Mechanisms of Nuclear Membrane Rupture and Repair
A general model of nuclear membrane rupture and repair is that it begins when nuclear lamina gaps form and leave areas of unprotected membrane that bleb and rupture when under tension, for instance due to actin-based nucleus compression or increased nuclear volume. After rupture, nuclear lamina proteins accumulate on the exposed chromatin and recruit membrane and membrane remodeling complexes to reduce and seal the large gap in nuclei, but not micronuclei. At this point, only a few proteins acting at each step have been identified and a comprehensive mechanistic picture is lacking.
A major challenge has been the necessity of using live-cell imaging to quantify changes in nuclear stability. To address this challenge, we developed a screening pipeline to quantify changes in rupture and repair frequency in fixed cells. This pipeline combines persistent mislocalization of a size-excluded cytoplasmic protein to the nucleus after a rupture event with CellProfiler (Carpenter et al., Genome Biology, 2016) and R to automatically calculate intracellular fluorescent ratios and quantify population-level rupture frequencies with high precision and sensitivity. Validation of this platform in a small siRNA screen identified over 20 new potential regulators of membrane stability (Gunn et al., bioRxiv, 2023), including many that increase rupture during mechanical stress, and work is ongoing in the lab to follow up on these proteins and expand the screen to comprehensively identify regulators of rupture and repair.
Projects
- What are the molecular mechanisms by which newly identified membrane rupture enhancers and suppressors act?
- What additional pathways regulate rupture and repair?
- Do specific factors regulate micronucleus versus nucleus rupture?
- Are membrane stability proteins misregulated in cancer, and does this correlate with specific disease outcomes?
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