Nuclear envelope rupture in laminopathy model cells
10 October 2018
UAntwerp, Stadscampus, Promotiezaal Grauwzusters - Lange Sint Annastraat 7 - 2000 Antwerpen (route: UAntwerpen, Stadscampus
4:00 PM - 6:00 PM
Winnok De Vos
PhD defence Joke Robijns - Department of Biomedical Sciences
A diverse group of diseases, called laminopathies, all range from mutations in the LMNA gene coding for A-type lamin proteins. These proteins are major constituents of the nuclear lamina which provides support to the nuclear envelope and is involved in gene regulations and organization. Until now, the exact principles of disease development and progression remain unknown. The observation of nuclear envelope ruptures (NER) in laminopathy patient cells has opened a new entry point for the laminopathy research field. NERs can lead to exchange of cytoplasmic and nuclear factors and although cells manage to repair, DNA damage can accumulate. Detailed investigation of the elicitors of NER can provide new therapeutic targets for the treatment of laminopathies. As patient cells are scarce, good cell models have to be constructed with isogenic background and limited side-effects to allow proper investigation of NERs. With the use of CRISPR/Cas9 genome editing technology, knockout model cells were created.
They recapitulated important features observed in patient cells including aberrant nuclear shape. The value of these knockout models cells in context of NERs was shown by subjecting them to automated live cell imaging and analysis. The analysis showed higher vulnerability of cells lacking A-type lamins, illustrated by a higher NER frequency. Also, the involvement of actomyosin contractility in NER induction was shown. More detailed investigation of NERs is hampered by their transient nature. This problem can be circumvented by targeted induction of NER. Atomic force microscopy and population wide cell confinement were used for NER induction. In combination with the previously constructed model cells, the effect of LMNA knockout and prelamin A accumulation on NER frequency and repair kinetics was elucidated. Finally, by assessment of changes in protein quantity and activity, interesting pathways related to A-type lamin loss and prelamin A accumulation were shown. In conclusion, the obtained insight and methods developed in this work contribute to the growing knowledge of NER.