Nuclear Lamina Protein Lamin Research Articles

Long-Axial-Range Double-Helix Point Spread Functions for 3D Volumetric Super-Resolution Imaging.

Single-molecule localization microscopy (SMLM) is a powerful tool for observing structures beyond the diffraction limit of light. Combining SMLM with engineered point spread functions (PSFs) enables 3D imaging over an extended axial range, as has been demonstrated for super-resolution imaging of various cellular structures. However, super-resolving structures in 3D in thick samples, such as whole mammalian cells, remains challenging as it typically requires acquisition and postprocessing stitching of multiple slices to cover the entire sample volume or more complex analysis of the data. Here, we demonstrate how the imaging and analysis workflows can be simplified by 3D single-molecule super-resolution imaging with long-axial-range double-helix (DH)-PSFs. First, we experimentally benchmark the localization precisions of short- and long-axial-range DH-PSFs at different signal-to-background ratios by imaging fluorescent beads. The performance of the DH-PSFs in terms of achievable resolution and imaging speed was then quantified for 3D single-molecule super-resolution imaging of mammalian cells by DNA-PAINT imaging of nuclear lamina protein lamin B1 in U-2 OS cells. Furthermore, we demonstrate how the use of a deep-learning-based algorithm allows the localization of dense emitters, drastically improving the achievable imaging speed and resolution. Our data demonstrate that using long-axial-range DH-PSFs offers stitching-free, 3D super-resolution imaging of whole mammalian cells, simplifying the experimental and analysis procedures for obtaining volumetric nanoscale structural information.

Oxidative stress and regulation of adipogenic differentiation capacity by sirtuins in adipose stem cells derived from female patients of advancing age

Patient age is critical for mesenchymal stem cell quality and differentiation capacity. We demonstrate that proliferation and adipogenic capacity of subcutaneous adipose stem cells (ASCs) from female patients declined with advanced age, associated with reduction in cell nucleus size, increase in nuclear lamina protein lamin B1/B2, and lamin A, upregulation of senescence marker p16INK4a and senescence-associated β-galactosidase activity. Adipogenic induction resulted in differentiation of adipocytes and upregulation of adipogenic genes CCAAT enhancer binding protein alpha, fatty acid binding protein 4, lipoprotein lipase, and peroxisome proliferator-activated receptor-γ, which was not affected by the Sirt-1 activator YK-3-237 or the Sirt-1 inhibitor EX-527. Protein expression of the stem cell markers Oct4 and Sox2 was not significantly downregulated with advanced patient age. Mitochondrial reactive oxygen species were increased in ASCs from old-aged patients, whereas protein expression of NADPH oxidases NOX1 and NOX4 was downregulated, and dual oxidase isoforms remained unchanged. Generation of nitric oxide and iNOS expression was downregulated. Protein expression of Sirt-1 and Sirt-3 decreased with patient age, whereas Sirt-2 and Sirt-5 remained unchanged. Induction of adipogenesis stimulated protein expression of Sirt-1 and Sirt-3, which was not affected upon pre-incubation with the Sirt-1-activator YK-3-237 or the Sirt-1-inhibitor EX-527. The Sirt-1 inhibitor Sirtinol downregulated adiponectin protein expression and the number of adipocytes, whereas YK-3-237 exerted stimulatory effects. In summary, our data demonstrate increased oxidative stress in ASCs of aging patients, and decline of adipogenic capacity due to Sirt-1- mediated adiponectin downregulation in elderly patients.

Abstract We030: Microtubule Organizing Centers in Cardiomyocyte Proliferation and Maturation

