| Popis: |
The centrosome is composed of numerous proteins that make up its core structure. Other proteins also transiently localise and interact with centrosomal proteins for functional modulation. Centrobin, one of the core centrosomal proteins, is involved in centriole biogenesis and elongation, ciliogenesis, and possibly, the DNA damage response. Although it was initially identified as a BRCA2-interacting protein that preferentially localises to the daughter centriole, its role in the DNA repair pathway or cilia formation has not been clearly defined. To investigate the functions of centrobin in centriole duplication, ciliogenesis, and DNA repair processes, we generated a novel monoclonal anti-centrobin antibody and used CRISPR/Cas9 to disrupt CNTROB gene in the immortalized, non-transformed hTERT-RPE1 cell line. We confirmed successful gene targeting by western blot, immunofluorescence microscopy and sequencing of the genomic locus and cDNA. We noticed that loss of centrobin had no impact on cell cycle progression but resulted in an increase in acentric and monocentric cells. We also used clonogenic survival assays to show that centrobin-deficient cells had a severe decrease in viability after exposure to ionising radiation (IR) and methyl methanosulfonate (MMS) but not ultraviolet radiation (UV). Interestingly, these sensitivities to genotoxic stress agents were rescued by stable transgenic expression of centrobin in our cell line. To further investigate the involvement of centrobin in the DNA damage response, we studied the kinetics of RAD51 recombinase and γ−H2AX IR-induced foci (IRIF) formation after IR or MMS treatment of centrobin-deficient cells. We observed that the percentage of cells with RAD51 and γ−H2AX IRIF was reduced in centrobin-deficient cells after exposure to IR or MMS. We also studied the roles of centrobin in primary cilia assembly and functions in vertebrate systems. Primary cilia are antenna-like microtubule-based sensory organelles for signal transduction and cell proliferation. Using immunofluorescence microscopy to study the localisation pattern of endogenous and Green Fluorescent Protein (GFP)-tagged centrobin, we noticed that, while centrobin was mainly retained on the daughter centrioles of proliferating cells, it localised to the mother centrioles of ciliated cells. We observed reduced ciliation frequency in CNTROB knockout cells, which was rescued by transient and stable transgenic centrobin. We also observed inappropriate accumulation of the negative regulators of ciliogenesis, CP110 and CEP97, in centrobin-deficient cells after serum deprivation. Further investigation revealed that centrobin and CP110 interact in vivo and that the microtubule binding- and CP110-interacting regions of centrobin fragments are required for ciliogenesis. Electron microscopy analysis of human centrobin-deficient cells showed that centrobin is crucial for axonemal extension from the ciliary bud. Finally, we used antisense-morpholino oligonucleotides (MO) technology to deplete centrobin in zebrafish and study its ciliary roles in this vertebrate system. Our investigation revealed that centrobin depletion causes reduced cilium number and length of nodal cilia in the Kupffer’s vesicle of zebrafish embryos. Furthermore, centrobin-depleted zebrafish embryos displayed microcephaly-like morphological abnormalities that are indicative of ciliary defects, thus identifying centrobin as a potential ciliopathy gene. To assess the roles of centrobin in organogenesis, we monitored left-right patterning and organ localisation in zebrafish embryos. We observed an increase in frequency of defective organ positioning which is a consequent of malfunctioning left-right patterning in the centrobin depleted embryos. Interestingly, these phenotypes were rescued by transgenic expression of MO-resistant transgenic centrobin. In summary, our data identify centrobin as a positive regulator of ciliogenesis and a participant in the DNA damage response in vertebrates. Therefore, centrobin may be a molecular link between centriole duplication, cilia formation and DNA damage responses. 2020-02-28 |
