Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Hoger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.     

Lamins A and C are not expressed at early stages of human lymphocyte differentiation

Lamins are major proteins of the nuclear envelope that are members of the intermediate filament protein family. In vertebrates, nuclei from differentiated tissues usually contain both lamins of the A and B subtypes, while embryonic tissues contain the B-type lamin only. We have examined the composition of the nuclear lamina in human B and T lymphocytes representative of distinct stages of lymphoid differentiation. We show here that, in both cell lineages, while lamin B is constitutively expressed at all stages of differentiation, A-type lamin expression is restricted to later developmental stages.     

The role of CaaX-dependent modifications in membrane association of Xenopus nuclear lamin B3 during meiosis and the fate of B3 in transfected mitotic cells

Recent evidence shows that the COOH-terminal CaaX motif of lamins is necessary to target newly synthesized proteins to the nuclear envelope membranes. Isoprenylation at the CaaX-cysteine has been taken to explain the different fates of A- and B-type lamins during cell division. A-type lamins, which loose their isoprenylation shortly after incorporation into the lamina structure, become freely soluble upon mitotic nuclear envelope breakdown. Somatic B-type lamins, in contrast, are permanently isoprenylated and, although depolymerized during mitosis, remain associated with remnants of nuclear envelope membranes. However, Xenopus lamin B3, the major B-type lamin of amphibian oocytes and eggs, becomes soluble after nuclear envelope breakdown in meiotic metaphase. Here we show that Xenopus lamin B3 is permanently isoprenylated and carboxyl methylated in oocytes (interphase) and eggs (meiotic metaphase). When transfected into mouse L cells Xenopus lamin B3 is integrated into the host lamina and responds to cell cycle signals in a normal fashion. Notably, the ectopically expressed Xenopus lamin does not form heterooligomers with the endogenous lamins as revealed by a coprecipitation experiment with mitotic lamins. In contrast to the situation in amphibian eggs, a significant portion of lamin B3 remains associated with membranes during mitosis. We conclude from these data that the CaaX motif-mediated modifications, although necessary, are not sufficient for a stable association of lamins with membranes and that additional factors are involved in lamin-membrane binding.     

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 thatXenopus laevis lamin L_II 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.     

Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.     

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.     

Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis

The cell type-specific expression of the major nuclear lamina polypeptides ("lamins") during development of Xenopus was studied using two monoclonal antibodies (L(0)46F7: specific for LIII, the single lamin of oocytes; PKB8: specific for LI and LII of some somatic cells). In the oocyte, LIII localizes in the nuclear polymer, but upon nuclear envelope breakdown it is solubilized to a form sedimenting at 9 S. In early embryos, LIII contributes to nuclear lamina formation until its depletion. Correspondingly, LI and LII begin to be expressed at a specific point in embryogenesis and appear to be integrated with LIII into a common lamina structure. Later in development, LIII reappears as a prominent nuclear lamina protein but only in certain cells (neurons, muscle cells, and diplotene oocytes). We conclude that amphibian lamins represent a family of proteins expressed in relation to certain programs of cell differentiation.     

Characterization of lamin mutation phenotypes in Drosophila and comparison to human laminopathies

Lamins are intermediate filament proteins that make up the nuclear lamina, a matrix underlying the nuclear membrane in all metazoan cells that is important for nuclear form and function. Vertebrate A-type lamins are expressed in differentiating cells, while B-type lamins are expressed ubiquitously. Drosophila has two lamin genes that are expressed in A- and B-type patterns, and it is assumed that similarly expressed lamins perform similar functions. However, Drosophila and vertebrate lamins are not orthologous, and their expression patterns evolved independently. It is therefore of interest to examine the effects of mutations in lamin genes. Mutations in the mammalian lamin A/C gene cause a range of diseases, collectively called laminopathies, that include muscular dystrophies and premature aging disorders. We compared the sequences of lamin genes from different species, and we have characterized larval and adult phenotypes in Drosophila bearing mutations in the lam gene that is expressed in the B-type pattern. Larvae move less and show subtle muscle defects, and surviving lam adults are flightless and walk like aged wild-type flies, suggesting that lam phenotypes might result from neuromuscular defects, premature aging, or both. The resemblance of Drosophila lam phenotypes to human laminopathies suggests that some lamin functions may be performed by differently expressed genes in flies and mammals. Such still-unknown functions thus would not be dependent on lamin gene expression pattern, suggesting the presence of other lamin functions that are expression dependent. Our results illustrate a complex interplay between lamin gene expression and function through evolution.     

Lco1 is a novel widely expressed lamin-binding protein in the nuclear interior

A-type lamins are localized at the nuclear envelope and in the nucleoplasm, and are implicated in human diseases called laminopathies. In a yeast two-hybrid screen with lamin C, we identified a novel widely expressed 171-kDa protein that we named Lamin companion 1 (Lco1). Three independent biochemical assays showed direct binding of Lco1 to the C-terminal tail of A-type lamins with an affinity of 700 nM. Lco1 also bound the lamin B1 tail with lower affinity (2 microM). Ectopic Lco1 was found primarily in the nucleoplasm and colocalized with endogenous intranuclear A-type lamins in HeLa cells. Overexpression of prelamin A caused redistribution of ectopic Lco1 to the nuclear rim together with ectopic lamin A, confirming association of Lco1 with lamin A in vivo. Whereas the major C-terminal lamin-binding fragment of Lco1 was cytoplasmic, the N-terminal Lco1 fragment localized in the nucleoplasm upon expression in cells. Furthermore, full-length Lco1 was nuclear in cells lacking A-type lamins, showing that A-type lamins are not required for nuclear targeting of Lco1. We conclude that Lco1 is a novel intranuclear lamin-binding protein. We hypothesize that Lco1 is involved in organizing the internal lamin network and potentially relevant as a laminopathy disease gene or modifier.     

