Investigating the molecular mechanisms of nuclear envelope stability and repair

Mechanical instability of the nucleus due to mutation or dysregulation of NE proteins is linked to cancer, muscular dystrophies and both physiological and accelerated ageing. At the nuclear envelope (NE), interactions between the nucleus and the cytoskeleton, sustained by LINC complexes, allow force transduction from the environment to the nucleus and cellular adaptation. However, it is not clear how NE instability in disease affects the structure and activity of LINC complexes, or their ability to adapt to increased extracellular forces. In Aim 1, I will investigate the effect of severe disease-associated NE instability in LINC organisation, force-sensing and in situ interactions and structure. I will use a multidisciplinary approach, from super-resolution microscopy (STORM) to in situ structural biology (cryo-ET) under normal and increased extracellular forces. At regions of extreme instability, NE ruptures occur. Repair occurs through the recruitment ESCRT-III membrane remodelling complexes. In Aim 2, I will investigate how ESCRT-III oligomerisation occurs at the NE and how these complexes might adapt different geometries to fit membrane topologies and close ruptures. I will develop new tools and use a cryo-confocal and cryo-ET pipeline to solve the in situ ultrastructure of ESCRT-III complexes at NE ruptures, during different stages of repair.