Proteins that bind A-type lamins: integrating isolated clues.
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What do such diverse molecules as DNA, actin, retinoblastoma protein and protein kinase Calpha all have in common? They and additional partners bind 'A-type' lamins, which form stable filaments in animal cell nuclei. Mutations in A-type lamins cause a bewildering range of tissue-specific diseases, termed 'laminopathies', including Emery-Dreifuss muscular dystrophy and the devastating Hutchinson-Gilford progeria syndrome, which mimics premature aging. Considered individually and collectively, partners for A-type lamins form four loose groups: architectural partners, chromatin partners, gene-regulatory partners and signaling partners. We describe 16 partners in detail, summarize their binding sites in A-type lamins, and sketch portraits of ternary complexes and functional pathways that might depend on lamins in vivo. On the basis of our limited current knowledge, we propose lamin-associated complexes with multiple components relevant to nuclear structure (e.g. emerin, nesprin 1alpha, actin) or signaling and gene regulation (e.g. LAP2alpha, retinoblastoma, E2F-DP heterodimers, genes) as 'food for thought'. Testing these ideas will deepen our understanding of nuclear function and human disease. (+info)
Disruption of spermatogenesis in mice lacking A-type lamins.
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Nuclear lamins are structural protein components of the nuclear envelope. Mutations in LMNA, the gene coding for A-type lamins, result in several human hereditary diseases, the laminopathies, which include Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, familial partial lipodystrophy and Hutchinson-Gilford progeria. Similar to the human conditions, it has been shown that Lmna(-/-) mice develop severe dystrophies of muscle and fat tissues. Here we report that Lmna(-/-) mice display impaired spermatogenesis, with a significant accumulation of spermatocytes I during early prophase I stages, while pachytene spermatocytes are severely defective in synaptic pairing of the sex chromosomes in particular, leading to massive apoptosis during the pachytene stage of meiosis I. In contrast, oogenesis remains largely unaffected in Lmna(-/-) mice. These results reveal A-type lamins as important determinants of male fertility. (+info)
A lamin A/C beta-strand containing the site of lipodystrophy mutations is a major surface epitope for a new panel of monoclonal antibodies.
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Using a phage-displayed peptide library, we have identified the epitope recognized by a new panel of five monoclonal antibodies (mAbs) raised against full-length recombinant human lamin A. The mAbs were found to recognize both lamin A and C by Western blotting and immunolocalization at the nuclear rim. A nine-amino acid consensus sequence PLLTYRFPP in the common immunoglobulin-like (Ig-like) domain of lamin A/C contains the binding site for all five mAbs. Three-dimensional structure of the Ig-like domain of lamin A/C shows this sequence is a complete beta-strand. This sequence includes arginine-482 (R482) which is mutated in most cases of Dunnigan-type familial partial lipodystrophy (FPLD). R482 may be part of an interaction site on the surface of lamin A/C for lamin-binding proteins associated with lipodystrophy. (+info)
Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation.
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BACKGROUND: The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene. RESULTS: Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates. CONCLUSIONS: From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that a rearrangement of the internal chromatin could lead to muscle-specific disease symptoms by interference with proper mRNA transcription. (+info)
A novel mutation, Ser143Pro, in the lamin A/C gene is common in finnish patients with familial dilated cardiomyopathy.
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AIMS: The mutations most frequently associated with dilated cardiomyopathy (DCM) have been reported in the lamin A/C gene. The role of variants of the lamin A/C gene was investigated in patients with DCM from eastern and southern Finland. METHODS AND RESULTS: All 12 exons of the lamin A/C gene were screened in 18 well-characterised familial DCM patients from eastern and southern Finland and in 72 sporadic DCM patients from eastern Finland using the PCR-SSCP method. A novel mutation, Ser143Pro (S143P), was detected in the lamin A/C gene in 24 subjects from 5 unrelated families and in one sporadic case of DCM. Sinus or atrioventricular nodal dysfunction occurred in the majority of the affected subjects, many of which required pacemaker implantation. Seven patients (28%) with the S143P mutation died suddenly or from progressive heart failure, or underwent heart transplantation. The haplotypes 5-5-5-3, 5-5-5-2, and 5-5-5-1 co-segregated with the Ser143Pro mutation, suggesting a founder effect of this mutation. CONCLUSIONS: A novel mutation S143P in the lamin A/C gene was found to be common among Finnish DCM patients. Haplotype analysis strongly suggests a founder effect of this mutation. The phenotype is characterised by severe heart failure, progressive atrioventricular conduction defects, and sudden death. Screening for the lamin A/C gene and, particularly, the S143P mutation seems warranted when patients with DCM have conduction system disturbances. (+info)
Conformational changes in the nuclear lamina induced by herpes simplex virus type 1 require genes U(L)31 and U(L)34.
