A high-content siRNA screen reveals new regulators of nuclear membrane stability.

Nuclear membrane rupture is a physiological response to in vivo situations, such as cell migration. When disregulated, it can have deleterious consequences for the cell including increased genome instability and inflammatory signaling. However, many of the molecular mechanisms underlying this process are unclear and only a few regulators have been uncovered. In this study, we developed a nuclear rupture reporter that is size-excluded from re-compartmentalization following nuclear rupture events. This allows for robust detection of factors influencing nuclear integrity in fixed cells. Additionally, we created and validated an automated pipeline for the analysis of fixed cell images that we applied in a high-content siRNA screen. Our screen identified conditions that led to both increased and decreased nuclear rupture frequency. Analysis of the hits demonstrated an enrichment in factors in the nuclear membrane and ER, and demonstrated that nuclear size is not strongly correlated with rupture frequency. Based on these results, we validated a new function for the protein phosphatase CTDNEP1 in maintaining nucleus stability. These findings provide new insights into the regulation of nuclear stability and define a novel method of rupture analysis that removes a substantial barrier to understanding the molecular mechanisms underlying this process.