Mechanisms of A-Type Lamin Targeting to Nuclear Ruptures Are Disrupted in LMNA- and BANF1-Associated Progerias

Abstract

Mutations in the genes LMNA and BANF1 can lead to accelerated aging syndromes called progeria. The protein products of these genes, A-type lamins and BAF, respectively, are nuclear envelope (NE) proteins that interact and participate in various cellular processes, including nuclear envelope rupture and repair. BAF localizes to sites of nuclear rupture and recruits NE-repair machinery, including the LEM-domain proteins, ESCRT-III complex, A-type lamins, and membranes. Here, we show that it is a mobile, nucleoplasmic population of A-type lamins that is rapidly recruited to ruptures in a BAF-dependent manner via BAF’s association with the Ig-like β fold domain of A-type lamins. These initially mobile lamins become progressively stabilized at the site of rupture. Farnesylated prelamin A and lamin B1 fail to localize to nuclear ruptures, unless that farnesylation is inhibited. Progeria-associated LMNA mutations inhibit the recruitment affected A-type lamin to nuclear ruptures, due to either permanent farnesylation or inhibition of BAF binding. A progeria-associated BAF mutant targets to nuclear ruptures but is unable to recruit A-type lamins. Together, these data reveal the mechanisms that determine how lamins respond to nuclear ruptures and how progeric mutations of LMNA and BANF1 impair recruitment of A-type lamins to nuclear ruptures.

Nucleoplasmic lamin C rapidly accumulates at sites of nuclear envelope rupture with BAF and cGAS

“…In the highly expressing cells, mEmerald-LA modestly accumulated at the rupture sites (∼1.2-fold enrichment 150 s after irradiation; Fig. 3, A–C ), in accordance with previous studies ( Denais et al, 2016 ; Sears and Roux, 2022 ; Young et al, 2020 ). Thus, the diffusible LA and LC in the nucleoplasm appear to be involved in the accumulation.…”

“…Next, we investigated the effect of the phosphorylation of LA/C at Ser 22 on accumulation at the rupture sites because previous studies have shown that this phosphorylation increases the nucleoplasmic pool ( Kochin et al, 2014 ) and is detected at the rupture sites ( Sears and Roux, 2022 ). To examine if the accumulating LC is phosphorylated, WT MEFs expressing sfGFP-DARPin-LA6 were laser-microirradiated, fixed within 10 min, and stained with an anti-phospho-Ser 22-LA/C antibody.…”

“…It has been reported that BAF is required for the recruitment of overexpressed LA to rupture sites ( Sears and Roux, 2022 ; Young et al, 2020 ). We also show that LC accumulation at rupture sites depends on BAF, supporting the idea that LA/C and p-BAF form diffusible complexes in the nucleus.…”

“…It has been also reported that some of laminopathy mutations associated with dilated cardiomyopathy (DCM), muscular dystrophy (MD), familial partial lipodystrophy (FPLD), Limb-girdle muscular dystrophy type 1B (LGMD1B), and Hutchinson-Gilford Progeria syndrome (HGPS) frequently cause spontaneous NE rupture ( De Vos et al, 2011 ; Earle et al, 2020 ; Kim et al, 2021 ). After NE rupture is induced by mechanical stress, LA/C but not LB1 appears to relocalize to the rupture sites ( Denais et al, 2016 ; Harada et al, 2014 ; Sears and Roux, 2022 ; Xia et al, 2019 ; Young et al, 2020 ). Remnant LC-rich nuclear blebs are formed in LA/C-KD cells expressing GFP-LA after constricted migration through narrow pores ( Cho et al, 2018 ).…”

A multiparametric anti-aging CRISPR screen uncovers a role for BAF in protein translation

“…NGPS remains poorly characterized compared to HGPS, and insights into the molecular mechanisms behind the disease have only recently started to emerge (14)(15)(16). Therefore, with the aim of identifying new genes and pathways relevant to NGPS, as well as potential targets for therapeutic avenues in the disease, we carried out a whole genome CRISPR/Cas9 arrayed microscopy screen in patient derived NGPS cells.…”