Nuclear membrane ruptures, cell death, and tissue damage in the setting of nuclear lamin deficiencies

Figures & data

Figure 1. Abnormal nuclear shape and nuclear membrane ruptures in mouse embryonic fibroblasts (MEFs) lacking all nuclear lamins. (a) Confocal micrographs of Lmna^+/+Lmnb1^+/+Lmnb2^+/+ (wild-type; WT), Lmna^–/–Lmnb1^–/–Lmnb2^–/– (triple knockout; TKO), and Lmna^+/+Lmnb1^–/–Lmnb2^+/+ (Lmnb1 knockout; B1KO) MEFs. Cells are stained with an antibody against LAP2β, an inner nuclear membrane protein (green). Arrowheads point to irregularly shaped nuclei; arrows point to nuclear blebs. Scale bars, 20 μm. (b) Bar graph showing numbers of nuclear membrane rupture events in WT, TKO, and B1KO MEFs. Black circles show the average number of nuclear membrane events (divided by numbers of cells examined) in three independent experiments. Numerical ratios show the total number of nuclear membrane rupture events in all three experiments divided by the total number of cells examined. **P < 0.001; ***P < 0.0001 by χ² test. (c) Confocal micrographs showing a nuclear membrane rupture (red arrows) in a TKO MEF [expressing a nuclear-localized green fluorescent protein (NLS-GFP)] as it migrated across the field (orange arrow depicts the direction of migration). NLS-GFP is in green; the differential interference contrast (DIC) image is in gray. Scale bar, 20 μm. Reproduced with permission from Chen et al. [16].

Figure 2. Effects of uniaxial stretching and actin depolymerization on nuclear membrane ruptures. (a) Bar graph showing percentages of wild-type (WT) mouse embryonic fibroblasts (MEFs) and triple-knockout (TKO) MEFs with nuclear membrane ruptures under static conditions and with uniaxial stretching. TKO MEFs lack all nuclear lamins. Black circles indicate percentages of cells with nuclear membrane ruptures in three independent experiments. Numerical ratios show the total number of cells with nuclear membrane ruptures in all three experiments over the total number of cells examined. ***P < 0.0005; ns, nonsignificant. (b) Bar graph showing that cytochalasin D reduces the percentage of nuclear membrane ruptures in TKO MEFs cultured under static conditions and with uniaxial stretching. Numerical ratios show the total number of cells with nuclear membrane ruptures over the total number of cells examined. ***P < 0.0001. Reproduced with permission from Chen et al. [16].

Figure 3. Nuclear membrane ruptures and cell death in the forebrain of E18.5 mouse embryos lacking lamin B1 expression in the forebrain. (a–b) Fluorescence micrographs of the cortical plate (CP) and ventricular zone (VZ) in the forebrain of (a) an E18.5 forebrain-specific Lmnb1 knockout (KO) embryo harboring a ROSA^nT-nG transgene (Lmnb1 KO ROSA) and (b) an E18.5 control embryo. The ROSA^nT-nG transgene encodes a fluorescent reporter that is normally confined to the nucleus. The fluorescent signal from the ROSA^nT-nG transgene is colored white. DNA was stained with DAPI (cyan). Yellow arrowheads point to Lmnb1 KO ROSA neurons in which the fluorescent reporter had escaped into the cytoplasm (indicating a nuclear membrane rupture). Scale bars, 10 μm. (c) Confocal micrographs showing caspase 3 expression (green) in the forebrain of an E18.5 control embryo and a littermate forebrain-specific Lmnb1 KO embryo. Caspase 3 is a marker of programmed cell death. DNA is stained with DAPI (blue). Scale bars, 50 μm. (d) Confocal micrographs showing the distribution of lamin B2 in forebrain neurons of a forebrain-specific Lmnb1 KO embryo, revealing an asymmetric distribution of lamin B2 along the nuclear rim in CP neurons but not VZ neurons. Lamin B2 was distributed evenly in CP neurons of a control embryo. Scale bars, 10 μm. Images in all four panels are reproduced, with permission, from Chen et al. [26].

Figure 4. Nuclear membrane ruptures are frequent but are repaired quickly in cultured B1KO neurons, whereas ruptures are infrequent and prolonged (never undergoing repair) in B2KO neurons. (a) Live-cell fluorescence microscopy images of B1KO and B2KO neurons that express a nuclear-localized green fluorescent protein (NLS-GFP). Red arrows point to cells with nuclear membrane ruptures. Scale bar, 20 μm. (b) Percentages of neurons with nuclear membrane (NM) ruptures in five independent experiments. Numerical ratios show the total numbers of cells with NM ruptures over the total numbers of cells examined. ***P < 0.0001 as defined by a χ² test. (c) NM rupture events in five independent experiments. Numerical ratios show the total numbers of NM ruptures over the total numbers of cells observed. **P < 0.001 as defined by a Student’s t test. (d) Average duration of NM ruptures. Repair of a NM rupture was never observed in a B2KO neuron; thus, the duration of 38.8 h represents the average length of time that ruptures were observed over the entire 50-h period of imaging. ***P < 0.0001 as defined by a Student’s t test. Reproduced with permission from Chen et al. [26].

Figure 5. Migration of lamin B1 knockout (B1KO) neurons into a field of narrowly spaced pillars subjects cells to constrictive forces and results in widespread cell death. (a) (left) Scanning electron micrograph (SEM) of a silicon wafer (with one side flat and the other side containing uniformly spaced silicon pillars (8 μm in diameter; 22 μm in height; spaced 4 μm apart). Scale bar, 30 μm. (middle) Higher magnification SEM of the silicon pillars. Scale bar, 3 μm. (right) Higher magnification SEM of the silicon pillars. Scale bar, 2 μm. (b) Immunofluorescence microscopy of wild-type (WT) and B1KO neurons that had been stained with a caspase 3–specific antibody (a marker of apoptotic cell death; red). DNA was stained with DAPI (blue). Only one WT neuron exhibited caspase 3 staining, whereas 21 B1KO neurons had caspase 3 staining. Scale bars, 50 μm. The panels below show caspase 3 staining in black; the edge of the field of pillars is marked by a yellow dashed line. Reproduced with permission from Chen et al. [26].

Figure 6. Identifying nuclear membrane ruptures in cultured cells does not always predict pathology in mouse models. (a) Confocal micrographs of NLS-GFP–expressing Lmnb1^+/+, Lmnb1^+/–, and Lmnb1^–/– neurons as they migrate away from cultured neurospheres, revealing nuclear membrane ruptures [escape of NLS-GFP (green) into the cytoplasm] in both Lmnb1^+/– and Lmnb1^–/– neurons. Ruptures were more frequent in Lmnb1^{–/ –} neurons than in Lmnb1^+/– neurons. The neurons were stained with antibodies against LAP2β (red), an inner nuclear membrane protein. White arrows point to cells with nuclear membrane ruptures. Scale bars, 10 μm. (b) Homozygous loss of Lmnb1 in the forebrain in forebrain-specific Lmnb1 knockout mice markedly reduces forebrain size; heterozygous loss of Lmnb1 in the forebrain does not [24]. Brackets indicate the forebrain (Fb). Scale bars, 200 μm. Images in panel b reproduced, with permission, from Coffinier et al. [24].

Figure 7. Expression of KASH2 in smooth muscle cells (SMCs) ameliorates aortic disease in a mouse model of HGPS. The HGPS mice (Lmna^G609G/G609G mice) were homozygous for the most common point mutation found in children with HGPS. KASH2 expression was from a Cre-activatable transgene (KASH2-EGFP); KASH2 expression was activated by an Sm22-Cre transgene. (a) Representative images of H&E-stained cross sections of the outer curvature of the ascending aorta in wild-type (WT) mice (Lmna^+/+KASH2-EGFP⁺Sm22-Cre⁺), HGPS mice (Lmna^G609G/G609GKASH2-EGFP⁺), and HGPS mice that expressed KASH2 in aortic SMCs (Lmna^G609G/G609GKASH2-EGFP⁺Sm22-Cre⁺). Dotted white lines outline the adventitial layer of the aorta. Colored yellow arrow indicates the medial layer of the aorta (m). Scale bars, 50 μm. (b) Bar graphs depicting medial SMCs (nuclei per μm²) and adventitial area as a percentage of total area of the cross section. n = 6/group; **P < 0.001 as defined by a Student’s t test. Reproduced with permission from Kim et al. [47].

Chen NY, Kim P, Weston TA, et al. Fibroblasts lacking nuclear lamins do not have nuclear blebs or protrusions but nevertheless have frequent nuclear membrane ruptures. Proc Natl Acad Sci U S A. 2018;115(40):10100–10105.

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