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At least 19 recordsLinked to original sources
PubMed1990

Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin.

Previous analyses of the nuclear lamina of mammalian cells have revealed three major protein components (lamins A, B and C) that have been identified by protein sequence homology as members of the intermediate filament (IF) protein family. It has been claimed that mammalian cells contain either all three lamins or lamin B alone. Using monoclonal antibodies specific for B-type lamins and cDNA cloning we identified a second major mammalian B-type lamin (murine lamin B2), thus showing that lamin composition in mammals is more complex than previously thought. Lamin B2 is coexpressed with lamin B1 (formerly termed lamin B) in all somatic cells and mammalian species that we analysed, including a variety of cells currently believed to contain only a single lamin. This suggests that two B-type lamins are necessary to form a functional lamina in mammalian somatic cells. By cDNA cloning we found that Xenopus laevis lamin LII is the amphibian homolog of mammalian lamin B2. Lamin expression during embryogenesis of amphibians and mammals shows striking similarities. The first lamins expressed in the early embryo are the two B-type lamins, while A-type lamins are only detected much later in development. These findings indicate that the genomic differentiation into two B-type lamins occurred early in vertebrate evolution and has been maintained in both their primary structure and pattern of expression.

Amino Acid Sequence↗
PubMed1998

Identification of the lamina-associated-polypeptide-2-binding domain of B-type lamin.

Lamina-associated polypeptide (LAP)2, which directly interacts with B-type lamins and chromosomes, is an integral membrane protein specifically distributed along the inner nuclear membrane of the nuclear envelope. Multiple regions of its large nucleoplasmic domain promote this localization, including the first (residues 1-296) and the second (residues 298-409) halves of the LAP2 N terminus. The second half is involved in LAP2 association with the nuclear lamina [Furukawa, K., Panté, N., Aebi, U. & Gerace, L. (1995) EMBO J. 14, 1626-1636]. In this study to further define its role, we examined which domain of B-type lamin interacts with LAP2 by means of a binding assay with bacterially expressed proteins and a yeast two-hybrid system. We found that amino acids in the region of residues 78-258 of the lamin B1 rod domain directly bound with LAP2. The data suggest that LAP2 may modulate the assembly of nuclear lamins.

Binding Sites↗
PubMed1989

Cloning and sequencing of cDNA clones encoding chicken lamins A and B1 and comparison of the primary structures of vertebrate A- and B-type lamins.

Nuclear lamins are intermediate-filament-type proteins forming a fibrillar meshwork underlying the inner nuclear membrane. The existence of multiple isoforms of lamin proteins in vertebrates is believed to reflect functional specializations during cell division and differentiation. Although biochemical criteria may be used to classify many lamin isoforms into A- and B-type subfamilies, the structural features distinguishing the members of these subfamilies remain to be characterized fully. Here, we report the complete primary structures of chicken lamins A and B1, as they are deduced from cloned cDNAs; in the accompanying paper we present the complete sequence of lamin B2, a second avian B-type lamin. Comparisons of the chicken lamin sequences with each other and with those of other lamins allow us to establish structural features that are common to members of both subfamilies. Conversely, multiple sequence alignments make it possible to identify a number of structural motifs that clearly differentiate B-type lamins from A-type lamins. With this information at hand, we attempt to correlate different biochemical properties of A- and B-type lamins with the presence or absence of specific sequence motifs.

Amino Acid Sequence↗
PubMed1997

Comparison of A and B-type lamin expression in reactive lymph nodes and nodular sclerosing Hodgkin's disease.

AIMS: In order to clarify the differentiation and proliferation status of the Reed-Sternberg and Hodgkin cells we studied A and B-type lamin expression with specific monoclonal antibodies in nodular sclerosing Hodgkin's disease. Its normal counterpart, the reactive lymph node, was also examined for lamin subtype expression. METHODS AND RESULTS: The CD20 positive centrocytes and centroblasts of the follicle centre in the reactive lymph nodes expressed lamin B1, but were not or only very weakly positive for lamin B2 or A-type lamin antibodies. Mantle zone lymphocytes displayed lamins B1 and B2, but were negative for A-type lamins. Furthermore, CD3- and CD20-positive lymphocytes in the medulla and paracortex lacked A-type lamins, but were positive for both B-type lamins. Finally, the proliferation marker Ki67 was mainly detected in the centroblasts, but also in a fraction of the A-type lamin negative cells in the paracortex and medulla. In Hodgkin's disease, all cells expressed lamins B1 and B2, whereas A-type lamins were primarily observed in CD30-positive Reed-Sternberg and Hodgkin cells. About 20% of the Reed-Sternberg and Hodgkin cells expressed Ki67, with co-expression of lamin A in most of these cells. CONCLUSIONS: Ki67 and A-type lamin staining were in general mutually exclusive in lymph nodes, indicating that A-type lamin positive cells are not proliferative. This suggests also that the A-type lamin expression in Reed-Sternberg and Hodgkin cells is correlated with a relatively mature phenotype of these malignant cells. However, some of these differentiated malignant cells still have a capacity to proliferate as indicated by Ki67 positivity. Our observation that lamin B2 expression in the follicle centre cells of the reactive lymph node is low or absent indicates that this lamin subtype is not always expressed in nucleated cells, which is in clear contrast to the results obtained in previous studies in other diseases and in normal tissues. Absence of lamin B2 expression may be associated with the follicle centre stage of B-cells.

