cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins.

The amino acid sequences deduced from cDNA clones of human lamin A and lamin C show identity between these two lamins except for an extra 9.0-kDa carboxyl-terminal tail that is present only in lamin A. Both lamins A and C contain an alpha-helical domain of approximately 360 residues that shows striking homology to a corresponding alpha-helical rod domain that is the structural hallmark of all intermediate filament proteins. However, the lamin alpha-helical domain is 14% larger than that of the intermediate filament proteins. In addition to the extensive homology to intermediate filament proteins as reported [McKeon, F., Kirschner, M. & Caput, D. (1986) Nature (London) 319, 463-468], a different 82-amino acid residue stretch at the carboxyl terminus of lamin A has been deduced and verified by amino acid sequencing. This region contains sequence homology to amino- and carboxylterminal domains of type I and type II epidermal keratins. Implications of the presence of these and other domains in lamins A and C for the assembly of the nuclear lamina are discussed.     

Lamin B1 is required for mouse development and nuclear integrity

Lamins are key structural components of the nuclear lamina, an intermediate filament meshwork that lies beneath the inner nuclear membrane. Lamins play a role in nuclear architecture, DNA replication, and gene expression. Mutations affecting A-type lamins have been associated with a variety of human diseases, including muscular dystrophy, cardiomyopathy, lipodystrophy, and progeria, but mutations in B-type lamins have never been identified in humans or in experimental animals. To investigate the in vivo function of lamin B1, the major B-type lamin, we generated mice with an insertional mutation in Lmnb1. The mutation resulted in the synthesis of a mutant lamin B1 protein lacking several key functional domains, including a portion of the rod domain, the nuclear localization signal, and the CAAX motif (the carboxyl-terminal signal for farnesylation). Homozygous Lmnb1 mutant mice survived embryonic development but died at birth with defects in lung and bone. Fibroblasts from mutant embryos grew under standard cell-culture conditions but displayed grossly misshapen nuclei, impaired differentiation, increased polyploidy, and premature senescence. Thus, the lamin B1 mutant mice provide evidence for a broad and nonredundant function of lamin B1 in mammalian development. These mutant mice and cell lines derived from them will be useful models for studying the role of the nuclear lamina in various cellular processes.     

A progeria mutation reveals functions for lamin A in nuclear assembly,architecture, and chromosome organization

Numerous mutations in the human A-type lamin gene (LMNA) cause the premature aging disease, progeria. Some of these are located in the α-helical central rod domain required for the polymerization of the nuclear lamins into higher order structures. Patient cells with a mutation in this domain, 433G>A (E145K) show severely lobulated nuclei, a separation of the A- and B-type lamins, alterations in pericentric heterochromatin, abnormally clustered centromeres, and mislocalized telomeres. The induction of lobulations and the clustering of centromeres originate during postmitotic nuclear assembly in daughter cells and this early G1 configuration of chromosomes is retained throughout interphase. In vitro analyses of E145K-lamin A show severe defects in the assembly of protofilaments into higher order lamin structures. The results show that this central rod domain mutation affects nuclear architecture in a fashion distinctly different from the changes found in the most common form of progeria caused by the expression of LAΔ50/progerin. The study also emphasizes the importance of lamins in nuclear assembly and chromatin organization.     

Antibodies to nuclear lamin C in chronic hepatitis delta virus infection

Sera of patients with chronic hepatitis delta virus infection stained the nuclear periphery in indirect immunofluorescence. Using proteins of isolated nuclei, isolated nuclear matrices, the nuclear pore complex-lamina fraction and purified lamins A and C as antigen source in immunoblotting experiments, nuclear lamin C was identified as the reactive antigen. Most sera tested (8 of 10) recognized nuclear lamin C exclusively, but not the nuclear lamins A and B. Antibodies reacting with both nuclear lamins A and C, which share extensive sequence homologies, have been reported to occur in autoimmune hepatitis and primary biliary cirrhosis. The present findings suggest that the novel autoantibody associated with chronic hepatitis delta virus infection recognizes an epitope localized in the short carboxyterminal region of nuclear lamin C.     

Decreased and aberrant nuclear lamin expression in gastrointestinal tract neoplasms

BACKGROUND: Altered expression of lamins A/C and B1, constituent proteins of the nuclear lamina, may occur during differentiation and has also been reported in primary lung cancer. AIMS: To examine the expression of these proteins in gastrointestinal neoplasms. PATIENTS: Archival human paraffin wax blocks and frozen tissue from patients undergoing surgical resection or endoscopic biopsy. METHODS: Immunohistochemistry and western blotting using polyclonal antisera against A type lamins and lamin B1. RESULTS: The expression of lamin A/C was reduced and was frequently undetectable by immunohistochemistry in all primary colon carcinomas and adenomas, and in 7/8 primary gastric cancers. Lamin B1 expression was reduced in all colon cancers, 16/18 colonic adenomas, and 6/8 gastric cancers. Aberrant, cytoplasmic labelling with both antibodies occurred in some colonic cancers and around one third of colonic adenomas. Cytoplasmic lamin A/C expression was detected in 3/8 gastric cancers. Lamin expression was reduced in gastric dysplasia, but not intestinal metaplasia, atrophy, or chronic gastritis. Lamin expression was low in carcinomas of oesophagus, prostate, breast, and uterus, but not pancreas. CONCLUSIONS: Reduced expression of nuclear lamins, sometimes together with aberrant, cytoplasmic immunoreactivity is common in gastrointestinal neoplasms. Altered lamin expression may be a biomarker of malignancy in the gastrointestinal tract.     

