The roles of centrins/caltractins at the centrosome and in genome maintenance
Dantas, Tiago Jorge O.G. de Almeida
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The principal microtubule-organising centre in animal cells, the centrosome, contains centrin, a small, highly-conserved calcium-binding protein that can be found throughout eukaryotes. Several centrin isoforms exist and have been implicated in various cellular processes including nuclear export and DNA repair. Although centrins play a major role in ensuring the assembly and appropriate functioning of the ciliary/flagellar basal bodies in protists and lower eukaryotes, their involvement in vertebrate centrosome duplication or centrosomal functions are less clear. Here we use the hyper-recombinogenic chicken DT40 cell line to dissect the roles of the various centrin isoforms. We found that all three centrin isoforms present in chicken, encoded by Cetn4, Cetn2 and Cetn3, are expressed in DT40 cells. We have successfully targeted all three Cetn loci, generating single, double and triple mutants of the centrin genes. RT-PCR, immunoblot and immunofluorescence microscopy have confirmed the absence of the centrins. Unexpectedly, centrin-deficient cells undergo normal cellular division with no significant differences in cell cycle progression. Light and electron microscopy analyses revealed no detectable defects in centrosome composition or ultrastructure in these cells. Additionally, microtubule re-nucleation analysis showed that the centrosomes retain function and cells are able to proceed through mitosis without any delay. Although centrin-deficient DT40 cells had normal survival rates following ionizing radiation (IR), they were highly sensitive to ultraviolet (UV) irradiation, with Cetn3 deficiency exacerbating the sensitivity of Cetn4/Cetn2 double mutants. DNA damage checkpoints were intact, but repair of UV-induced DNA damage was delayed in centrin nulls. To further investigate centrins' activity in nucleotide excision repair, we performed mutagenic analysis of centrin2. Surprisingly, we found that neither key Mps1, CK2 and Aurora A phosphorylation sites nor the calcium binding ability of centrin2 are required for survival following UV treatment. However, we show that the mutation of all four EF hands impacts centrin2 localisation to the centrosome and dramatically decreases the formation of defined, linear centrosomal structures induced upon cPOC5 overexpression. Finally, we show that centrin's activity in NER is mainly downstream of XPC recruitment to UV-induced DNA lesions. Overall, our results demonstrate that centrins are dispensable for centrosome biogenesis/duplication in DT40 cells but have a major role in the repair of UV induced DNA lesions through nucleotide excision repair.
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