A new microscopy pipeline for studying the initial stages of nuclear and micronuclear rupture and repair Journal Article
| Authors: | Di Bona, M.; Bakhoum, S. F. |
| Article Title: | A new microscopy pipeline for studying the initial stages of nuclear and micronuclear rupture and repair |
| Abstract: | 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 toward 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. In this study, 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 and showed how BAF is differentially recruited at sites of primary and micronuclear rupture. Additionally, we applied this method to study the recruitment at primary nuclei of the inner nuclear membrane protein LEM-domain 2 (LEMD2) and Charged Multivesicular Protein 7 (CHMP7), the scaffolding protein of the endosomal sorting complex required for transport III (ESCRT-III) membrane remodeling complex. 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 protein dynamics at sites of nuclear and micronuclear envelope rupture and paves the way to more time-resolved studies of nuclear envelope repair. Copyright © 2024 Di Bona and Bakhoum. |
| Keywords: | protein expression; unclassified drug; human cell; microscopy; cell nucleus membrane; endoplasmic reticulum; lentivirus vector; dna binding; cytosol; scaffold protein; escrt protein; micronucleus; cell membrane protein; cytometry; three-dimensional imaging; human; article; barrier to autointegration factor; endoplasmic reticulum membrane; dwell time; high-resolution microscopy; micronuclear repair impairment; micronuclear rupture; nuclear envelope repair; nuclear envelope rupture; real-time protein recruitment analysis; spatiotemporal protein recruitment analysis; charged multivesicular protein 7; lem domain 2; membrane rupture |
| Journal Title: | Frontiers in Cell and Developmental Biology |
| Volume: | 12 |
| ISSN: | 2296-634X |
| Publisher: | Frontiers Media S.A. |
| Date Published: | 2024-09-18 |
| Start Page: | 1475095 |
| Language: | English |
| DOI: | 10.3389/fcell.2024.1475095 |
| PROVIDER: | scopus |
| PMCID: | PMC11445188 |
| PUBMED: | 39359718 |
| DOI/URL: | |
| Notes: | Article -- MSK corresponding author is Melody Di Bona -- Source: Scopus |
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