Mammalian cardiomyocytes undergo a loss of proliferative capacity due to cell division failure, leading to polyploidy (genome duplication) during postnatal maturation. Centrosomes, primary microtubule organizing centers (MTOCs) in animal cells comprised of centrioles and pericentriolar matrix (PCM), play a crucial role in cell division. In differentiated cells, non-centrosomal (nc) MTOCs can reorganize microtubules, with cellular localization varying between cell types to mediate specialized functions. The specific role of ncMTOCs in cardiomyocyte division and maturation remains unknown. This study investigates the formation and function of ncMTOCs in cardiomyocytes, aiming to elucidate the regulating mechanism. Our results demonstrate that during the maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), centrosomes disassemble, and their PCM translocates to the nuclear envelope, establishing perinuclear ncMTOCs. Furthermore, maturing CMs with ncMTOCs often exhibit polyploidy, indicating incomplete cell division. Microtubule dynamic experiments reveal that ncMTOCs serve as organizing centers for microtubules, promoting their assembly around the nucleus in maturing cardiomyocytes. To understand the mechanism regulating the transition from centrosomes to ncMTOCs in cardiomyocytes, we identified a significant downregulation of the nuclear lamina protein Lamin B2 (LMNB2) during CM maturation. Our results indicate that LMNB2 is essential for mitotic spindle formation in CMs but not in stem cells, suggesting a distinct role for LMNB2 in CMs. Our results also suggest an increased centrosome to ncMTOC transition in LMNB2-depleted CMs at an early differentiation stage. Ongoing research aims to investigate the impact of perinuclear ncMTOCs on cardiomyocyte proliferation through pericentriolar matrix knockdown in hiPSC-CMs. Additionally, the study seeks to further elucidate the role of Lamin B2 in the transition of centrosome proteins and its underlying mechanism.

Abstract Tu086: Repairing nuclear envelope ruptures to ameliorate Lamin-related cardiomyopathy

Heterozygous loss-of-function mutations in LMNA , encoding nuclear lamina protein Lamin A/C, cause severe adult-onset dilated cardiomyopathy. A prevailing hypothesis posits that LMNA insufficiency causes nuclear envelope structural defects that ultimately cause the disease. However, the mechanisms linking defective nuclear envelopes to cardiomyopathy remain undefined. To determine specific nuclear envelope defects and their consequences, we deleted Lmna in cardiomyocytes in adult mice ( Lmna CKO ). Strikingly, a modest (50%) reduction of Lamin A/C caused widespread localized ruptures of the nuclear envelope in cardiomyocytes (En et al. bioRxiv 2023). The nuclear envelope ruptures did not cause immediate cell death, but accompanied a strong inflammatory response in the heart, prior to fatal cardiomyopathy. We hypothesized that DNA leaked from ruptured nuclei might elicit the cGAS-STING cytosolic DNA sensing pathway of innate immunity. Contrary to this hypothesis, we did not observe cGAS-STING activation in Lmna CKO cardiomyocytes. This lack of cGAS-STING activation was likely due to the near absence of cGAS protein in adult cardiomyocytes. To investigate the mechanism underlying cardiac inflammation in Lmna CKO mice, we conducted time-course single-nucleus RNA-seq. This analysis nominated cardiac fibroblasts as the central mediator of the inflammatory response, receiving ECM-mediated signaling from Lmna CKO cardiomyocytes and recruiting immune cells to the mutant hearts. Finally, we found evidence suggesting that nuclear envelope repair activity counteracts nuclear envelope ruptures in Lmna CKO mice. We found that the envelope ruptured sites co-localized with the ESCRT-III membrane remodeling complex, previously implicated in nuclear envelope repair. We further found that overexpression of DNA-binding protein BANF1 promoted ESCRT-III recruitment to the ruptured sites. We are currently investigating whether facilitated nuclear envelope repair ameliorates cardiomyopathy in Lmna CKO mice. If it does, nuclear envelope repair may be a potential therapeutic strategy for LMNA -related dilated cardiomyopathy.

UBC9-mediated SUMOylation of Lamin B1 enhances DNA-damage-induced nuclear DNA leakage and autophagy after spinal cord injury.

Recent studies have shown that nucleophagy can mitigate DNA damage by selectively degrading nuclear components protruding from the nucleus. However, little is known about the role of nucleophagy in neurons after spinal cord injury (SCI).Western blot analysis and immunofluorescence were performed to evaluate the nucleophagy after nuclear DNA damage and leakage in SCI neurons in vivo and NSC34 expression in primary neurons cultured with oxygen-glucose deprivation (OGD) in vitro, as well as the interaction and colocalization of autophagy protein LC3 with nuclear lamina protein Lamin B1. The effect of UBC9, a Small ubiquitin-related modifier (SUMO) E2 ligase, on Lamin B1 SUMOylation and nucleophagy was examined by siRNA transfection or 2-D08 (a small-molecule inhibitor of UBC9), immunoprecipitation, and immunofluorescence.In SCI and OGD injured NSC34 or primary cultured neurons, neuronal nuclear DNA damage induced the SUMOylation of Lamin B1, which was required by the nuclear Lamina accumulation of UBC9. Furthermore, LC3/Atg8, an autophagy-related protein, directly bound to SUMOylated Lamin B1, and delivered Lamin B1 to the lysosome. Knockdown or suppression of UBC9 with siRNA or 2-D08 inhibited SUMOylation of Lamin B1 and subsequent nucleophagy and protected against neuronal death.Upon neuronal DNA damage and leakage after SCI, SUMOylation of Lamin B1 is induced by nuclear Lamina accumulation of UBC9. Furthermore, it promotes LC3-Lamin B1 interaction to trigger nucleophagy that protects against neuronal DNA damage.

