Effects of lamin A / C structure on 53BP1 foci kinetics

Abstract

In the last 15 years, mechanobiology rose to an important field in biology. Taking the surrounding of a cell into consideration when studying responses to internal and external stimuli is a crucial step on the way to advanced cell culture models. In cancer research, cell culture conditions play an important role when investigating the response of cells to ionizing radiation. The so- called cell adhesion mediated radio resistance (CAM-RR) was shown to protect cancer cells from radiotherapy when cultured in 3D hydrogels, simulating the extracellular matrix (ECM). CAM-RR was later attributed to chromatin density changes in cells cultured in 3D hydrogels, compared to cells cultured on flat and rigid 2D surfaces. Chromatin density is controlled by the nuclear structural proteins lamin A/C, which regulates (de-)condensation of chromatin into (eu-)heterochromatin. Lamin A/C is directly linked to the actin cytoskeleton through the LINC complex, which is in turn linked to the ECM by integrins. Therefore lamin A/C represents the nuclear part of a direct mechanical link, spanning through the whole cell, from cell surrounding to chromatin. Other groups showed that lamin A/C acts as a binder for important DNA double strand break (DSB) repair proteins such as 53BP1 and Rad51 and that this binding prevents proteasomal degradation. This interaction leads to reduced DSB repair when Lamin A/C is knocked down, which can be rescued by an overexpression of 53BP1. These findings inspired lamin A/C distribution and its effect on 53BP1 foci kinetics to be the main focus of this thesis. The aim was to create a system to specifically disturb the lamin A/C nucleoskeleton in the nucleoplasm without disturbing the structural integrity of the lamina. The idea was to pull the lamin A/C from the nucleoplasm to the lamina without changing overall lamin A/C levels to show the importance of lamin A/C presence in the nucleoplasm for successful DNA double strand break repair. Therefore a mClover3-lamin A/C reporter cell line was created using CRISPR/Cas9. This cell line was then transfected with a lamin B1-mCherry-GFPNanoTrap fusion protein. The lamin B1 acts as an anchor in the lamina for the GFP NanoTrap to pull the mClover3-labeled lamin A/C from the nucleoplasm to the lamina. The fluorophore mCherry acts as a transfection marker. Transfected cells imported the fusion protein into the nucleus and incorporated it correctly into the lamina. Binding of the mClover3 label of the lamin A/C with the GFP NanoTrap resulted in decreased levels of lamin A/C in the nucleoplasm. With the reduction in nucleoplasmic lamin A/C came a reduction in 53BP1 foci after X-ray treatment, but almost no difference in repair efficiency was detected in the first 6 hours after IR. After 12 hours 53BP1 foci numbers were similar again in transfected and untransfected cells. Also no difference in residual 53BP1 foci numbers after 24 hours was detectable. Experiments were done in mouse embryonic fibroblasts (MEF), cultured in 2D. Next, effects of the lamin B1-mCherry-GFPNanoTrap on MEF cells cultured in a 3D collagen hydrogel were observed. Sadly, the number and quality of cells growing in 3D did not result in a big enough number for statistically significant results. For other experiments with 3D collagen hydrogel cultured cells, hTERT immortalized normal human fibroblasts (82-6) were used to great success. However the attempt to create a lamin A/C reporter cell line with the 82-6 hTERT fibroblasts was not successful. A comparison between 2D and 3D cultured cells was, unfortunately, not possible, but the system to reduce lamin A/C in the nucleoplasm was successfully established and tested.