Fluorescent Antibody Technique, Indirect↗
PubMed1989

A second higher vertebrate B-type lamin. cDNA sequence determination and in vitro processing of chicken lamin B2.

The chicken nuclear lamina is composed of at least three proteins called lamins A, B1 and B2. In addition, putative precursors are transiently expressed during in vivo synthesis of lamins A and B2. Here we report the complete sequence of lamin B2 as it is deduced from a cloned cDNA. Comparison of lamin B2 with lamins A and B1 in the accompanying paper provides definitive proof for the existence of two structurally distinct chicken B-type lamins. Furthermore, we show that in vitro translation of transcripts derived from lamin A and lamin B2 cDNAs yielded polypeptides that were indistinguishable, by two-dimensional gel electrophoresis, from the putative in vivo precursors of lamins A and B2 respectively. However, whereas the lamin A precursor was stable, the translation product of the lamin B2 transcript was processed in the reticulocyte lysate to a polypeptide comigrating on two-dimensional gels with authentic mature lamin B2. This processing event could be inhibited by chelators of divalent cations, i.e. o-phenanthroline and EDTA. Our results indicate that the transiently expressed variant of lamin B2 represent a bonafide precursor, and that two distinct activities are involved in processing of newly synthesized lamins A and B2. Lamin precursors processing is discussed in relation to characteristic differences in the interactions of A and B-type lamins with the nuclear membrane.

Amino Acid Sequence↗
PubMed1997

A- and B-type lamins are differentially expressed in normal human tissues.

A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies.

Antibodies, Monoclonal↗
PubMed1997

The inner nuclear membrane protein LAP1 forms a native complex with B-type lamins and partitions with spindle-associated mitotic vesicles.

We have examined the in situ organization and nearest neighbours of the 'lamina-associated polypeptide-1' (LAP1), a type II membrane protein and a major constituent of the mammalian nuclear envelope. We show here that, during interphase, LAP1 forms multimeric assemblies which are suspended in the inner nuclear membrane and are specifically associated with B-type lamins. The LAP1-lamin B complex is distinct from analogous complexes formed by the 'lamina-associated polypeptide-2' (LAP2), another inner nuclear membrane protein, and includes a protein kinase. Upon nuclear envelope breakdown, LAP1 partitions with mitotic vesicles which carry nuclear lamin B. The LAP1 vesicles can be distinguished from fragments of the nuclear envelope containing LAP2 and exhibit a striking co-alignment with spindle microtubules. These observations suggest that the inner nuclear membrane comprises discrete territories which accommodate specific integral membrane proteins and are differentially disassembled during mitosis.

Amino Acid Sequence↗
PubMed1997

Assembly of A- and B-type lamins studied in vivo with the baculovirus system.

We have expressed an A-type lamin (Xenopus lamin A), a probable A-type lamin (Drosophila lamin C), two B-type lamins (Xenopus lamin LI, Drosophila lamin Dmo), and two mutants of Xenopus lamin A in Sf9 cells. All proteins were synthesized at high levels resulting in formation of paracrystals with an axial repeat of 18.5-20.0 nm by A-type lamins; in contrast B-type lamins assembled into aggregates with a fibrillar ultrastructure. Of the four wild-type proteins analyzed only lamin Dmo was found in the nuclear compartment of Sf9 cells in association with the lamina whereas the three other lamins assembled into polymers localized in the cytoplasm as well as the nucleoplasm. The Xenopus lamin A mutant lacking the complete carboxy-terminal tail assembled in the cytoplasm into long filament bundles consisting of fibrils of less than 6 nm diameter. In vitro the non-helical amino-terminal head domain of lamins is required for the formation of 'head-to-tail' polymers. A lamin A mutant lacking this domain could be efficiently extracted from Sf9 cells with physiological buffers containing Triton X-100, demonstrating the importance of this domain for lamin assembly in vivo.

