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

Nuclear membrane rupture is a physiological response to multiple in vivo processes, such as cell migration, that can cause extensive genome instability and upregulate invasive and inflammatory pathways. However, the underlying molecular mechanisms of rupture are unclear and few regulators have been identified. In this study, we developed a 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. We combined this with an automated image analysis pipeline in a high-content siRNA screen to identify new proteins that both increase and decrease nuclear rupture frequency in cancer cells. Pathway analysis identified an enrichment of nuclear membrane and ER factors in our hits and we demonstrate that one of these, the protein phosphatase CTDNEP1, is required for nuclear stability. Analysis of known rupture determinants, including an automated quantitative analysis of nuclear lamina gaps, are consistent with CTDNEP1 acting independently of actin and nuclear lamina organization. Our findings provide new insights into the molecular mechanism of nuclear rupture and define a highly adaptable program for rupture analysis that removes a substantial barrier to new discoveries in the field.