Effect of morphological change on the maturation of human induced pluripotent stem cell-derived cardiac tissue in rotating flow culture

IntroductionUnderstanding the critical factors for the maturation of human induced pluripotent stem cell (hiPSC)-derived cardiac tissue is important for further development of culture techniques. Rotating flow culture, where the tissues float in the culture medium by balancing its gravitational settling and the medium flow generated in rotating disk-shaped culture vessels, is one of culture systems used for tissue engineering. It has previously been demonstrated that rotating flow culture leads to the formation of matured cardiac tissue with higher levels of function and structure than the other culture systems. However, the detailed mechanisms underlying the maturation of cardiac tissue remain unclear. This study investigated the maturation process of hiPSC-derived cardiac tissue in rotating flow culture with a focus on morphological changes in the tissue, which is a trigger for maturation. MethodsThe cardiac tissue, which consisted of cardiomyocytes derived from hiPSCs, was cultured on the 3D scaffold of poly (lactic-co-glycolic) acid (PLGA)-aligned nanofibers, in rotating flow culture for 5 days. During the culture, the time profile of projected area of tissue and formation of maturation marker proteins (β-myosin heavy chain and Connexin-43), tissue structure, and formation of nuclear lamina proteins (Lamin A/C) were compared with that in static suspension culture. ResultsThe ratio of the projected area of tissue significantly decreased from Day 0 to Day 3 due to tissue shrinkage. In contrast, Western blot analysis revealed that maturation protein markers of cardiomyocytes significantly increased after Day 3. In addition, in rotating flow culture, flat-shaped nuclei and fiber-like cytoskeletal structures were distributed in the surface region of tissue where medium flow was continuously applied. Moreover, Lamin A/C, which are generally formed in differentiated cells owing to mechanical force across the cytoskeleton and critically affect the maturation of cardiomyocytes, were significantly formed in the tissue of rotating flow culture. ConclusionsIn this study, we found that spatial heterogeneity of tissue structure and tissue shrinkage occurred in rotating flow culture, which was not observed in static suspension culture. Moreover, from the quantitative analysis, it was also suggested that tissue shrinkage in rotating flow culture contributed its following tissue maturation. These findings showed one of the important characteristics of rotating flow culture which was not revealed in previous studies.

Myonuclear alterations associated with exercise are independent of age in humans.

Age-related decline in skeletal muscle structure and function can be mitigated by regular exercise. However, the precise mechanisms that govern this are not fully understood. The nucleus plays an active role in translating forces into biochemical signals (mechanotransduction), with the nuclear lamina protein lamin A regulating nuclear shape, nuclear mechanics and ultimately gene expression. Defective lamin A expression causes muscle pathologies and premature ageing syndromes, but the roles of nuclear structure and function in physiological ageing and in exercise adaptations remain obscure. Here, we isolated single muscle fibres and carried out detailed morphological and functional analyses on myonuclei from young and older exercise-trained individuals. Strikingly, myonuclei from trained individuals were more spherical, less deformable, and contained a thicker nuclear lamina than those from untrained individuals. Complementary to this, exercise resulted in increased levels of lamin A and increased myonuclear stiffness in mice. We conclude that exercise is associated with myonuclear remodelling, independently of age, which may contribute to the preservative effects of exercise on muscle function throughout the lifespan. KEY POINTS: The nucleus plays an active role in translating forces into biochemical signals. Myonuclear aberrations in a group of muscular dystrophies called laminopathies suggest that the shape and mechanical properties of myonuclei are important for maintaining muscle function. Here, striking differences are presented in myonuclear shape and mechanics associated with exercise, in both young and old humans. Myonuclei from trained individuals were more spherical, less deformable and contained a thicker nuclear lamina than untrained individuals. It is concluded that exercise is associated with age-independent myonuclear remodelling, which may help to maintain muscle function throughout the lifespan.