Autoimmune response directed against conserved determinants of nuclear envelope proteins in a patient with linear scleroderma.

We have studied the autoantibodies in the serum of a patient with linear scleroderma that specifically recognize the nuclear envelope of cultured cells. These antibodies bind to conserved determinants of nuclear lamins, the predominant mammalian nuclear envelope proteins. Of the three mammalian nuclear lamin proteins (970, P68, and P60), only P70 and P60 bind the autoantibodies. In addition, two proteins of the Drosophila embryonic nuclear matrix, P70 and P68, bind these autoantibodies. We have used nuclear matrices to isolate the autoantibodies from the patient's serum that react to the nuclear lamins. At least three different IgG heavy chains were found to be involved in this autoimmune response to nuclear lamins, indicating that this response is not due to the expansion of a single B-cell clone.     

Biogenesis of the nuclear lamina: in vivo synthesis and processing of nuclear protein precursors.

Utilizing antibodies against lamins A, B1, and B2, we have studied the biogenesis of the nuclear lamina in chicken embryo fibroblasts. (Lamins B1 and B2 have been identified recently as structurally distinct "lamin B" proteins.) We demonstrate that, unique among the nuclear proteins studied to date, lamin A is synthesized as a higher molecular mass precursor. A short-lived higher molecular mass variant (t 1/2 approximately equal to 3 min) accompanying the mature-size protein was also detected in the case of lamin B2 biosynthesis, but no precursor was found for lamin B1. By combining pulse-chase experiments with subcellular fractionation, we provide evidence that synthesis of lamin proteins occurs on free polysomes; subsequently, the newly synthesized proteins become rapidly associated with a crude nuclear fraction. The lamin A precursor is processed within the nucleus with a half-time of about 30 min. Concomitantly, lamin proteins acquire a characteristic resistance to detergent extraction, suggesting their insertion into a submembraneous protein network. The described biogenetic pathway involving precursor synthesis and processing is very unusual for nuclear proteins; it may have interesting implications for the mechanisms of transport and assembly of poorly soluble nuclear proteins.     

Genomic instability and innate immune responses to self-DNA in progeria

In the last decade, we have seen increasing evidence of the importance of structural nuclear proteins such as lamins in nuclear architecture and compartmentalization of genome function and in the maintenance of mechanical stability and genome integrity. With over 400 mutations identified in the LMNA gene (encoding for A-type lamins) associated with more than ten distinct degenerative disorders, the role of lamins as genome caretakers and the contribution of lamins dysfunction to disease are unarguable. However, the molecular mechanisms whereby lamins mutations cause pathologies remain less understood. Here, we review pathways and mechanisms recently identified as playing a role in the pathophysiology of laminopathies, with special emphasis in Hutchinson Gilford Progeria Syndrome (HGPS). This devastating incurable accelerated aging disease is caused by a silent mutation in the LMNA gene that generates a truncated lamin A protein “progerin” that exerts profound cellular toxicity and organismal decline. Patients usually die in their teens due to cardiovascular complications such as myocardial infarction or stroke. To date, there are no efficient therapies that ameliorate disease progression, stressing the need to understand molecularly disease mechanisms that can be targeted therapeutically. We will summarize data supporting that replication stress is a major cause of genomic instability in laminopathies, which contributes to the activation of innate immune responses to self-DNA that in turn accelerate the aging process.

     

Heterotypic and homotypic associations between the nuclear lamins: site-specificity and control by phosphorylation.

Using purified components in affinity chromatography and blot binding assays, we have found that rat liver lamins A, B, and C can associate in homotypic and heterotypic fashions. Heterotypic A-B and C-B complexes are unusually stable and involve the common amino-terminal domain of lamins A and C, but not their helical "rod" domain. A synthetic peptide, comprising the first 32 amino acid residues of lamins A and C, is able to fully compete with the intact molecules for binding to lamin B. Conversely, heterotypic A-C associations and homotypic A-A and C-C interactions appear significantly weaker than A/C-B binding and do not involve the lamin A and C amino-terminal domain. Homotypic B-B complexes are not formed to any considerable extent unless isolated lamin B subunits are "superphosphorylated" in vitro with protein kinase A. However, when lamins A and C are similarly modified, no changes in their binding specificity can be detected. These data suggest that the nuclear lamina, unlike other multicomponent intermediate filaments, constitutes a nonobligatory heteropolymer. They also indicate that cAMP-dependent phosphorylation of interphase lamin B could cause remodeling of the lamina and establishment of homopolymeric domains.     

Change of karyoskeleton during spermatogenesis of Xenopus: expression of lamin LIV, a nuclear lamina protein specific for the male germ line.