Animals↗
PubMed2003

Lamina-associated polypeptide 2beta (LAP2beta) is contained in a protein complex together with A- and B-type lamins.

Lamina-associated polypeptide 2beta (LAP2beta) of vertebrates is an integral membrane protein of the inner nuclear membrane that is generated by alternative splicing from the LAP2 gene. In the majority of Xenopus somatic cells including cultured kidney epithelial cells (A6 cells) there is only one major LAP2 isoform expressed that has the highest similarities with the mammalian LAP2beta whereas isoforms corresponding in size to the mammalian LAP2gamma and alpha are not detectable. We selected A6 cells and A6 cells stably expressing GFP fusion proteins of Xenopus LAP2beta (XLAP2Pbeta) as a model system to study interactions between LAP2beta and lamins. In vitro binding experiments with GST-XLAP2beta fusion proteins and immunoprecipitations with antibodies to GFP revealed that XLAP2beta is part of a complex that contains A- and B-type lamins. For the targeting to the nuclear envelope and the in vivo formation of this complex, GFP fusion proteins were sufficient comprising only the carboxyterminal 135 amino acids of XLAP2beta or the comparable region of zebrafish LAP2beta. A highly conserved 36 amino acids long sequence is located in this region of LAP2beta that is part of the lamina-binding domain previously identified in rat LAP2beta. GFP-LAP2beta fusion proteins of Xenopus, zebrafish, and rat that contained this sequence do compete with endogenous LAP2 in transfected cells for the same binding sites in the lamina. Our data indicate that the lamina-binding site of LAP2beta has been highly conserved during vertebrate evolution and suggests that this region of LAP2beta mediates the interactions between polymers of A- and B-type lamins.

Amino Acid Sequence↗
PubMed1992

The gene structure of Xenopus nuclear lamin A: a model for the evolution of A-type from B-type lamins by exon shuffling.

Nuclear lamins are intermediate filament (IF) type proteins that form a fibrillar network underlying the inner nuclear membrane. The existence of multiple subtypes of lamins in vertebrates has been interpreted in terms of functional specialization during cell division and differentiation. The structure of a gene encoding an A-type lamin of Xenopus laevis was analysed. Comparison with that of a B-type lamin of the same species shows remarkable conservation of the exon/intron pattern. In both genes the last exon, only 9-12 amino acids in length, encodes the complete information necessary for membrane targeting of lamins, i.e. a ras-related CaaX motif. The lamin A specific extension of the tail domain is encoded by a single additional exon. The 5' boundary of this exon coincides with the sequence divergence between human lamins A and C, for which an alternative splice mechanism had previously been suggested. Arguments are presented suggesting that B-type lamins represent the ancestral type of lamins and that A-type lamins derived there from by exon shuffling. The acquisition of the new exon might explain the different fates of A- and B-types lamins during cell division.

Amino Acid Sequence↗
PubMed2006

Nuclear titin interacts with A- and B-type lamins in vitro and in vivo.

Lamins form structural filaments in the nucleus. Mutations in A-type lamins cause muscular dystrophy, cardiomyopathy and other diseases, including progeroid syndromes. To identify new binding partners for lamin A, we carried out a two-hybrid screen with a human skeletal-muscle cDNA library, using the Ig-fold domain of lamin A as bait. The C-terminal region of titin was recovered twice. Previous investigators showed that nuclear isoforms of titin are essential for chromosome condensation during mitosis. Our titin fragment, which includes two regions unique to titin (M-is6 and M-is7), bound directly to both A- and B-type lamins in vitro. Titin binding to disease-causing lamin A mutants R527P and R482Q was reduced 50%. Studies in living cells suggested lamin-titin interactions were physiologically relevant. In Caenorhabditis elegans embryos, two independent C. elegans (Ce)-titin antibodies colocalized with Ce-lamin at the nuclear envelope. In lamin-downregulated [lmn-1(RNAi)] embryos, Ce-titin was undetectable at the nuclear envelope suggesting its localization or stability requires Ce-lamin. In human cells (HeLa), antibodies against the titin-specific domain M-is6 gave both diffuse and punctate intranuclear staining by indirect immunofluorescence, and recognized at least three bands larger than 1 MDa in immunoblots of isolated HeLa nuclei. In HeLa cells that transiently overexpressed a lamin-binding fragment of titin, nuclei became grossly misshapen and herniated at sites lacking lamin B. We conclude that the C-terminus of nuclear titin binds lamins in vivo and might contribute to nuclear organization during interphase.

Amino Acid Sequence↗
PubMed2001

Mistargeting of B-type lamins at the end of mitosis: implications on cell survival and regulation of lamins A/C expression.

We previously showed that targeting of protein phosphatase 1 (PP1) to the nuclear envelope (NE) by the A-kinase anchoring protein, AKAP149, correlates with nuclear assembly of B-type lamins in vitro. We demonstrate here that failure of AKAP149-mediated assembly of B-type lamins into the nuclear lamina at the end of mitosis is followed by apoptosis, and induces expression of the gene encoding A-type lamins in cells that normally do not express lamins A/C. In HeLa cells, inhibition of PP1 association with the NE mediated by a peptide containing the PP1-binding domain of AKAP149 results in failure of B-type lamins to assemble, and in their rapid caspase-dependent proteolysis. However, assembly of lamins A/C is not affected. Nonetheless, apoptosis follows within hours of nuclear reformation after mitosis. In lymphoid KE37 cells, which do not express lamins A/C, inhibition of B-type lamin assembly triggers rapid synthesis and nuclear assembly of both lamins A and C before apoptosis takes place. The results indicate that nuclear assembly of B-type lamins is essential for cell survival. They also suggest that mistargeting of B-type lamins at the end of mitosis elicits a tentative rescue process to assemble a nuclear lamina in lymphoid cells that normally do not express lamins A/C.

A Kinase Anchor Proteins↗
PubMed1996

Characterization and quantitation of three B-type lamins in Xenopus oocytes and eggs: increase of lamin LI protein synthesis during meiotic maturation.

We have previously shown that Xenopus oocytes, eggs, and early embryos contain lamins LII and LIII, and that portions of each are associated with distinct egg vesicle populations. We now report that a lamin similar or identical to the B-type lamin LI is also present in oocyte nuclei and in egg extracts. We have quantitated the three B-type lamins per oocyte nucleus, and have calculated relative ratios of LI:LIII = 1:100, and LII:LIII = 1:10. Similar to lamin LII, 5-15% of lamin LI is associated with egg membranes in a biochemically stable manner. Egg vesicles absorbed with lamin isoform-specific antibodies to magnetic beads indicate that lamin LI-associated egg membranes are of heterogenous morphology, and are independent from the lamin LII and LIII vesicle populations. Compared to other nuclear envelope proteins, the synthesis of lamin LI protein is specifically elevated during meiotic maturation, resulting in a 4- to 12-fold higher amount of lamin LI in eggs than is present in oocyte nuclei. Immunofluorescence and immunoblot analysis demonstrated that lamins LI, LII, and LIII are associated with the nuclear envelope formed on demembranated sperm when added to activated egg extract. These results strongly suggest that three different lamin-associated vesicle populations are involved in the formation of a nuclear envelope in egg extracts.

Animals↗
PubMed1994

Type B lamins remain associated with the integral nuclear envelope protein p58 during mitosis: implications for nuclear reassembly.

p58 (also referred to as the lamin B receptor) is an integral membrane protein of the nuclear envelope known to form a multimeric complex with the lamins and other nuclear proteins during interphase. To examine the fate of this complex during mitosis, we have investigated the partitioning and the molecular interactions of p58 in dividing chicken hepatoma (DU249) cells. Using confocal microscopy and double immunolabelling, we show here that lamins B1 and B2 co-localize with p58 during all phases of mitosis and co-assemble around reforming nuclei. A close juxtaposition of p58/lamin B-containing vesicles and chromosomes is already detectable in metaphase; however, p58 and lamin reassembly proceeds slowly and is completed in late telophase--G1. Flotation of mitotic membranes in sucrose density gradients and analysis of mitotic vesicles by immunoelectron microscopy confirms that p58 and most of the type B lamins reside in the same compartment. Co-immunoprecipitation of both proteins by affinity-purified anti-p58 antibodies shows that they are physically associated in the context of a mitotic p58 'sub-complex'. This sub-assembly does not include the type A lamins which are fully solubilized during mitosis. Our data provide direct, in vivo and in vitro evidence that the majority of type B lamins remain connected to nuclear membrane 'receptors' during mitosis. The implications of these findings in nuclear envelope reassembly are discussed below.

Animals↗
PubMed2005

Null mutants of Drosophila B-type lamin Dm(0) show aberrant tissue differentiation rather than obvious nuclear shape distortion or specific defects during cell proliferation.

