Lamin b1 polymorphism influences morphology of the nuclear envelope, cell cycle progression, and risk of neural tube defects in mice.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects whose complex multigenic causation has hampered efforts to delineate their molecular basis. The effect of putative modifier genes in determining NTD susceptibility may be investigated in mouse models, particularly those that display partial penetrance such as curly tail, a strain in which NTDs result from a hypomorphic allele of the grainyhead-like-3 gene. Through proteomic analysis, we found that the curly tail genetic background harbours a polymorphic variant of lamin B1, lacking one of a series of nine glutamic acid residues. Lamins are intermediate filament proteins of the nuclear lamina with multiple functions that influence nuclear structure, cell cycle properties, and transcriptional regulation. Fluorescence loss in photobleaching showed that the variant lamin B1 exhibited reduced stability in the nuclear lamina. Genetic analysis demonstrated that the variant also affects neural tube closure: the frequency of spina bifida and anencephaly was reduced three-fold when wild-type lamin B1 was bred into the curly tail strain background. Cultured fibroblasts expressing variant lamin B1 show significantly increased nuclear dysmorphology and diminished proliferative capacity, as well as premature senescence, associated with reduced expression of cyclins and Smc2, and increased expression of p16. The cellular basis of spinal NTDs in curly tail embryos involves a proliferation defect localised to the hindgut epithelium, and S-phase progression was diminished in the hindgut of embryos expressing variant lamin B1. These observations indicate a mechanistic link between altered lamin B1 function, exacerbation of the Grhl3-mediated cell proliferation defect, and enhanced susceptibility to NTDs. We conclude that lamin B1 is a modifier gene of major effect for NTDs resulting from loss of Grhl3 function, a role that is likely mediated via the key function of lamin B1 in maintaining integrity of the nuclear envelope and ensuring normal cell cycle progression.

Nuclear envelope invaginations and cancer.

The nuclear envelope (NE) surrounds the nucleus and separates it from the cytoplasm. The NE is not a passive structural component, but rather contributes to various cellular processes such as genome organization, transcription, signaling, and stress responses. Although the NE is mostly a smooth surface, it also forms invaginations that can reach deep into the nucleoplasm and may even traverse the nucleus completely. Cancer cells are generally characterized by irregularities and invaginations of the NE that are of diagnostic and prognostic significance. In the current chapter, we describe the link between nuclear invaginations and irregularities with cancer and explore possible mechanistic roles they might have in tumorigenesis.

Repetitive disruptions of the nuclear envelope invoke temporary loss of cellular compartmentalization in laminopathies.

The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.

The induction of a nucleoplasmic reticulum by prelamin A accumulation requires CTP:phosphocholine cytidylyltransferase-α.

Farnesylated prelamin A accumulates when the final endoproteolytic maturation of the protein fails to occur and causes a dysmorphic nuclear phenotype; however, the morphology and mechanisms of biogenesis of these changes remain unclear. We show here that acute prelamin A accumulation after reduction in the activity of the ZMPSTE24 endoprotease by short interfering RNA knockdown, results in the generation of a complex nucleoplasmic reticulum that depends for its formation on the enzyme CTP:phosphocholine-cytidylyltransferase-α (CCT-α, also known as choline-phosphate cytidylyltransferase A). This structure can form during interphase, confirming that it is independent of mitosis and therefore not a consequence of disordered nuclear envelope assembly. Serial-section dual-axis electron tomography reveals that these invaginations can take two forms: one in which the inner nuclear membrane infolds alone with an inter membrane space interior, and the other in which an invagination of both nuclear membranes occurs, enclosing a cytoplasmic core. Both types of invagination can co-exist in one nucleus and both are frequently studded with nuclear pore complexes (NPC), which reduces NPC abundance on the nuclear surface.

Is LMNB1 a susceptibility gene for neural tube defects in humans?

BACKGROUND: Lamins are intermediate filament proteins that form a major component of the nuclear lamina, a protein complex at the surface of the inner nuclear membrane. Numerous clinically diverse conditions, termed laminopathies, have been found to result from mutation of LMNA. In contrast, coding or loss of function mutations of LMNB1, encoding lamin B1, have not been identified in human disease. In mice, polymorphism in Lmnb1 has been shown to modify risk of neural tube defects (NTDs), malformations of the central nervous system that result from incomplete closure of the neural folds. METHODS: Mutation analysis by DNA sequencing was performed on all exons of LMNB1 in 239 samples from patients with NTDs from the United Kingdom, Sweden, and United States. Possible functional effects of missense variants were analyzed by bioinformatics prediction and fluorescence in photobleaching. RESULTS: In NTD patients, we identified two unique missense variants that were predicted to disrupt protein structure/function and represent putative contributory mutations. Fluorescence loss in photobleaching analysis showed that the A436T variant compromised stability of lamin B1 interaction within the lamina. CONCLUSION: The genetic basis of human NTDs appears highly heterogenous with possible involvement of multiple predisposing genes. We hypothesize that rare variants of LMNB1 may contribute to susceptibility to NTDs.

Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression.

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.

The nuclear envelope and its involvement in cellular stress responses.

The nuclear envelope is not only important for the structural integrity of the nucleus, but also involved in a number of cellular functions. It has been shown to be important for maintaining and controlling chromatin organization, sequestering transcription factors, replication, transcription and signalling. The nuclear envelope is thus important for development and differentiation, and some of its components are essential for cell viability. Among the many functions which are emerging for the nuclear envelope is its involvement in protecting the cell against different types of cellular stress. In the present paper, we review key findings which describe the roles of nuclear envelope components in responses to common types of stress conditions.

Stress-induced release of Oct-1 from the nuclear envelope is mediated by JNK phosphorylation of lamin B1.

The nuclear lamina can bind and sequester transcription factors (TFs), a function lost if the lamina is abnormal, with missing or mutant lamin proteins. We now show that TF sequestration is not all-or-nothing, but a dynamic physiological response to external signals. We show that the binding of the ubiquitous TF, Oct-1, to lamin B1 was reversed under conditions of cellular stress caused, inter alia, by the chemical methylating agent methylmethanesulfonate (MMS). A search for lamin B1 post-translational modifications that might mediate changes in Oct-1 binding using kinase inhibitors uncovered a role for c-Jun N-terminal kinase (JNK). Phosphoproteomic and site-directed mutagenesis analyses of lamin B1 isolated from control and MMS-treated nuclei identified T575 as a JNK site phosphorylated after stress. A new phospho-T575 specific anti-peptide antibody confirmed increased interphase cellular T575 phosphorylation after cell exposure to certain stress conditions, enabling us to conclude that lamin B1 acts as an interphase kinase target, releasing Oct-1 to execute a protective response to stress.

Structure-function relationships of the extracellular domain of the autosomal dominant polycystic kidney disease-associated protein, polycystin-1.

Polycystin-1 (PC-1) is a member of a novel family of proteins that have a multidomain structure. Although the C-terminal intracellular segments have been extensively studied, mainly with respect to their putative involvement in cell signalling, the potential function of the extracellular domains has received less attention. Mutations in PC-1 result in autosomal dominant polycystic kidney disease (ADPKD) which is characterised by perturbation of transport resulting in fluid accumulation, cell proliferation and modification of the extracellular matrix. The possibility that the interaction of a component of the extracellular matrix or some external factor with PC-1 may be important in the initiation or progression of ADPKD cannot currently be ruled out. The purpose of this review is to assess current evidence for the function of the PC-1 extracellular domains, and their potential implications for ADPKD.

Removal of detergents from protein extracts using activated charcoal prior to immunological analysis.

The use of dextran-coated activated charcoal (DCC) powder to absorb solubilising detergents from cell lysates is described. Normal embryonic epithelial cells were lysed in the presence of sodium dodecyl sulphate (SDS). The detergent was then absorbed with DCC to facilitate analysis of polycystin-1 with antibody-based methods. Polycystin-1 is a membrane protein that is involved in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The adverse effect of SDS on antibody-polycystin-1 binding was studied and the improvement resulting from its removal demonstrated using enzyme-linked immunosorbent assays (ELISAs). The results indicate that DCC can be used in a simple manner to remove highly reactive membrane-solubilising reagents from protein mixtures prior to immunological analysis. This procedure may be relevant to a variety of other techniques that are normally affected by detergents.

Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells.

Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness.

Influence of particle size and reactive oxygen species on cobalt chrome nanoparticle-mediated genotoxicity.

Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQ sensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential.

The nucleoplasmic reticulum: form and function.

The nuclear envelope (NE) physically separates nucleoplasm and cytoplasm, contributes to nuclear structural integrity, controls selective bidirectional transport of ions and macromolecular cargo, regulates gene expression, and acts as a mechanotransducer and a platform for signalling. It is noteworthy however that the NE is not simply a smooth-surfaced outer boundary but is interrupted by invaginations that reach deep within the nucleoplasm and could even traverse the nucleus completely. The existence of such a complex branched network of invaginations forming a nucleoplasmic reticulum (NR) provides sites that are capable of carrying out the 'conventional' NE functions deep within the nucleus in regions that would otherwise be remote from the nuclear periphery. In this review, we describe the structural features of NR in normal and pathological states and discuss the current understanding of their functional and possible pathological roles.

A novel role for BRCA1 in regulating breast cancer cell spreading and motility.