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The herpes simplex virus type 1 (HSV-1) U(L)31 and U(L)34 proteins are dependent on each other for proper targeting to the nuclear membrane and are required for efficient envelopment of nucleocapsids at the inner nuclear membrane. In this work, we show that whereas the solubility of lamins A and C (lamin A/C) was not markedly increased, HSV induced conformational changes in the nuclear lamina of infected cells, as viewed after staining with three different lamin A/C-specific antibodies. In one case, reactivity with a monoclonal antibody that recognizes an epitope in the lamin tail domain was greatly reduced in HSV-infected cells. This apparent HSV-induced epitope masking required both U(L)31 and U(L)34, but these proteins were not sufficient to mask the epitope in uninfected cells, indicating that other HSV proteins are also required. In the second case, staining with a rabbit polyclonal antibody that primarily recognizes epitopes in the lamin A/C rod domain revealed that U(L)34 is required for HSV-induced decreased availability of epitopes for reaction with the antibody, whereas U(L)31 protein was dispensable for this effect. Still another polyclonal antibody indicated virtually no difference in lamin A/C staining in infected versus uninfected cells, indicating that the HSV-induced changes are more conformational than the result of lamin depletion at the nuclear rim. Further evidence supporting an interaction between the nuclear lamina and the U(L)31/U(L)34 protein complex includes the observations that (i) overexpression of the U(L)31 protein in uninfected cells was sufficient to relocalize lamin A/C from the nuclear rim into nucleoplasmic aggregates, (ii) overexpression of U(L)34 was sufficient to relocalize some lamin A/C into the cytoplasm, and (iii) both U(L)31 and U(L)34 could directly bind lamin A/C in vitro. These studies suggest that the U(L)31 and U(L)34 proteins modify the conformation of the nuclear lamina in infected cells, possibly by direct interaction with lamin A/C, and that other proteins are also likely involved. Given that the nuclear lamina potentially excludes nucleocapsids from envelopment sites at the inner nuclear membrane, the lamina alteration may reflect a role of the U(L)31/U(L)34 protein complex in perturbing the lamina to promote nucleocapsid egress from the nucleus. Alternatively, the data are compatible with a role of the lamina in targeting the U(L)31/U(L)34 protein complex to the nuclear membrane. (+info)
Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome.
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Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication. (+info)
Cell-type-specific interactions at regulatory motifs in the first intron of the lamin A gene.
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Lamins A, C and C2 are alternatively spliced products of the LMNA gene; lamins A and C are expressed in differentiated somatic cells, whereas lamin C2 is expressed in germ cells. We have analyzed a segment of the first intron of the LMNA gene for cell-type-specific regulatory elements. We identified a 420-bp fragment that increased promoter activity in lamin A-expressing cells but repressed activity in undifferentiated cells. DNase I footprinting and electrophoretic mobility shift assays revealed two binding motifs, footprinted region A (FPRA) and FPRB. The hepatocyte nuclear factor-3beta was bound to FPRA only in somatic cell extracts and this motif had an inhibitory effect on promoter activity. The retinoic X receptor beta, RXRbeta, bound near FPRB with extracts from lamin A- or C2-expressing cells, and this site enhanced promoter activity. We have, thus, identified two novel binding sites for transcription factors in a region likely to function as an important regulatory element for the cell-type-specific transcription of A-type lamins. (+info)