Declining lamin B1 expression mediates age-dependent decreases of hippocampal stem cell activity.

Neural stem cells (NSCs) generate neurons throughout life in the hippocampal dentate gyrus. With advancing age, levels of neurogenesis sharply drop, which has been associated with a decline in hippocampal memory function. However, cell-intrinsic mechanisms mediating age-related changes in NSC activity remain largely unknown. Here, we show that the nuclear lamina protein lamin B1 (LB1) is downregulated with age in mouse hippocampal NSCs, whereas protein levels of SUN-domain containing protein 1 (SUN1), previously implicated in Hutchinson-Gilford progeria syndrome (HGPS), increase. Balancing the levels of LB1 and SUN1 in aged NSCs restores the strength of the endoplasmic reticulum diffusion barrier that is associated with segregation of aging factors in proliferating NSCs. Virus-based restoration of LB1 expression in aged NSCs enhances stem cell activity invitro and increases progenitor cell proliferation and neurogenesis invivo. Thus, we here identify a mechanism that mediates age-related decline of neurogenesis in the mammalian hippocampus.

Linking skeletal muscle aging with osteoporosis by lamin A/C deficiency.

The nuclear lamina protein lamin A/C is a key component of the nuclear envelope. Mutations in the lamin A/C gene (LMNA) are identified in patients with various types of laminopathy-containing diseases, which have features of accelerated aging and osteoporosis. However, the underlying mechanisms for laminopathy-associated osteoporosis remain largely unclear. Here, we provide evidence that loss of lamin A/C in skeletal muscles, but not osteoblast (OB)-lineage cells, results in not only muscle aging-like deficit but also trabecular bone loss, a feature of osteoporosis. The latter is due in large part to elevated bone resorption. Further cellular studies show an increase of osteoclast (OC) differentiation in cocultures of bone marrow macrophages/monocytes (BMMs) and OBs after treatment with the conditioned medium (CM) from lamin A/C-deficient muscle cells. Antibody array screening analysis of the CM proteins identifies interleukin (IL)-6, whose expression is markedly increased in lamin A/C-deficient muscles. Inhibition of IL-6 by its blocking antibody in BMM-OB cocultures diminishes the increase of osteoclastogenesis. Knockout (KO) of IL-6 in muscle lamin A/C-KO mice diminishes the deficits in trabecular bone mass but not muscle. Further mechanistic studies reveal an elevation of cellular senescence marked by senescence-associated beta-galactosidase (SA-β-gal), p16Ink4a, and p53 in lamin A/C-deficient muscles and C2C12 muscle cells, and the p16Ink4a may induce senescence-associated secretory phenotype (SASP) and IL-6 expression. Taken together, these results suggest a critical role for skeletal muscle lamin A/C to prevent cellular senescence, IL-6 expression, hyperosteoclastogenesis, and trabecular bone loss, uncovering a pathological mechanism underlying the link between muscle aging/senescence and osteoporosis.

Vitamin C Treatment Rescues Prelamin A-Induced Premature Senescence of Subchondral Bone Mesenchymal Stem Cells.

Aging is a predominant risk factor for many chronic conditions. Stem cell dysfunction plays a pivotal role in the aging process. Prelamin A, an abnormal processed form of the nuclear lamina protein lamin A, has been reported to trigger premature senescence. However, the mechanism driving stem cell dysfunction is still unclear. In this study, we found that while passaging subchondral bone mesenchymal stem cells (SCB-MSCs) in vitro, prelamin A accumulation occurred concomitantly with an increase in senescence-associated β-galactosidase (SA-β-Gal) expression. Unlike their counterparts, SCB-MSCs with prelamin A overexpression (MSC/PLA) demonstrated decreased proliferation, osteogenesis, and adipogenesis but increased production of inflammatory factors. In a hind-limb ischemia model, MSC/PLA also exhibited compromised therapy effect. Further investigation showed that exogenous prelamin A triggered abnormal nuclear morphology, DNA and shelterin complex damage, cell cycle retardation, and eventually cell senescence. Changes in gene expression profile were also verified by microarray assay. Interestingly, we found that ascorbic acid or vitamin C (VC) treatment could inhibit prelamin A expression in MSC/PLA and partially reverse the premature aging in MSC/PLA, with reduced secretion of inflammatory factors and cell cycle arrest and resistance to apoptosis. Importantly, after VC treatment, MSC/PLA showed enhanced therapy effect in the hind-limb ischemia model. In conclusion, prelamin A can accelerate SCB-MSC premature senescence by inducing DNA damage. VC can be a potential therapeutic reagent for prelamin A-induced aging defects in MSCs.