To elucidate the function of metazoan B-type lamins during development, new null mutations of the Drosophila B-type lamin gene, lamDm(0), were analyzed in parallel with the misg(sz18) mutation, a lamDm(0) allele reported previously. Although in all these mutants, lamin Dm(0) protein was undetectable in neuroblasts and imaginal disc cells from the second instar larval stage onward, cells continued to proliferate. In contrast to the embryonic lethality of another Drosophila lamDm(0) allele, lam(PM15), reported previously, lethality did not occur until late pupal stages. Chromosomal structure and the overall nuclear shape remained normal even at these late pupal stages, although obviously abnormal nuclear pore complex distribution was observed concomitant with the loss of lamin Dm(0) protein. Compensating expression of lamin C was not induced in the absence of lamin Dm(0). Thus, no lamin-containing nuclear structures were found in proliferating larval neuroblasts. We did find that developmental abnormalities appeared in specific organs during the late pupal stage, preceding lethality. Surprisingly, coordinated size increase (hypertrophy) of the ventriculus was observed accompanied by cell division and muscle layer formation. Hypertrophy of the ventriculus correlated with a decrease in ecdysteroid hormone receptor B1 (EcRB1) protein, and furthermore could be suppressed by a heat-inducible EcRB1 transgene. In contrast, both gonadal and CNS tissues exhibited underdevelopment.

Animals↗
PubMed2006

The truncated prelamin A in Hutchinson-Gilford progeria syndrome alters segregation of A-type and B-type lamin homopolymers.

Hutchinson-Gilford progeria syndrome (HGPS) is a dominant autosomal premature aging syndrome caused by the expression of a truncated prelamin A designated progerin (Pgn). A-type and B-type lamins are intermediate filament proteins that polymerize to form the nuclear lamina network apposed to the inner nuclear membrane of vertebrate somatic cells. It is not known if in vivo both type of lamins assemble independently or co-assemble. The blebbing and disorganization of the nuclear envelope and adjacent heterochromatin in cells from patients with HGPS is a hallmark of the disease, and the ex vivo reversal of this phenotype is considered important for the development of therapeutic strategies. Here, we investigated the alterations in the lamina structure that may underlie the disorganization caused in nuclei by Pgn expression. We studied the polymerization of enhanced green fluorescent protein- and red fluorescent protein-tagged wild-type and mutated lamins in the nuclear envelope of living cells by measuring fluorescence resonance energy transfer (FRET) that occurs between the two fluorophores when tagged lamins interact. Using time domain fluorescence lifetime imaging microscopy that allows a quantitative analysis of FRET signals, we show that wild-type lamins A and B1 polymerize in distinct homopolymers that further interact in the lamina. In contrast, expressed Pgn co-assembles with lamin B1 and lamin A to form a mixed heteropolymer in which A-type and B-type lamin segregation is lost. We propose that such structural lamina alterations may be part of the primary mechanisms leading to HGPS, possibly by impairing functions specific for each lamin type such as nuclear membrane biogenesis, signal transduction, nuclear compartmentalization and gene regulation.

Animals↗
PubMed1995

Vimentin-associated mitotic vesicles interact with chromosomes in a lamin B- and phosphorylation-dependent manner.

We have assessed the involvement of the nuclear lamins in nuclear envelope reassembly. Analysis of perforated mitotic cells shows that A-type lamins are partly cytosolic and partly chromosome-bound, whereas B-type lamins are associated with vesicular structures throughout cell division. Lamin B-containing vesicles appear to dock on vimentin intermediate filaments during prometaphase, but dissociate from the cytoskeleton and assemble around chromatin at later phases of mitosis. Mitotic vesicles isolated from prometaphase cells en bloc with vimentin filaments can specifically capture chromosomes. Efficient chromosome capturing requires cytosolic factors and a dephosphorylating environment. Urea-stripping of the vesicles abolishes binding to chromosomes. However, reconstitution of the stripped membranes with purified B-type lamins restores their ability to bind to chromosomes in a cytosol- and dephosphorylation-dependent fashion. Vesicles reconstituted with B-type lamins form membraneous 'crescents' on the surfaces of chromosomes, but, unlike native vesicles, do not fuse into large sheets. From these observations we conclude that the initial targeting of mitotic vesicles to chromosomes is dependent on B-type lamins and on factors present in the mitotic cytoplasm. Apparently, further recruitment of membranes and fusion of chromosome-bound vesicles onto chromatin involves non-lamin peripheral membrane proteins.

Animals↗