BRCA1 C-terminal (BRCT) domains in BRCA1 are essential for tumor suppressor function, though the underlying mechanisms remain unclear. We identified ezrin, radixin, and moesin as BRCA1 BRCT domain-interacting proteins. Ezrin-radixin-moesin (ERM) and F-actin colocalized with BRCA1 at the plasma membrane (PM) of cancer cells, especially at leading edges and focal adhesion sites. In stably expressing cancer cells, high levels of enhanced green fluorescent protein (EGFP)-BRCA1(1634-1863) acted as a dominant-negative factor, displacing endogenous BRCA1 from the PM. This led to delayed cell spreading, increased spontaneous motility, and irregular monolayer wound healing. MCF-7 cells (intact BRCA1) showed lower motility than HCC1937 cells (truncated BRCA1), but expression of EGFP-BRCA1(1634-1863) in MCF-7 increased motility. Conversely, full-length BRCA1 expression in HCC1937 decreased motility but only if the protein retained ubiquitin ligase activity. We conclude that full-length BRCA1 is important for complete tumor suppressor activity via interaction of its BRCT domains with ERM at the PM, controlling spreading and motility of cancer cells via ubiquitin ligase activity.

The nuclear envelope can control gene expression and cell cycle progression via miRNA regulation.

The nuclear envelope can regulate gene expression through its interaction with chromatin and by the sequestration of specific transcription factors. In this study, we show that such regulation can be achieved via microRNA regulation. We identify a set of miRNAs that are dysregulated in the absence of a fully functional nuclear lamina. We then focus on miRNA-31 and experimentally confirm its targets. The target set identified is significantly enriched in genes involved in controlling progress through the cell cycle such as Cdkn2a. Normalizing miRNA-31 levels, either using a specific inhibitor or by restoration of the nuclear lamina, also normalizes cell cycle distribution and cell proliferation rates. We show that the 3'UtR of p16(Ink4a)/p19(Arf) has a functional miRNA-31 binding site which contributes to the observed regulation of cell cycle progression. Our findings are the first demonstration that the nuclear envelope can control gene expression by regulating specific miRNA levels, and that miRNA-31 is involved in the regulation of cell proliferation and progress through the cell cycle at least in part by regulating the levels of p16(Ink4a)/p19(Arf).

Lamin B1 controls oxidative stress responses via Oct-1.

Interaction of lamins with chromatin and transcription factors regulate transcription. Oct-1 has previously been shown to colocalize partly with B-type lamins and is essential for transcriptional regulation of oxidative stress response genes. Using sequential extraction, co-immunoprecipitation (IP), fluorescence loss in photobleaching, and fluorescence resonance energy transfer, we confirm Oct-1-lamin B1 association at the nuclear periphery and show that this association is lost in Lmnb1(Delta/Delta) cells. We show that several Oct-1-dependent genes, including a subset involved in oxidative stress response, are dysregulated in Lmnb1(Delta/Delta) cells. Electrophoretic mobility shift assay and chromatin IP reveal that Oct-1 binds to the putative octamer-binding sequences of the dysregulated genes and that this activity is increased in cells lacking functional lamin B1. Like Oct1(-/-) cells, Lmnb1(Delta/Delta) cells have elevated levels of reactive oxygen species and are more susceptible to oxidative stress. Sequestration of Oct-1 at the nuclear periphery by lamin B1 may be a mechanism by which the nuclear envelope can regulate gene expression and contribute to the cellular response to stress, development, and aging.

Interaction of the leucine-rich repeats of polycystin-1 with extracellular matrix proteins: possible role in cell proliferation.

Polycystin-1, the product of the PKD1 gene, is a membrane-bound multidomain protein with a unique structure and a molecular weight of approximately 460 kD. The purpose of this study is to investigate the binding of the cystein-flanked leucine-rich repeats (LRR) of polycystin-1 to extracellular matrix (ECM) components. These interactions may play a role in normal renal development as well as the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). In vitro assays were used to assess the binding of a fusion protein containing the LRR of polycystin-1 and that of affinity purified polycystin-1 to a number of ECM components. The results showed that the LRR modulate the binding of polycystin-1 to collagen I, fibronectin, laminin, and cyst fluid-derived laminin fragments. The addition of the LRR fusion protein to cells in culture resulted in a significant dose-dependent reduction in the rate of proliferation. Cyst fluid-derived laminin fragments had a stimulatory effect on cell proliferation, which was reversed by the LRR fusion protein. These results suggest that the LRR of polycystin-1 act as mediators of the polycystin-1 interaction with the ECM. The observed suppression effect of the LRR on cell proliferation suggests a functional role of the LRR-mediated polycystin-1 involvement in cell-matrix and cell-cell interactions. These interactions may result in the enhanced cell proliferation that is a characteristic feature of ADPKD.