2035 – AGE-RELATED DOWNREGULATION OF LMNA IMPACTS HUMAN HEMATOPOIETIC STEM CELL FUNCTION

Hematopoietic stem cells, essential for the production of blood cells, decline in function with aging. Among these changes with aging are increased frequency of mutations in epigenetic modifiers. We analyzed the epigenetic landscape of Lineage-CD34+CD38- human HSC-enriched fraction (HSCe) from young (18-30 years), middle-aged (45-55 years), and aged (65-75 years) individuals by comparing changes in gene expression by RNA sequencing and histone marks (H3K4me3, H3K4me1, H3K27ac, H3K27me3) by ChIP sequencing. Integrative analysis of the histone profiles by k-means clustering identified 12 clusters of genes defined by their specific pattern of age-associated epigenetic changes, which were accompanied by changes in gene expression affecting 1,133 genes between young and aged HSCe. The LMNA gene, which encodes the nuclear lamina protein Lamin A/C, was one of the most downregulated genes with aging (7.9-fold, p=1.9 × 10−13). This downregulation was mediated by loss of active histone marks H3K4me3 at the LMNA gene promoter, as well as loss of H3K27ac at two putative enhancer regions. ShRNA-mediated knockdown of LMNA in young, CD34+ hematopoietic stem and progenitor cells (HSPCs) resulted in an increase in myeloid colony formation (n=7 biological replicates with an average of 33% in total colony increase), consistent with the aging phenotype. Moreover, knockdown of LMNA in CD34+ cells impaired myeloid differentiation in liquid culture as determined by persistence of CD34 expression (n=5, p≤0.0001) with a delay in CD11b expression (n=5, p≤0.05). Initial results by STORM super resolution imaging show loss of LMNA leads to enlarged nuclear size and changes in chromatin localization. In conclusion, loss of LMNA recapitulates features of HSC aging, and restoring LMNA levels may be important for restoring normal function in aged HSCs.

Modeling of LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells.

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the LMNA gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling LMNA-related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in LMNA were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on β-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca2+ dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of LMNA-related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease.

Nuclear mechanosensing.

Structural links from the nucleus to the cytoskeleton and to the extracellular environment play a role in direct mechanosensing by nuclear factors. Here, we highlight recent studies that illustrate nuclear mechanosensation processes ranging from DNA repair and nuclear protein phospho-modulation to chromatin reorganization, lipase activation by dilation, and reversible rupture with the release of nuclear factors. Recent progresses demonstrate that these mechanosensing processes lead to modulation of gene expression such as those involved in the regulation of cytoskeletal programs and introduce copy number variations. The nuclear lamina protein lamin A has a recurring role, and various biophysical analyses prove helpful in clarifying mechanisms. The various recent observations provide further motivation to understand the regulation of nuclear mechanosensing pathways in both physiological and pathological contexts.

Extreme nuclear branching in healthy epidermal cells of the Xenopus tail fin.

Changes in nuclear morphology contribute to the regulation of complex cell properties, including differentiation and tissue elasticity. Perturbations of nuclear morphology are associated with pathologies that include progeria, cancer and muscular dystrophy. The mechanisms governing nuclear shape changes in healthy cells remain poorly understood, partially because there are few models of nuclear shape variation in healthy cells. Here, we introduce nuclear branching in epidermal fin cells of Xenopus tropicalis as a model for extreme variation of nuclear morphology in a diverse population of healthy cells. We found that nuclear branching arises within these cells and becomes more elaborate during embryonic development. These cells contain broadly distributed marks of transcriptionally active chromatin and heterochromatin, and have active cell cycles. We found that nuclear branches are disrupted by loss of filamentous actin and depend on epidermal expression of the nuclear lamina protein Lamin B1. Inhibition of nuclear branching disrupts fin morphology, suggesting that nuclear branching may be involved in fin development. This study introduces the nuclei of the Xenopus fin as a powerful new model for extreme nuclear morphology in healthy cells to complement studies of nuclear shape variation in pathological contexts.This article has an associated First Person interview with the first author of the paper.