Lamins are the major constituent proteins of the nuclear lamina. In the frog, Xenopus laevis, they are the products of a multigene family whose expression can be correlated to certain routes of cell differentiation. For example, lamins LI (Mr, 72,000) and LII (Mr, 68,000) is expressed, together with LI/LII, in certain highly differentiated cell types such as neurons and muscle cells and is the only lamin present in diplotene oocytes. Here we report the identification by means of two monoclonal antibodies of a fourth lamin (LIV) of Mr 75,000, which is expressed specifically during the later stages of spermatogenesis. In the seminiferous tubules, Sertoli cells contain LI/LII and LIII whereas, among the spermatogenic cells, spermatogonia contain only LI and LII. In contrast, in spermatids and sperm cells these lamins are completely replaced by lamin LIV. Primary spermatocytes are negative with both antibodies, indicating that a switch in the expression of lamins occurs early in spermatogenesis. Lamin LIV is distributed in patches along the nuclear envelopes of elongated spermatids and sperm cells rather than in the characteristic continuous lamina pattern found in most other cells. We hypothesize that the specific expression of lamin LIV is related to the conspicuous changes of nuclear architecture and chronmatin composition that are known to take place during the late stages of sperm development.     

The tail domain of lamin Dm0 binds histones H2A and H2B

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm0 were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0 tail domain. The apparent Kd of binding of the lamin Dm0 tail domain was found to be approximately 1 microM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm0 tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm0 tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo.     

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins.

Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.     

Nesprin-1 mutations in human and murine cardiomyopathy

Mutations in LMNA, the gene encoding the nuclear membrane proteins, lamins A and C, produce cardiac and muscle disease. In the heart, these autosomal dominant LMNA mutations lead to cardiomyopathy frequently associated with cardiac conduction system disease. Herein, we describe a patient with the R374H missense variant in nesprin-1α, a protein that binds lamin A/C. This individual developed dilated cardiomyopathy requiring cardiac transplantation. Fibroblasts from this individual had increased expression of nesprin-1α and lamins A and C, indicating changes in the nuclear membrane complex. We characterized mice lacking the carboxy-terminus of nesprin-1 since this model expresses nesprin-1 without its carboxy-terminal KASH domain. These Δ/ΔKASH mice have a normally assembled but dysfunctional nuclear membrane complex and provide a model for nesprin-1 mutations. We found that Δ/ΔKASH mice develop cardiomyopathy with associated cardiac conduction system disease. Older mutant animals were found to have elongated P wave duration, elevated atrial and ventricular effective refractory periods indicating conduction defects in the myocardium, and reduced fractional shortening. Cardiomyocyte nuclei were found to be elongated with reduced heterochromatin in the Δ/ΔKASH hearts. These findings mirror what has been described from lamin A/C gene mutations and reinforce the importance of an intact nuclear membrane complex for a normally functioning heart.     

Protein kinase activity associated with the nuclear lamina.

A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on serine residues. Their two-dimensional phosphopeptide maps show multiple phosphorylation sites and a considerable similarity to the phosphopeptide maps of lamins labeled in vivo. Photoaffinity labeling experiments revealed several polypeptide fractions in the nuclear lamina fraction that are candidates for the protein kinase(s).     

Some HIV protease inhibitors alter lamin A/C maturation and stability, SREBP-1 nuclear localization and adipocyte differentiation

OBJECTIVES: To study whether HIV protease inhibitors could induce nuclear lamina alterations in adipocytes as observed in a genetic form of lipodystrophy due to lamin A/C mutation. DESIGN: We have previously observed that indinavir (IDV) impairs adipocyte differentiation and sterol regulatory element-binding protein-1 (SREBP-1) nuclear localization in 3T3-F442A adipocytes. We compared here the effects of IDV with that produced by two other PIs, nelfinavir (NFV) and amprenavir (APV) on adipose conversion, cellular localization of SREBP-1, nuclear morphology, and maturation and stability of the lamina network. RESULTS: IDV and NFV, but not APV, altered adipose cell differentiation, as shown by lipid staining and protein expression of SREBP-1, CAAAT/enhancer binding protein (C/EBP)alpha and fatty acid synthase (FAS). In IDV-treated cells, 50-60 % of the nuclei could not accumulate SREBP-1. Twenty percent of these SREBP-negative nuclei were grossly dysmorphic, with blebs and prominent herniations, and showed an altered distribution of lamin A/C and lamin B. In IDV-treated cells, nuclear fragilization was shown by the abnormal extractibility of lamina proteins and SREBP-1, and the accumulation of prelamin A. NFV similarly altered lamin A/C maturation whereas APV was almost ineffective. CONCLUSIONS: We show in an adipose cell line that IDV and NFV induced alterations at the nuclear level by promoting defects in lamin A/C maturation, organization and stability. We suggest that these lamina network alterations might be responsible for SREBP-1 nuclear mislocalization therefore resulting in altered adipocyte differentiation.     

Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae.

We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin B was correctly targeted in yeast. Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane. Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks. Cell fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes. The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins. The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane. The observed colocalization and cofractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast.