Transient nuclear lamin A/C accretion aids in recovery from vapor nanobubble-induced permeabilisation of the plasma membrane

Vapor nanobubble (VNB) photoporation is a physical method for intracellular delivery that has gained significant interest in the past decade. It has successfully been used to introduce molecular cargo of diverse nature into different cell types with high throughput and minimal cytotoxicity. For translational purposes, it is important to understand whether and how photoporation affects cell homeostasis. To obtain a comprehensive view on the transcriptional rewiring that takes place after VNB photoporation, we performed a longitudinal shotgun RNA-sequencing experiment. Six hours after photoporation, we found a marked upregulation of LMNA transcripts as well as their protein products, the A-type lamins. At the same time point, we observed a significant increase in several heterochromatin marks, suggesting a global stiffening of the nucleus. These molecular features vanished 24 h after photoporation. Since VNB-induced chromatin condensation was prolonged in LMNA knockout cells, A-type lamins may be required for restoring the nucleus to its original state. Selective depletion of A-type lamins reduced cell viability after VNB photoporation, while pharmacological stimulation of LMNA transcription increased the percentage of successfully transfected cells that survived after photoporation. Therefore, our results suggest that cells respond to VNB photoporation by temporary upregulation of A-type lamins to facilitate their recovery.

Scaling concepts in ‘omics: Nuclear lamin-B scales with tumor growth and often predicts poor prognosis, unlike fibrosis

Physicochemical principles such as stoichiometry and fractal assembly can give rise to characteristic scaling between components that potentially include coexpressed transcripts. For key structural factors within the nucleus and extracellular matrix, we discover specific gene-gene scaling exponents across many of the 32 tumor types in The Cancer Genome Atlas, and we demonstrate utility in predicting patient survival as well as scaling-informed machine learning (SIML). All tumors with adjacent tissue data show cancer-elevated proliferation genes, with some genes scaling with the nuclear filament LMNB1, including the transcription factor FOXM1 that we show directly regulates LMNB1. SIML shows that such regulated cancers cluster together with longer overall survival than dysregulated cancers, but high LMNB1 and FOXM1 in half of regulated cancers surprisingly predict poor survival, including for liver cancer. COL1A1 is also studied because it too increases in tumors, and a pan-cancer set of fibrosis genes shows substoichiometric scaling with COL1A1 but predicts patient outcome only for liver cancer—unexpectedly being prosurvival. Single-cell RNA-seq data show nontrivial scaling consistent with power laws from bulk RNA and protein analyses, and SIML segregates synthetic from contractile cancer fibroblasts. Our scaling approach thus yields fundamentals-based power laws relatable to survival, gene function, and experiments.

VP8, the Major Tegument Protein of Bovine Herpesvirus-1, Is Partially Packaged during Early Tegument Formation in a VP22-Dependent Manner

Bovine herpesvirus-1 (BoHV-1) is a major cause of rhinotracheitis and vulvovaginitis in cattle. VP8, the major tegument protein of BoHV-1, is essential for viral replication in the host. VP8 is phosphorylated by the viral kinase US3, mediating its translocation to the cytoplasm. VP8 remains nuclear when not phosphorylated. Interestingly, VP8 has a significant presence in mature BoHV-1YmVP8, in which the VP8 phosphorylation sites are mutated. This suggests that VP8 might be packaged during primary envelopment of BoHV-1. This was investigated by mass spectrometry and Western blotting, which showed VP8, as well as VP22, to be constituents of the primary enveloped virions. VP8 and VP22 were shown to interact via co-immunoprecipitation experiments, in both BoHV-1-infected and VP8-transfected cells. VP8 and VP22 also co-localised with one another and with nuclear lamin-associated protein 2 in BoHV-1-infected cells, suggesting an interaction between VP8 and VP22 in the perinuclear region. In cells infected with VP22-deleted BoHV-1 (BoHV-1ΔUL49), VP8 was absent from the primary enveloped virions, implying that VP22 might be critical for the early packaging of VP8. In conclusion, a novel VP22-dependent mechanism for packaging of VP8 was identified, which may be responsible for a significant amount of VP8 in the viral particle.

Mitochondrial fatty acid utilization increases chromatin oxidative stress in cardiomyocytes

The inability of adult mammalian cardiomyocytes to proliferate underpins the development of heart failure following myocardial injury. Although the newborn mammalian heart can spontaneously regenerate for a short period of time after birth, this ability is lost within the first week after birth in mice, partly due to increased mitochondrial reactive oxygen species (ROS) production which results in oxidative DNA damage and activation of DNA damage response. This increase in ROS levels coincides with a postnatal switch from anaerobic glycolysis to fatty acid (FA) oxidation by cardiac mitochondria. However, to date, a direct link between mitochondrial substrate utilization and oxidative DNA damage is lacking. Here, we generated ROS-sensitive fluorescent sensors targeted to different subnuclear compartments (chromatin, heterochromatin, telomeres, and nuclear lamin) in neonatal rat ventricular cardiomyocytes, which allowed us to determine the spatial localization of ROS in cardiomyocyte nuclei upon manipulation of mitochondrial respiration. Our results demonstrate that FA utilization by the mitochondria induces a significant increase in ROS detection at the chromatin level compared to other nuclear compartments. These results indicate that mitochondrial metabolic perturbations directly alter the nuclear redox status and that the chromatin appears to be particularly sensitive to the prooxidant effect of FA utilization by the mitochondria.

Nuclear lamin A in rotator cuff tear margin tenocytes: an antiapoptotic and cell mechanostat factor

Background The network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamin. A- and B-type lamins are the major components of the nuclear lamina. Lamins function in many nuclear activities. The role of lamin A and transcription factors (NF-kB) as anti-apoptotic is well documented. Recently, lamin A has also been considered as a mechanosensor protein that is able to maintain nuclear integrity from mechanical insults. We aimed to verify how lamin A expression varies in healthy cuff cells and in those with different-sized tears where various mechanical stresses are present. Methods Forty-three patients with rotator cuff tear (RCT) [23M–20F, mean age (SD): 63.5 (6.1)] were enrolled. Tissue samples excised from the most medial point of tear margins were analyzed for lamin A expression by immunohistochemistry. Controls were represented by samples obtained by normal supraspinatus tendons excised from patients submitted to reverse shoulder prosthesis implant [8M–7F, mean age (SD): 67.9 (7.1)]. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed. Results Our study revealed a moderate intensity of lamin A in the healthy cuff tendons, a higher expression of this protein in the small tears, and a significant decrease of lamin A with increasing tear size (p < 0.0001). Conclusions Our study emphasizes the importance of early repair of small RCTs since nuclear stability is maintained, and the cellular function is protected by lamin A overexpression. High re-tear of massive cuff repair could be due to cellular apoptosis and nuclear modifications induced by lamin A lack. Level of evidence III

Increased expression of LAP2β eliminates nuclear membrane ruptures in nuclear lamin–deficient neurons and fibroblasts

Defects or deficiencies in nuclear lamins cause pathology in many cell types, and recent studies have implicated nuclear membrane (NM) ruptures as a cause of cell toxicity. We previously observed NM ruptures and progressive cell death in the developing brain of lamin B1–deficient mouse embryos. We also observed frequent NM ruptures and DNA damage in nuclear lamin–deficient fibroblasts. Factors modulating susceptibility to NM ruptures remain unclear, but we noted low levels of LAP2β, a chromatin-binding inner NM protein, in fibroblasts with NM ruptures. Here, we explored the apparent link between LAP2β and NM ruptures in nuclear lamin–deficient neurons and fibroblasts, and we tested whether manipulating LAP2β expression levels would alter NM rupture frequency. In cortical plate neurons of lamin B1–deficient embryos, we observed a strong correlation between low LAP2β levels and NM ruptures. We also found low LAP2β levels and frequent NM ruptures in neurons of cultured Lmnb1−/− neurospheres. Reducing LAP2β expression in Lmnb1−/− neurons with an siRNA markedly increased the NM rupture frequency (without affecting NM rupture duration), whereas increased LAP2β expression eliminated NM ruptures and reduced DNA damage. Consistent findings were observed in nuclear lamin–deficient fibroblasts. Reduced LAP2β expression increased NM ruptures, whereas increased LAP2β expression virtually abolished NM ruptures. Increased LAP2β expression nearly abolished NM ruptures in cells subjected to mechanical stress (an intervention that increases NM ruptures). Our studies showed that increasing LAP2β expression bolsters NM integrity in nuclear lamin–deficient cells and markedly reduces NM rupture frequency.

Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole cell mechanics

The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we show that A- and B-type nuclear lamin isoforms distinctively modulate both nuclear and cellular volume and selectively stabilize the linker of nucleoskeleton and cytoskeleton (LINC) complexes that couple the nucleus to cytoskeletal actin and vimentin. We reveal, further, that loss of each of the four-known lamin isoforms in the mouse embryonic fibroblasts differentially affects cortical and cytoplasmic stiffness as well as cellular contractility, and then propose a LINC complex mediated model that explains these impaired mechanical phenotypes. Finally, we demonstrate that loss of each lamin isoform softens the nucleus in a manner that correlates with loss of heterochromatin. Together, these findings uncover distinctive roles for each lamin isoform in maintaining cellular and nuclear mechanics.

Nuclear Lamin A/C Expression Is a Key Determinant of Paclitaxel Sensitivity

Paclitaxel is a key member of the Taxane (Taxol/paclitaxel, docetaxel/taxotere) family of successful drugs used in the current treatment of several solid tumors, including ovarian cancer. The molecular target of Taxol/paclitaxel has been identified as tubulin, and paclitaxel binding alters the dynamics and thus stabilizes microtubule bundles. Traditionally, the anti-cancer mechanism of paclitaxel has been thought to originate from its interfering with the role of microtubules in mitosis, resulting in mitotic arrest and subsequent apoptosis. However, recent evidence suggests that paclitaxel operates in cancer therapies via an as-yet-undefined mechanism rather than as a mitotic inhibitor. We found that paclitaxel caused a striking break up of nuclei (referred to as multimicronucleation) in malignant ovarian cancer cells but not in normal cells, and susceptibility to undergo nuclear fragmentation and cell death correlated with a reduction in nuclear lamina proteins, Lamin A/C. Lamin A/C proteins are commonly lost, reduced, or heterogeneously expressed in ovarian cancer, accounting for the aberration of nuclear shape in malignant cells. Mouse ovarian epithelial cells isolated from Lamin A/C null mice were highly sensitive to paclitaxel and underwent nuclear breakage, compared to control wildtype cells. Forced over-expression of Lamin A/C led to resistance to paclitaxel-induced nuclear breakage in cancer cells. Additionally, paclitaxel-induced multimicronucleation occurred independently of cell division that was achieved either by the withdrawal of serum or addition of mitotic inhibitors. These results provide a new understanding for the mitotic-independent mechanism for paclitaxel killing of cancer cells, where paclitaxel induces nuclear breakage in malignant cancer cells that have a malleable nucleus, but not in normal cells that have a stiffer nuclear envelope. As such, we identify that reduced nuclear Lamin A/C protein levels correlate with nuclear shape deformation and is a key determinant of paclitaxel sensitivity of cancer cells.