Abstract 11: Lamin A/C Mutations Epigenetically Dysregulate Scn5a Gene Expression, Perturbing Action Potential Properties in IPSC-derived Cardiomyocytes

Mutations of the LMNA gene, encoding the nuclear lamina proteins Lamin A/C, are a common cause of dilated cardiomyopathy, typically manifesting in association with cardiac conduction defects. LaminA/C regulate various nuclear activities, including maintenance of the nuclear structure, gene transcription and chromatin organization. Most studies on the consequences of Lamin A/C defects were conducted on fibroblasts, while studies on human cardiomyocytes (CMs) are scarce. We therefore generated a cardiac model of laminopathy obtained by differentiation of CMs from induced pluripotent stem cells (iPSCs) of patients carrying the K219T Lamin A/C mutation. In vitro, these cells recapitulate the morphological features of dilated cardiomyopathy, specifically sarcomeric disorganization and increased size. Using this model, we performed a comprehensive analysis of the electrophysiological properties of LMNA-CMs both at single cell level and in a multi-cellular setting. Using patch-clamp technique, results revealed significant changes in maximal upstroke velocity (dV/dt max ), action potential amplitude (APA) and overshoot (OV) in LMNA-CMs compared to those obtained from family-matched healthy controls (CNTR); these defects were associated with a reduction of the peak sodium currents and a diminished conduction velocity, measured in strands of electrically-coupled CMs. Biochemical studies showed a significant reduction of both the sodium channel Nav1.5 protein and its transcript in LMNA-CMs, accompanied by an increased binding of LaminA/C to the promoter of its coding gene, SCN5A. Binding of the Polycomb group protein SUZ12 and of the H3K27me3 histone repressive mark was also increased. Consistently, 3D-FISH experiments also indicated a preferential localization of SCN5A genomic loci at the nuclear periphery in LMNA-CMs. As a whole, our findings support a model in which mutated Lamin A/C perturb SCN5A gene expression by favouring PRC2 (Polycomb Repressive Complex 2) binding to its promoter, leading to decreased sodium current peak and slower conduction velocity. This mechanism may eventually sustain the conduction abnormalities inevitably occurring in patients with LMNA-cardiomyopathy.

Nuclear envelope structural proteins facilitate nuclear shape changes accompanying embryonic differentiation and fidelity of gene expression

BackgroundNuclear size and shape are specific to a cell type, function, and location, and can serve as indicators of disease and development. We previously found that lamin A/C and associated nuclear envelope structural proteins were upregulated when murine embryonic stem (ES) cells differentiated to primitive endoderm cells. Here we further investigated the morphological changes of nuclei that accompany this differentiation.ResultsThe nuclei of undifferentiated wild type cells were found shaped as flattened, irregular ovals, whereas nuclei of Gata4-positive endoderm cells were more spherical, less flattened, and with a slightly reduced volume. The morphological change was confirmed in the trophectoderm and primitive endoderm lineages of E4.5 blastocysts, compared to larger and more irregularly shaped of the nuclei of the inner cell mass. We established ES cells genetically null for the nuclear lamina proteins lamin A/C or the inner nuclear envelope protein emerin, or compound mutant for both lamin A/C and emerin. ES cells deficient in lamin A/C differentiated to endoderm but less efficiently, and the nuclei remained flattened and failed to condense. The size and shape of emerin-deficient nuclei also remained uncondensed after treatment with RA. The emerin/lamin A/C double knockout ES cells failed to differentiate to endoderm cells, though the nuclei condensed but retained a generally flattened ellipsoid shape. Additionally, ES cells deficient for lamin A/C and/or emerin had compromised ability to undergo endoderm differentiation, where the differentiating cells often exhibited coexpression of pluripotent and differentiation markers, such as Oct3/4 and Gata4, respectively, indicating an infidelity of gene regulation.ConclusionsThe results suggest that changes in nuclear size and shape, which are mediated by nuclear envelope structural proteins lamin A/C and/or emerin, also impact gene regulation and lineage differentiation in early embryos. Nevertheless, mice lacking both lamin A/C and emerin were born at the expected frequency, indicating their embryonic development is completed despite the observed protein deficiency.

