An intact nuclear envelope (NE) prevents the mixing of cytoplasmic and nuclear contents and protects the genome from damage. Work has shown that NE ruptures during interphase (NERDI) cause transient loss of nuclear compartmentalisation and failure to efficiently repair these ruptures contributes to genome instability and pro-inflammatory responses. Thus, uncovering the mechanisms that underpin rupture repair is a fundamental question in cell biology, and a better understanding of this process has broad implications for human diseases. NERDIs can occur due to cytoskeletal compressive forces at sites of weakened lamina. Exposure of inner nuclear membrane proteins triggers the recruitment of the endosomal sorting complex required for transport (ESCRT) machinery, a membrane remodelling pathway highly conserved in eukaryotes which mediates NE sealing. A key unanswered question is how local compressive forces are counteracted to allow efficient membrane resealing. Here, we identify the ESCRT-associated and poorly understood protein BROX as a crucial factor required to maintain NE integrity. BROX depletion leads to abnormal nuclear morphology, increased frequency of NERDI events that take longer to recover and higher levels of DNA damage. Critically, we show that BROX binds Nesprin-2-giant, a component of the linker of nucleoskeleton and cytoskeleton complex (LINC). This interaction promotes Nesprin-2-giant ubiquitination and local removal from compression sites, facilitating the relaxation of mechanical stress imposed by compressive actin fibres at the rupture site. The increase in NERDI events could also be rescued by the partial depletion of Nesprin-2-giant and demonstrated that BROX specifically counteracts the compressive forces applied by nesprin-2-giant during NERDI repair. Thus, BROX rebalances excessive cytoskeletal forces in cells experiencing NE instability to promote effective NERDI repair. We then demonstrate that the interaction between BROX and the ESCRT-III protein CHMP5 is important for the function of both BROX and CHMP5 in NE repair. The results here demonstrate that BROX coordinates mechanosensing with membrane remodelling to ensure the maintenance of nuclear-cytoplasmic compartmentalisation and genomic stability.
The role of the ESCRT-associated protein BROX at the nuclear envelope
Wallis, S. (Author). 1 Sept 2021
Student thesis: Doctoral Thesis › Doctor of Philosophy