Reorganization of the nuclear lamina and cytoskeleton in adipogenesis

A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the ‘linker of nucleus and cytoskeleton’ (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy

Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells

The nuclear lamina and the cytoskeleton form an integrated structure that warrants proper mechanical functioning of cells. We have studied the correlation between structural alterations and migrational behaviour in fibroblasts with and without A-type lamins. We show that loss of A-type lamins causes loss of emerin and nesprin-3 from the nuclear envelope, concurring with a disturbance in the connection between the nucleus and the cytoskeleton in A-type lamin-deficient (lmna -/-) cells. In these cells functional migration assays during in vitro wound healing revealed a delayed reorientation of the nucleus and the microtubule-organizing center during migration, as well as a loss of nuclear oscillatory rotation. These observations in fibroblasts isolated from lmna knockout mice were confirmed in a 3T3 cell line with stable reduction of lmna expression due to RNAi approach. Our results indicate that A-type lamins play a key role in maintaining directional movement governed by the cytoskeleton, and that the loss of these karyoskeletal proteins has important consequences for functioning of the cell as a mechanical entity

Cellular strain avoidance is mediated by a functional actin cap - observations in an Lmna-deficient cell model

In adherent cells, the relevance of a physical mechanotransduction pathway provided by the perinuclear actin cap stress fibers has recently emerged. Here, we investigate the impact of a functional actin cap on the cellular adaptive response to topographical cues and uniaxial cyclic strain. Lmna-deficient fibroblasts are used as a model system because they do not develop an intact actin cap, but predominantly form a basal layer of actin stress fibers underneath the nucleus. We observe that topographical cues induce alignment in both normal and Lmna-deficient fibroblasts, suggesting that the topographical signal transmission occurs independently of the integrity of the actin cap. By contrast, in response to cyclic uniaxial strain, Lmna-deficient cells show a compromised strain avoidance response, which is completely abolished when topographical cues and uniaxial strain are applied along the same direction. These findings point to the importance of an intact and functional actin cap in mediating cellular strain avoidanc

Lmna knockout mouse embryonic fibroblasts are less contractile than their wild-type counterparts

\u3cp\u3eIn order to maintain tissue homeostasis and functionality, adherent cells need to sense and respond to environmental mechanical stimuli. An important ability that adherent cells need in order to properly sense and respond to mechanical stimuli is the ability to exert contractile stress onto the environment via actin stress fibers. The actin stress fibers form a structural chain between the cells' environment via focal adhesions and the nucleus via the nuclear lamina. In case one of the links in this chain is missing or aberrant, contractile stress generation will be affected. This is especially the case in laminopathic cells, which have a missing or mutated form of the LMNA gene encoding for part of the nuclear lamina. Using the thin film method combined with sample specific finite element modeling, we quantitatively showed a fivefold lower contractile stress generation of Lmna knockout mouse embryonic fibroblasts (MEFs) as compared to wild-type MEFs. Via fluorescence microscopy it was demonstrated that the lower contractile stress generation was associated with an impaired actin stress fiber organization with thinner actin fibers and smaller focal adhesions. Similar experiments with wild-type MEFs with chemically disrupted actin stress fibers verified these findings. These data illustrate the importance of an organized actin stress fiber network for contractile stress generation and demonstrate the devastating effect of an impaired stress fiber organization in laminopathic fibroblasts. Next to this, the thin film method is expected to be a promising tool in unraveling contractility differences between fibroblasts with different types of laminopathic mutations.\u3c/p\u3

Soft substrates normalize nuclear morphology and prevent nuclear rupture in fibroblasts from a laminopathy patient with compound heterozygous LMNA mutations

\u3cp\u3eLaminopathies, mainly caused by mutations in the LMNA gene, are a group of inherited diseases with a highly variable penetrance; i.e., the disease spectrum in persons with identical LMNA mutations range from symptom-free conditions to severe cardiomyopathy and progeria, leading to early death. LMNA mutations cause nuclear abnormalities and cellular fragility in response to cellular mechanical stress, but the genotype/phenotype correlations in these diseases remain unclear. Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease.   Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome. We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures. No malformations were seen in healthy control cells at any substrate stiffness. In addition, analysis of the actin cytoskeleton organization in this laminopathy cells demonstrates that the onset of nuclear abnormalities correlates to an increase in cytoskeletal tension. Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility. This assay may be useful in predicting patient-specific phenotypic development and in investigations on the underlying mechanisms of nuclear and cellular fragility in laminopathies.\u3c/p\u3

Cellular strain avoidance is mediated by a functional actin cap - observations in an Lmna-deficient cell model

In adherent cells, the relevance of a physical mechanotransduction pathway provided by the perinuclear actin cap stress fibers has recently emerged. Here, we investigate the impact of a functional actin cap on the cellular adaptive response to topographical cues and uniaxial cyclic strain. Lmna-deficient fibroblasts are used as a model system because they do not develop an intact actin cap, but predominantly form a basal layer of actin stress fibers underneath the nucleus. We observe that topographical cues induce alignment in both normal and Lmna-deficient fibroblasts, suggesting that the topographical signal transmission occurs independently of the integrity of the actin cap. By contrast, in response to cyclic uniaxial strain, Lmna-deficient cells show a compromised strain avoidance response, which is completely abolished when topographical cues and uniaxial strain are applied along the same direction. These findings point to the importance of an intact and functional actin cap in mediating cellular strain avoidance.</p

Assessment of fibroblast nuclear morphology aids interpretation of LMNA variants

The phenotypic heterogeneity of Lamin A/C (LMNA) variants renders it difficult to classify them. As a consequence, many LMNA variants are classified as variant of unknown significance (VUS). A number of studies reported different types of visible nuclear abnormalities in LMNA-variant carriers, such as herniations, honeycomb-like structures and irregular Lamin staining. In this study, we used lamin A/C immunostaining and nuclear DAPI staining to assess the number and type of nuclear abnormalities in primary dermal fibroblast cultures of laminopathy patients and healthy controls. The total number of abnormal nuclei, which includes herniations, honeycomb-structures, and donut-like nuclei, was found to be the most discriminating parameter between laminopathy and control cell cultures. The percentage abnormal nuclei was subsequently scored in fibroblasts of 28 LMNA variant carriers, ranging from (likely) benign to (likely) pathogenic variant. Using this method, 27 out of 28 fibroblast cell cultures could be classified as either normal (n = 14) or laminopathy (n = 13) and no false positive results were obtained. The obtained specificity was 100% (CI 40-100%) and sensitivity 77% (46-95%). We conclude that assessing the percentage of abnormal nuclei is a quick and reliable method, which aids classification or confirms pathogenicity of identified LMNA variants causing formation of aberrant lamin A/C protein