Autophagy deficient keratinocytes display increased DNA damage, senescence and aberrant lipid composition after oxidative stress in vitro and in vivo

Autophagy allows cells fundamental adaptations to metabolic needs and to stress. Using autophagic bulk degradation cells can clear crosslinked macromolecules and damaged organelles that arise under redox stress. Accumulation of such debris results in cellular dysfunction and is observed in aged tissue and senescent cells. Conversely, promising anti-aging strategies aim at inhibiting the mTOR pathway and thereby activating autophagy, to counteract aging associated damage. We have inactivated autophagy related 7 (Atg7), an essential autophagy gene, in murine keratinocytes (KC) and have found in an earlier study that this resulted in increased baseline oxidative stress and reduced capacity to degrade crosslinked proteins after oxidative ultraviolet stress. To investigate whether autophagy deficiency would promote cellular aging, we studied how Atg7 deficient (KO) and Atg7 bearing cells (WT) would respond to stress induced by paraquat (PQ), an oxidant drug commonly used to induce cellular senescence.Atg7 deficient KC displayed increased prostanoid signaling and a pro- mitotic gene expression signature as compared to the WT. After exposure to PQ, both WT and KO cells showed an inflammatory and stress-related transcriptomic response. However, the Atg7 deficient cells additionally showed drastic DNA damage- and cell cycle arrest signaling. Indeed, DNA fragmentation and –oxidation were strongly increased in the stressed Atg7 deficient cells upon PQ stress but also after oxidizing ultraviolet A irradiation. Damage associated phosphorylated histone H2AX (γH2AX) foci were increased in the nuclei, whereas expression of the nuclear lamina protein lamin B1 was strongly decreased. Similarly, in both, PQ treated mouse tail skin explants and in UVA irradiated mouse tail skin, we found a strong increase in γH2AX positive nuclei within the basal layer of Atg7 deficient epidermis.Atg7 deficiency significantly affected expression of lipid metabolic genes. Therefore we performed lipid profiling of keratinocytes which demonstrated a major dysregulation of cellular lipid metabolism. We found accumulation of autophagy agonisitic free fatty acids, whereas triglyceride levels were strongly decreased. Together, our data show that in absence of Atg7/autophagy the resistance of keratinocytes to intrinsic and environmental oxidative stress was severely impaired and resulted in DNA damage, cell cycle arrest and a disturbed lipid phenotype, all typical for premature cell aging.

Critical Roles of Α-Type Lamins in Neutrophil Differentiation Revealed By Analyses of Lmna Trancriptional Regulation and Myeloid Models with Aberrant Lamin Α Expression

