM in centrin2-deficent cells and centrin2 EF-hand domain mutant cells, we observed a defect in TTBK2 localization but not in CEP164, suggest its defects in ciliogenesis. Moreover, the interaction of centrin2 with its partners SFI1 and POC5 is depended on its EF-hand domains. Centrin2 EF-hand domains were able to rescue the UV sensitivity of centrin2 null cells upon UV-induced damage. Our data show that centrin2 is functionally dependent on its calcium-binding domains for its roles at the centrosome but not for its NER activity, suggesting these functions are separable and that centrin2 plays distinct roles in these processes.
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