Microtubules (MTs) are tubulin polymers that play vital roles in cells, such as chromosome segregation or transport of cargo. The imbalance in MT dynamics might lead to neurodegenerative diseases or cancers, which can also be a result of excessive DNA damage. Hence, Lottersberger, et al., 2015 and Lawrimore, et al., 2017 started studying that connection. Both groups showed that dynamic MTs are required for DNA damage repair. The presented work in this thesis aims to reveal the link between posttranslational modifications (PTMs) and dynamics of MTs and DNA damage response signalling using cellular assays. Initially, non-synchronised and G1 synchronised RPE-1 cells cultured without serum for 48 hours as well as G1 synchronised RPE-1 and then re-stimulated with 10 % serum were treated with DNA damaging agents producing double strand breaks: neocarzinostatin (NCS) (radiomimetic,acts via oxidative stress), phleomycin (acts via oxidative stress) or etoposide (integrates with a DNA strand and topoisomerase II preventing DNA strands re-ligation) to establish the most effective DNA damaging drug that allowed to observe any alterations in the PTMs of α-tubulin overtime. Immunoblotting and immunofluorescence results demonstrated that the NCS acts most rapidly and efficiently among tested DNA damaging drugs. Thus, time course experiments were performed, determining whether DNA damage can affect PTMs and dynamics of MTs. The immunoblotting and immunofluorescence analyses indicated a significant decrease in PTMs of acetylated, tyrosinated and polyglutamylated α-tubulin and simultaneously MT motion assay demonstrated an increase in MT dynamics in response to DNA damage induced by NCS at a 30 minute time point, suggesting that DNA damage affects MTs. Therefore, the next set of experiments was performed to establish whether perturbation of MTs could affect DNA damage response (DDR) signalling. Hence, the MTs were hyperstabilised or destabilised using taxol (time course 1 hour to 7 hours) or nocodazole (time course 1 hour to 7 hours), respectively, followed by NCS-induced DNA damage. After hyperstabilisation of MTs and DNA damage by neocarzinostatin, immunoblotting of G1 synchronised cells that were stimulated with 10 % serum shows that the level of DNA damage marker, KAP1 phosphorylated on serine 824 decreased compared to control. Interestingly, the level of another DNA damage marker- γH2AX was not altered. These preliminary results might imply that MTs affect DNA damage in a non-γH2AX-dependent manner. Nevertheless, these results should be further validated in order to unequivocally claim the correlation between PTMs and dynamics of MTs and DDR signalling.
|Date of Award||19 Mar 2019|
|Supervisor||Rafael E Carazo Salas (Supervisor) & Abderrahmane Kaidi (Supervisor)|