Mechanisms of A-type lamin targeting to nuclear ruptures are disrupted in LMNA- and BANF1- associated progerias

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.

Gaussian curvature dilutes the nuclear lamina, favoring nuclear rupture, especially at high strain rate

Nuclear rupture has long been associated with deficits or defects in lamins, with recent results also indicating a role for actomyosin stress, but key physical determinants of rupture remain unclear. Here, lamin-B stably interacts with the nuclear membrane at sites of low Gaussian curvature yet dilutes at high-curvature to favor rupture, whereas lamin-A depletes similarly but only at high strain-rates. Live cell imaging of lamin-B1 gene-edited cancer cells is complemented by fixed-cell imaging of ruptured nuclei in: iPS-derived cells from progeria patients, cells within beating chick embryo hearts, and cancer cells that develop multiple ruptures in migrating through small pores. Dilution and curvature-dependent rupture fit a parsimonious model of a stiff filament that detaches from a curved surface, suggesting an elastic-type response of lamin-B, but rupture is also modestly suppressed by inhibiting myosin-II and by hypotonic stress, which slow the strain rates. Lamin-A dilution and nuclear rupture likelihood indeed increase above a threshold rate of pulling into small pipettes, suggesting a viscoplastic coupling to the envelope for protection against nuclear rupture.

Abstract MP230: Nuclear Rupture In A Mouse Model Of Lmna -related Cardiomyopathy Causes Cytoplasmic Exposure Of The Proinflammatory Signaling Protein Hmgb1

Cardiomyopathies caused by mutations in LMNA, encoding nuclear Lamin A/C, are highly malignant and prevalent. How LMNA mutations cause cardiomyopathies remains unknown. We characterized cellular, molecular, and pathological evolution of mouse models of LMNA -related cardiomyopathy and provide evidence for a model in which nuclear rupture generates nuclear-localized proinflammatory signaling as a candidate molecular mechanism underlying disease pathogenesis. We observed that cardiomyocyte-specific, tamoxifen-inducible deletion of Lmna in adult mice ( Lmna CMKO ) caused a gradual reduction of Lamin A/C protein at the nuclear lamina, reflecting the slow turnover of Lamin A/C. A modest reduction of Lamin A/C in Lmna CMKO was sufficient to cause extensive fibrosis, reduced ejection fraction, and chamber dilation by 3 weeks after Lmna gene deletion. Lmna CMKO cardiomyocytes exhibited localized rupture of the nuclear envelope 2 weeks prior to the development of fibrosis and reduction of ejection fraction. Nuclear rupture in Lmna CMKO was immediately followed by an extensive upregulation of pro-inflammatory gene expression programs. We hypothesized that nuclear rupture might expose nuclear DNA to the cytoplasm thereby activating the pro-inflammatory cGas-STING cytosolic DNA sensing pathway. However, we did not observe localization of the cytosolic DNA sensor cGas to cytoplasmic DNA protruded from the ruptured nuclei in Lmna CMKO cardiomyocytes. Instead, we found that HMGB1, a potent proinflammatory protein normally sequestered in the nucleus, was released from the ruptured nuclei in Lmna CMKO cardiomyocytes. Mass spectrometry identified a strong interaction between Lamin A/C and HMGB1 in normal human fibroblast cells. Our data suggested that Lamin A/C tethers HMGB1 to the nuclear periphery by direct interaction and that reduction of Lamin A/C unleashes HMGB1 to the cytoplasm upon nuclear rupture. Future work will examine the hypothesis that cytoplasmic HMGB1 triggers pathogenic sterile inflammation leading to dilated cardiomyopathies in Lmna CMKO mice. In conclusion, we identified the nuclear rupture-induced cytoplasmic release of HMGB1 as a candidate mechanism underlying LMNA -related cardiomyopathies.

Actin assembly ruptures the nuclear envelope by prying the lamina away from nuclear pores and nuclear membranes in starfish oocytes

The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) that are stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin ‘shell’ in starfish oocytes, in contrast to microtubule-driven tearing in mammalian fibroblasts. Here, we address the mechanism of F-actin-driven NE rupture by correlated live-cell, super-resolution and electron microscopy. We show that actin is nucleated within the lamina, sprouting filopodia-like spikes towards the nuclear membranes. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining stretches of membrane accumulate NPCs that are associated with the still-intact lamina. Packed NPCs sort into a distinct membrane network, while breaks appear in ER-like, pore-free regions. We reveal a new function for actin-mediated membrane shaping in nuclear rupture that is likely to have implications in other contexts, such as nuclear rupture observed in cancer cells.

Vimentin protects cells against nuclear rupture and DNA damage during migration

Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear size and shape are important factors in regulating the mechanical properties of cells during their migration through such tight spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein that polymerizes into networks extending from a juxtanuclear cage to the cell periphery. However, the role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics remains unknown. Here, we use wild-type and vimentin-null mouse embryonic fibroblasts to show that VIFs regulate nuclear shape and perinuclear stiffness, cell motility in 3D, and the ability of cells to resist large deformations. These changes increase nuclear rupture and activation of DNA damage repair mechanisms, which are rescued by exogenous reexpression of vimentin. Our findings show that VIFs provide mechanical support to protect the nucleus and genome during migration.

Constricted migration modulates stem cell differentiation

Tissue regeneration at an injured site depends on proliferation, migration, and differentiation of resident stem or progenitor cells, but solid tissues are often sufficiently dense and constricting that nuclei are highly stressed by migration. In this study, constricted migration of myoblastic cell types and mesenchymal stem cells (MSCs) increases nuclear rupture, increases DNA damage, and modulates differentiation. Fewer myoblasts fuse into regenerating muscle in vivo after constricted migration in vitro, and myodifferentiation in vitro is likewise suppressed. Myosin II inhibition rescues rupture and DNA damage, implicating nuclear forces, while mitosis and the cell cycle are suppressed by constricted migration, consistent with a checkpoint. Although perturbed proliferation fails to explain defective differentiation, nuclear rupture mislocalizes differentiation-relevant MyoD and KU80 (a DNA repair factor), with nuclear entry of the DNA-binding factor cGAS. Human MSCs exhibit similar damage, but osteogenesis increases—which is relevant to bone and to calcified fibrotic tissues, including diseased muscle. Tissue repair can thus be modulated up or down by the curvature of pores through which stem cells squeeze.

Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation.