Nuclear envelope organization in papillary thyroid carcinoma

Papillary thyroid carcinomas (PTCs) have characteristic nuclear shape changes compared to follicular-type thyroid epithelium. We tested the hypothesis that the altered nuclear shape results from altered distribution or expression of the major structural proteins of the nuclear envelope. Lamin A, lamin B1, lamin C, lamin B receptor (LBR), lamina-associated polypeptide 2 (LAP2), emerin, and nuclear pores were examined. PTC's with typical nuclear features by H&E were compared to non-neoplastic thyroid and follicular neoplasms using confocal microscopy, and semi-quantitative immunoblotting. Lamin A/C, lamin B1, LAP2, emerin, and nuclear pores all extend throughout the grooves and intranuclear inclusions of PTC. Their distribution and fluorescent intensity is not predictably altered relative to nuclear envelope irregularities. By immunoblotting, the abundance (per cell) and electrophoretic mobilities of lamin A, lamin B1, lamin C, emerin, and LAP2 proteins do not distinguish PTC, normal thyroid, or follicular neoplasms. These results do not support previously published predictions that lamin A/C expression is related to a loss of proliferative activity. At least three LAP2 isoforms are identified in normal and neoplastic thyroid. LBR is sparse or undetectable in all the thyroid samples. The results suggest that the irregular nuclear shape of PTC is not determined by these nuclear envelope structural proteins per se. We review the structure of the nuclear envelope, the major factors that determine nuclear shape, and the possible functional consequences of its alteration in PTC.     

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.     

Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis

The nuclear envelope is a dynamic structure that completely disassembles in response to MPF/cdc2 activity in mitosis. A key feature of this process is the hyperphosphorylation of the major structural proteins of the envelope, the nuclear lamins A, B, and C. Two highly conserved serine residues of the lamin protein (Ser-22 and Ser-392 of lamins A and C) are symmetrically positioned 5 amino acids from the ends of the large alpha-helical domain and are shown in the accompanying paper by Ward and Kirschner to be among four sites phosphorylated during nuclear envelope breakdown. Mutations in Ser-22 and Ser-392 that prevent phosphorylation at these sites block the disassembly of the nuclear lamina during mitosis. We propose a model for the regulation of lamin assembly in which phosphorylation just outside the ends of the alpha-helical domain controls the assembly dynamics of the lamin coiled-coil dimers.     

The nuclear envelope, muscular dystrophy and gene expression

Lamins and other nuclear envelope proteins organize nuclear architecture through structural attachments that vary dynamically during the cell cycle and cell differentiation. Genetic studies have now shown that people with mutations in either lamins A/C or emerin, a nuclear membrane protein, develop Emery-Dreifuss muscular dystrophy. A mouse model for this rare disease has been created by knocking out the gene that encodes lamin A/C. This article discusses these and other recent results in the wider context of nuclear envelope function, as a framework for thinking about the possible ways in which defects in nuclear envelope proteins can lead to disease.     

Identification of lamin B2 as a substrate of protein kinase C in BALB/MK-2 mouse keratinocytes

Protein phosphorylation by activation of protein kinase C was examined using quiescent cultures of the mouse epidermal keratinocyte line BALB/MK-2. Treatment with phorbol ester caused rapid phosphorylation of five proteins with molecular weights of 80,000, 70,000, 40,000, 34,000, 28,000. Of these proteins, the 70,000 molecular weight one (p70) was studied further. Its position on two-dimensional gel suggested that p70 is nuclear envelope lamin B. This possibility was confirmed by the co-migration of p70 with the lamin fraction of mouse liver and its immunoprecipitation with antinuclear lamina antibody. The lamin B fraction consists of lamin B1 and lamin B2. Evidence that p70 is lamin B2 was obtained by peptide mapping and amino acid sequencing. Lamin B2 is the only lamin that shows a substantial increase in phosphorylation on treatment of BALB/MK-2 cells with phorbol ester.     

Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm.

We have constructed a series of mutations in the human A lamin cDNA to identify and alter the nuclear localization signal using an in vivo functional assay system. The nuclear localization signal in the lamin proteins has both structural and functional similarities with that of the SV40 large T-antigen. Mutations within this functional domain result in the assembly of cytoplasmic tubular structures, and the behavior of these mutants suggests a post-translational dimerization of the lamin proteins prior to their transport into the nucleus. In the course of this work other regions of the carboxy terminus of the A/C lamin proteins have been implicated in the proper assembly and structure of the nuclear envelope.     