Neutrophils differentiate from common myeloid progenitors in the bone marrow where they form uniquely lobulated nuclei while acquiring multiple functions critical to their innate immune responses. We are investigating the roles of nuclear envelope (NE)-associated proteins, including the lamin B receptor (Lbr) and the nuclear lamina proteins lamin A/C, in regulating these nuclear changes, along with determining if these proteins are important to neutrophil functions including chemotaxis, phagocytosis, or reactive oxygen species (ROS) production. We previously reported that Lbr expression increases during neutrophil differentiation while lamin A/C expression decreases. We also used immortalized mouse myeloid progenitors to show that either loss of Lbr or increased lamin A/C expression caused nuclear hypolobulation similar to Pelger-Huët anomaly in humans, plus aberrant chemotaxis. Interestingly, cells lacking Lbr produced less ROS, whereas those overexpressing the lamins exhibited deficient phagocytosis. These studies suggest that the functions of these nuclear proteins go beyond simply supporting nuclear structural changes. Since our previous studies relied on the use of immortalized cell lines, which may not exhibit the full spectrum of normal neutrophil functional responses, we have focused on using techniques to derive and characterize neutrophils with aberrant NE protein expression from ex vivo cultured bone marrow. We also examined whether loss of both A-type lamins affects myeloid progenitor growth and differentiation.Here we show that functionally active neutrophils can be generated by ex vivo culture of bone marrow progenitors from cryopreserved stocks. Imaging flow cytometry, which combines the spatial resolution of fluorescence microscopy with the high throughput format of flow cytometry, was used to demonstrate that the ex vivo cultures at progressive stages of neutrophil maturation exhibit characteristic changes to cell surface marker expression. Customized gating and masking strategies were applied using the image analysis software to quantitatively measure phagocytosis of Escherichia coli particles and release of neutrophil extracellular traps (NETs). We also identified a method to evaluate NET release as a function of cell activation during phagocytosis.We next transduced our ex vivo cultured progenitors with retroviral vectors providing ectopic lamin A expression, and found that derived neutrophils exhibited hypolobulated nuclei morphologically resembling our in vitro models. We also knocked out Lmna expression in the immortalized myeloid progenitors by CRISPR-Cas9 mediated gene disruption, and identified defective growth of the progenitors plus aberrant ROS production in derived neutrophils, similar to the phenotypes observed in cells lacking Lbr.The transcriptional control of the Lmna gene during neutrophil differentiation also was investigated. In contrast to our previous report showing that the ETS factor GABP cooperates with PU.1 or C/EBPε to regulate the Lbr promoter, none of these factors affected background levels of Lmna promoter activities in COS cell reporter assays despite identified ETS and C/EBP consensus binding sites. However, addition of C/EBPα expression caused a greater than 50% decrease in Lmna promoter activities. Furthermore, quantitative ChIP assays demonstrated that C/EBPα most efficiently bound to identified C/EBP binding sequences in the Lmna promoter, indicating that C/EBPα inhibits Lmna expression in differentiating neutrophils.Combined our results demonstrate that precisely regulated levels of NE-associated proteins are critical to neutrophil nuclear maturation and their acquisition of certain functional responses. Our continued studies will utilize imaging flow cytometry to characterize the functions of ex vivo culture-derived neutrophils with aberrant A-type lamin expression, as well as identify roles of additional NE-associated proteins during neutrophil maturation. We are also poised to further characterize the control of genes encoding such NE proteins by various combinations of ETS and C/EBP transcription factors known to regulate myelopoiesis. We predict these studies will provide important insight into the molecular mechanisms governing neutrophil nuclear changes, and why aberrant lobulation is commonly associated with certain hematopoietic disorders. DisclosuresNo relevant conflicts of interest to declare.

Nuclear envelope structural defect underlies the main cause of aneuploidy in ovarian carcinogenesis.

BackgroundThe Cancer Atlas project has shown that p53 is the only commonly (96 %) mutated gene found in high-grade serous epithelial ovarian cancer, the major histological subtype. Another general genetic change is extensive aneuploidy caused by chromosomal numerical instability, which is thought to promote malignant transformation. Conventionally, aneuploidy is thought to be the result of mitotic errors and chromosomal nondisjunction during mitosis. Previously, we found that ovarian cancer cells often lost or reduced nuclear lamina proteins lamin A/C, and suppression of lamin A/C in cultured ovarian epithelial cells leads to aneuploidy. Following up, we investigated the mechanisms of lamin A/C-suppression in promoting aneuploidy and synergy with p53 inactivation.ResultsWe found that suppression of lamin A/C by siRNA in human ovarian surface epithelial cells led to frequent nuclear protrusions and formation of micronuclei. Lamin A/C-suppressed cells also often underwent mitotic failure and furrow regression to form tetraploid cells, which frequently underwent aberrant multiple polar mitosis to form aneuploid cells. In ovarian surface epithelial cells isolated from p53 null mice, transient suppression of lamin A/C produced massive aneuploidy with complex karyotypes, and the cells formed malignant tumors when implanted in mice.ConclusionsBased on the results, we conclude that a nuclear envelope structural defect, such as the loss or reduction of lamin A/C proteins, leads to aneuploidy by both the formation of tetraploid intermediates following mitotic failure, and the reduction of chromosome (s) following nuclear budding and subsequent loss of micronuclei. We suggest that the nuclear envelope defect, rather than chromosomal unequal distribution during cytokinesis, is the main cause of aneuploidy in ovarian cancer development.

Autophagy mediates degradation of nuclear lamina.

Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents, and is associated with human diseases1–3. While extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known regarding the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery4–6, is present in the nucleus and directly interacts with the nuclear lamina protein Lamin B1, and binds to lamin-associated domains (LADs) on chromatin. This LC3-Lamin B1 interaction does not downregulate Lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated Ras. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers Lamin B1 to the lysosome. Inhibiting autophagy or the LC3-Lamin B1 interaction prevents activated Ras-induced Lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests this new function of autophagy as a guarding mechanism protecting cells from tumorigenesis.