BAF facilitates interphase nuclear membrane repair through recruitment of nuclear transmembrane proteins

Young, Alexandra M.; Gunn, Amanda; Hatch, Emily M.

doi:10.1091/mbc.e20-01-0009

Cited by 79 publications

(83 citation statements)

References 35 publications

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“…BROX depletion significantly delayed mCherry-NLS nuclear re-accumulation after NERDI as compared with control, while stable expression of siRNA-resistant BROX (GFP-L-BROX r ) restored repair times to control levels ( Figures 1 A–1C and S1 A; Video S1 ). Importantly, GFP-L-BROX r transiently accumulated at rupture sites, as denoted by DNA herniations and co-localization with mCherry-BAF foci ( Denais et al., 2016 ; Halfmann et al., 2019 ; Young et al., 2020 ), supporting a direct role of BROX in NE repair ( Figures 1 D–1F; Video S2 ). Mutation of BROX farnesylation site (GFP-L-BROX r C408S) ( Ichioka et al., 2008 ) disrupted BROX association with the NE ( Figures S1 B and S1C), abolished its recruitment to rupture sites and repair function ( Figures 1 F–1H, S1 D, and S1E; Video S2 ).…”

Section: Resultsmentioning

confidence: 67%

“…NE ruptures expose the chromatin-associated protein barrier to autointegration factor (BAF) and inner nuclear membrane proteins such as LEMD2, which recruits endosomal sorting complex required for transport (ESCRT) machinery to remodel the damaged membranes ( Halfmann et al., 2019 ; Penfield et al., 2018 ; Thaller et al., 2019 ; Young et al., 2020 ). NERDI repair is completed by local polymerization of ESCRT-III filaments and subsequent constriction by the AAA-ATPase VPS4 seals the NE gap ( Denais et al., 2016 ; Raab et al., 2016 ).…”

Section: Introductionmentioning

confidence: 99%

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The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair

et al. 2021

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Summary Transient nuclear envelope ruptures during interphase (NERDI) occur due to cytoskeletal compressive forces at sites of weakened lamina, and delayed NERDI repair results in genomic instability. Nuclear envelope (NE) sealing is completed by endosomal sorting complex required for transport (ESCRT) machinery. A key unanswered question is how local compressive forces are counteracted to allow efficient membrane resealing. Here, we identify the ESCRT-associated protein BROX as a crucial factor required to accelerate repair of the NE. Critically, BROX binds Nesprin-2G, a component of the linker of nucleoskeleton and cytoskeleton complex (LINC). This interaction promotes Nesprin-2G ubiquitination and facilitates the relaxation of mechanical stress imposed by compressive actin fibers at the rupture site. Thus, BROX rebalances excessive cytoskeletal forces in cells experiencing NE instability to promote effective NERDI repair. Our results demonstrate that BROX coordinates mechanoregulation with membrane remodeling to ensure the maintenance of nuclear-cytoplasmic compartmentalization and genomic stability.

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Section: Resultsmentioning

confidence: 67%

Section: Introductionmentioning

confidence: 99%

The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair

et al. 2021

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“…Resealing of the NE typically occurs within minutes but can occasionally last for hours. The repair process is mediated by multiple proteins including barrier-to-autointegration factor (BAF), LEM domain proteins and ESCRT-III components [6,7,[83][84][85] and is thought to involve the recruitment of ER sheets to the sites of rupture to reseal the membrane.…”

Section: Nuclear Envelope Blebs and Rupturesmentioning

confidence: 99%

Current Methods and Pipelines for Image-Based Quantitation of Nuclear Shape and Nuclear Envelope Abnormalities

Janssen

Breusegem

Larrieu

2022

Cells

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Any given cell type has an associated “normal” nuclear morphology, which is important to maintain proper cellular functioning and safeguard genomic integrity. Deviations from this can be indicative of diseases such as cancer or premature aging syndrome. To accurately assess nuclear abnormalities, it is important to use quantitative measures of nuclear morphology. Here, we give an overview of several nuclear abnormalities, including micronuclei, nuclear envelope invaginations, blebs and ruptures, and review the current methods used for image-based quantification of these abnormalities. We discuss several parameters that can be used to quantify nuclear shape and compare their outputs using example images. In addition, we present new pipelines for quantitative analysis of nuclear blebs and invaginations. Quantitative analyses of nuclear aberrations and shape will be important in a wide range of applications, from assessments of cancer cell anomalies to studies of nucleus deformability under mechanical or other types of stress.

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“… Alternatives: Other fluorescently tagged proteins can be overexpressed and used to sort micronuclei. For example, GFP-BAF ( Halfmann et al., 2019 ; Liu et al., 2018 ; Young et al., 2020 ), which localizes to sites of nuclear envelope rupture, can – in principle – also be used to mark and sort ruptured micronuclei. Generate a cell line overexpressing the fluorescent protein Histone H2B-mCherry to mark chromatin.…”

Section: Before You Beginmentioning

confidence: 99%

Purification of micronuclei from cultured cells by flow cytometry

Toufektchan

Maciejowski

2021

STAR Protocols

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Summary Micronuclei are aberrant nuclear compartments that form when chromosomes or chromosome fragments fail to incorporate into a primary nucleus during mitotic exit. Ruptures at the micronuclear envelope are associated with DNA damage and activation of immune sensing pathways. To gain insights into these processes, we have developed a method to purify ruptured micronuclei. This method paves the way toward understanding the consequences of micronuclear envelope rupture. For complete details on the use and execution of this protocol, please refer to Mohr et al. (2021) .

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BAF facilitates interphase nuclear membrane repair through recruitment of nuclear transmembrane proteins

Cited by 79 publications

References 35 publications

The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair

The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair

Current Methods and Pipelines for Image-Based Quantitation of Nuclear Shape and Nuclear Envelope Abnormalities

Purification of micronuclei from cultured cells by flow cytometry

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