Distribution of emerin during the cell cycle

Human emerin is a nuclear membrane protein that is lost or altered in patients with Emery-Dreifuss muscular dystrophy (EMD). While the protein is expressed in the majority of human tissues analyzed, the pathology predominates in cardiac and skeletal muscles of patients with EMD. Our results show that emerin can be detected by immunocytochemistry and immunoblotting in the nuclear envelope of all vertebrates studied from man to Xenopus. Immunolocalizations and nuclear envelope extraction experiments confirm that emerin possesses properties characteristic for integral membrane proteins of the inner nuclear membrane. Some nuclear envelope proteins are localized also in annulate lamellae (AL), i.e. cytoplasmic flattened membrane cisternae penetrated by pore complexes. To verify whether emerin is contained in these membrane stacks, we have induced the formation of AL by exposure of rat cells (line RV-SMC) to sublethal doses of the antimitotic drug vinblastine sulfate and found that emerin is present in the nuclear envelope, but is absent from AL. In contrast to the homogeneous distribution of emerin in the nuclear envelope of interphase cells, this protein shows a focal accumulation in the nuclear membranes of late telophase cells. During early reassembly of the nuclear envelope at this mitotic stage emerin colocalizes with lamin A/C but not with lamin B and LAP2 proteins. Confocal laser scanning microscopy after double-labeling experiments with emerin and tubulin shows that emerin is concentrated in areas of the mitotic spindle and in the midbody of mitotic cells suggesting a close interaction of these proteins. Our data suggest that emerin participates in the reorganisation of the nuclear envelope at the end of mitosis.     

Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope.

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.     

Nuclear envelope defects in muscular dystrophy

Muscular dystrophies are a heterogeneous group of disorders linked to defects in 20-30 different genes. Mutations in the genes encoding a pair of nuclear envelope proteins, emerin and lamin A/C, have been shown to cause the X-linked and autosomal forms respectively of Emery-Dreifuss muscular dystrophy. A third form of muscular dystrophy, limb girdle muscular dystrophy 1b, has also been linked to mutations in the lamin A/C gene. Given that these two genes are ubiquitously expressed, a major goal is to determine how they can be associated with tissue specific diseases. Recent results suggest that lamin A/C and emerin contribute to the maintenance of nuclear envelope structure and at the same time may modulate the expression patterns of certain mechanosensitive and stress induced genes. Both emerin and lamin A/C may play an important role in the response of cells to mechanical stress and in this way may help to maintain muscle cell integrity.     

The myristoylation site of meiotic lamin C2 promotes local nuclear membrane growth and the formation of intranuclear membranes in somatic cultured cells

Lamin C2 is a splice product of the mammalian lamin A gene and expressed in primary spermatocytes where it is distributed in the form of discontinuous plaques at the nuclear envelope. We have previously shown that the aminoterminal hexapetide GNAEGR of lamin C2 following the start methionine is essential for its association with the nuclear envelope and that the aminoterminal glycine of the hexapeptide is myristoylated. Here we have analyzed the ultrastructural changes induced in COS-7 and Xenopus A6 cells by overexpressing rat lamin C2 or a human lamin C mutant possessing the lamin C2-specific hexapeptide at its aminoterminus. Both lamins were targeted to the nuclear envelope of mammalian and amphibian cells and induced the formation of intranuclear membranes, whereas wild-type human lamin C and a lamin C2 mutant, that both lack this lipid moiety, did not. Our data indicate that the myristoyl group of lamin C2 has besides its demonstrated role in nuclear envelope association additional functions during spermatogenesis. Our present study complements previously published results where we have shown that the CxxM motif of lamins promotes nuclear membrane growth (Prüfert et al., 2004. J. Cell Sci. 117, 6105-6116).     

Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma

Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.Subject terms: Nuclear organization, Cancer     

Characterization of lamin proteins in BHK cells

Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.     

Nuclear envelope proteins: Identification of lamin B subtypes

The lamins are a group of nuclear envelope proteins thought to form a structural layer at the nuclear periphery. Lamins A, B and C occur in many cell types although lamin C, a subtype of lamin A, is relatively decreased in chicken cells. A subtype of lamin B has been found in chicken cells. This subtype, called lamin B1, is slightly larger and more acidic than the quantitatively major subtype now called lamin B2. The lamin B subtypes have very similar primary sequences and share a distinctive topology. Two lamin B subtypes have not been observed in mammalian tissues but have been found in three avian tissues, erythrocytes of mature chickens and liver and erythrocytes of 11- to 13-day-old embryos. As these tissues differ widely in metabolic activity, both subtypes appear to be constitutive nuclear proteins.     

The nuclear lamin protein family in higher vertebrates. Identification of quantitatively minor lamin proteins by monoclonal antibodies

The nuclear lamina, a structure closely apposed to the inner nuclear membrane, is believed to provide a framework important for nuclear envelope integrity and interphase chromatin organization. So far, in mammalian and avian species three major constituents of the lamina, lamins A, B, and C, have been identified. These proteins migrate to characteristic positions on two-dimensional gels, lamin B being more acidic than lamins A and C. Here, we show that the composition of the nuclear lamina in avian and mammalian cells is more complex than previously assumed. When analyzed on two-dimensional gels, the major 66-kDa chicken "lamin B" protein can readily be identified. However, an additional 68-kDa protein migrates to a similarly acidic position. Based on the following evidence, both proteins can be considered as two distinct members of the lamin protein family. First, peptide mapping experiments and immunological criteria demonstrate that these two proteins are not related to each other or to lamin A via postsynthetic modifications or precursor-product relationships. Second, as determined by immunocytochemical techniques, both proteins are located exclusively at the nuclear periphery. Third, both proteins display the biochemical properties characteristic of lamin proteins, i.e. they are resistant to extraction of nuclei with nonionic detergents, nucleases, and high salt. Fourth, both proteins are immunologically related to previously characterized lamin proteins: the major 66-kDa chicken "lamin B" protein shares at least two epitopes with lamin A. However, contrary to what current nomenclature might suggest, this 66-kDa chicken "lamin B" protein is not related to rat liver lamin B, but to a minor component of rat liver pore-complex lamina preparations that had not previously been recognized as a lamin protein. Conversely, the minor 68-kDa component of chicken lamina preparations that had not previously been considered to be a lamin protein is immunologically related to rat liver lamin B. Thus, in addition to demonstrating the existence of quantitatively minor lamin proteins in higher vertebrates, our results caution against assigning structural homologies between lamin proteins from different species on the basis of gel electrophoresis analyses.     

The conserved carboxy-terminal cysteine of nuclear lamins is essential for lamin association with the nuclear envelope

We have analyzed the interaction of soluble nuclear lamins with the nuclear envelope by microinjection of normal and mutated lamins into the cytoplasm of Xenopus laevis oocytes. Our results demonstrate that the conserved cysteine of the carboxy-terminal tetrapeptide Cys Ala/Ser Ile Met of lamins is essential for their association with the nuclear envelope. Removal of this sequence or replacement of the cysteine by serine resulted in Xenopus lamin L1 remaining in a soluble, non-envelope-associated state within the nucleus. Similar mutations of Xenopus lamin A resulted in only partial reduction of nuclear envelope association, indicating that lamin A contains additional signals that can partially compensate for the lack of the cysteine. Mammalian lamin C lacks this tetrapeptide and is not associated with the nuclear envelope in our experimental system. Cloning of the tetrapeptide Cys Ala Ile Met to the carboxy terminus of human lamin C resulted in lamin being found in a nuclear envelope-associated form in oocytes. Mutations at the amino terminus and in the alpha-helical region of lamin L1 revealed that the carboxy terminus mediates the association of lamins with the nuclear envelope; however, this alone is insufficient for maintenance of a stable association with the nuclear envelope.     

Epstein-Barr virus BGLF4 kinase induces disassembly of the nuclear lamina to facilitate virion production

DNA viruses adopt various strategies to modulate the cellular environment for efficient genome replication and virion production. Previously, we demonstrated that the BGLF4 kinase of Epstein-Barr virus (EBV) induces premature chromosome condensation through the activation of condensin and topoisomerase IIα (C. P. Lee, J. Y. Chen, J. T. Wang, K. Kimura, A. Takemoto, C. C. Lu, and M. R. Chen, J. Virol. 81:5166-5180, 2007). In this study, we show that BGLF4 interacts with lamin A/C and phosphorylates lamin A protein in vitro. Using a green fluorescent protein (GFP)-lamin A system, we found that Ser-22, Ser-390, and Ser-392 of lamin A are important for the BGLF4-induced disassembly of the nuclear lamina and the EBV reactivation-mediated redistribution of nuclear lamin. Virion production and protein levels of two EBV primary envelope proteins, BFRF1 and BFLF2, were reduced significantly by the expression of GFP-lamin A(5A), which has five Ser residues replaced by Ala at amino acids 22, 390, 392, 652, and 657 of lamin A. Our data indicate that BGLF4 kinase phosphorylates lamin A/C to promote the reorganization of the nuclear lamina, which then may facilitate the interaction of BFRF1 and BFLF2s and subsequent virion maturation. UL kinases of alpha- and betaherpesviruses also induce the disassembly of the nuclear lamina through similar sites on lamin A/C, suggesting a conserved mechanism for the nuclear egress of herpesviruses.     

The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum

In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.     

Identification and Cloning of an mRNA Coding for a Germ Cell-Specific A-Type Lamin in Mice

The nuclear lamins are karyoskeletal proteins which have important functions, such as maintaining nuclear envelope integrity and organizing high order nuclear structure during mitosis in higher eukaryotes. In somatic mammalian cells, the A-type and B-type lamins, composed of lamins A and C and lamins B1 and B2, are major components of the nuclear lamina. However, A-type lamins have as yet not been identified in germ cells and undifferentiated embryonic cells. Here we report the cloning of a new 52-kDa A-type lamin from mouse pachytene spermatocytes, termed lamin C2 because of its similarities with lamin C. It has a sequence identical to that of lamin C except that the N -terminal segment, containing the head and the α-helical coil 1A domains, is replaced with a short non-α-helical stretch of amino acids. In mice, lamin C2 was found to be specifically expressed in germ cells. This specific expression and unique structure suggests a role for lamin C2 in determining the organization of nuclear and chromosomal structures during spermatogenesis.