Melody Di Bona ^* Samuel F. Bakhoum

• Memorial Sloan Kettering Cancer Center, New York, United States

Nuclear envelope repair is a fundamental cellular response to stress, especially for cells experiencing frequent nuclear ruptures, such as cancer cells. Moreover, for chromosomally unstable cancer cells, characterized by the presence of micronuclei, the irreversible rupture of these structures constitutes a fundamental step towards cancer progression and therapy resistance. For these reasons, the study of nuclear envelope rupture and repair is of paramount importance. Nonetheless, due to the constraint imposed by the stochastic nature of rupture events, a precise characterization of the initial stage of nuclear repair remains elusive. Here, we overcame this limitation by developing a new imaging pipeline that deterministically induces rupture while simultaneously imaging fluorescently-tagged repair proteins. We provide a detailed step-by-step protocol to implement this method on any confocal microscope, and applied it to study the major nuclear repair protein, Barrier-to-Autointegration Factor (BAF). As a proof of principle, we demonstrated two different downstream analysis methods, through which we showed how BAF is differentially recruited at sites of primary and micronuclear rupture. Additionally, we applied this method to study the recruitment of the inner nuclear membrane protein LEM-domain 2 (LEMD2) and the scaffolding protein of the Endosomal Sorting Complex Required for Transport III (ESCRT-III) membrane remodeling complex, Charged Multivesicular Protein 7 (CHMP7) at primary nuclei. The CHMP7-LEMD2 binding is the fundamental step allowing the recruitment of ESCRT-III, which represents the other major nuclear repair mechanism. This demonstrates the method's applicability for investigating proteins dynamic at sites of nuclear and micronuclear envelope rupture and paves the way to more time-resolved studies of nuclear envelope repair.