Nuclear lamin antigens are developmentally regulated during porcine and bovine embryogenesis

The nuclear lamins, proteins that reside on the inner face of the nuclear envelope, are thought to provide attachment sites for anchoring the chromatin to the nuclear envelope, thus facilitating the overall organization of the nucleus. The composition of the nuclear lamin proteins changes during differentiation and development in a variety of mammalian and nonmammalian tissues. Bovine and porcine oocytes and early embryos were prepared for immunocytochemical detection of nuclear lamins using three different antibodies (recognizing lamin B, lamins A/B/C, or lamins A/C). In both species, germinal vesicle nuclei and early cleavage stage nuclei react positively with the antibodies. However, on nuclei of bovine embryos, the A/C epitope was not detectable at the 16-cell stage, compact morula, spherical blastocyst, or the chorionic cell nuclei of a Day 35 conceptus, but was detectable on both amniotic and embryonic ectodermal cell nuclei of a Day 35 conceptus. All three antibodies reacted with nuclei from two bovine tissue culture cell lines (bovine embryonic cells and Madin-Darby bovine kidney cells) and one porcine kidney cell line. Nuclei in porcine embryos followed a similar pattern, except the loss of the A/C epitope occurred at the 8-cell stage and the epitope was absent from compact morula and spherical blastocyst stage nuclei. All interphase nuclei in both species reacted with both anti-lamin A/B/C and anti-lamin B antibodies, whereas metaphase chromosomes did not react with any of the lamin antibodies tested. The change in recognizing the lamin epitope occurred one cell cycle after the expected transition from maternal control to zygotic control of development. Nuclear transplantation showed that 16-cell stage porcine nuclei, which are lamin A/C negative, acquired the A/C epitope after transfer to an enucleated metaphase II oocyte. These results suggest that the A/C epitope is developmentally regulated.     

Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins

Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (?607–656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.     

Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.     

Nuclear lamins are differentially expressed in retinal neurons of the adult rat retina

Lamins are type V intermediate filament proteins that support nuclear membranes. They are divided into A-type lamins, which include lamin A and C, and B-type lamins, which include lamin B1 and B2. In the rat brain, lamin A and C are expressed in relatively equal amounts, while the expressions of lamin B1 and B2 vary depending on the cell type. Lamins play important roles in normal morphogenesis and function. In the nervous system, their abnormal expression causes several neurodegenerative diseases such as peripheral neuropathy, leukodystrophy and lissencephaly. The retina belongs to the central nervous system (CNS) and has widely been used as a source of CNS neurons. We investigated the expression patterns of lamin subtypes in the adult rat retina by immunohistochemistry and found that the staining patterns differed when compared with the brain. All retinal neurons expressed lamin B1 and B2 in relatively equal amounts. In addition, horizontal cells and a subpopulation of retinal ganglion cells expressed lamin A and C, while photoreceptor cells expressed neither lamin A nor C, and all other retinal neurons expressed lamin C only. This differential expression pattern of lamins in retinal neurons suggests that they may be involved in cellular differentiation and expression of cell-specific genes in individual retinal neurons.     

Teratocarcinoma stem cells and early mouse embryos contain only a single major lamin polypeptide closely resembling lamin B

The nuclear lamina in adult mammalian somatic cells is composed of three major proteins, lamins A, B, and C. The expression of these proteins during the differentiation of teratocarcinomas and mouse embryogenesis is described. Embryos up to day 8 of gestation and embryonal carcinoma (EC) cells express only a single lamin species closely resembling, if not identical to, lamin B. Lamins A and/or C were detected in fertilized eggs, but disappear during the first 2-4 cleavage divisions, only reappearing in 8 day post-implantation embryos. These two lamins are absent from EC cells, but are strongly expressed in some of their derivatives. These results show that cells of the early mouse embryo do not have a functional requirement for lamins A and C and imply that the structural organization of the nucleus may change fundamentally during embryogenesis.     

Detection of nuclear lamin B epitopes in oocyte nuclei from mice, sea urchins, and clams using a human autoimmune serum

Somatic nuclei typically contain two or three major proteins, the lamins A, B, and C or their antigenically related equivalents, interspersed between the chromatin and its attachment site, the inner nuclear membrane. The late oocyte nuclear envelopes of the previously investigated Xenopus and Spisula germinal vesicles, however, have no chromatin attached and only one lamin-like protein. Since mouse and sea urchin germinal vesicles have chromatin attached, we tested them for the possible presence of more than one lamin. In both species we found two different lamins incorporated in their nuclear envelope structure. One lamin is recognized by anti-lamin B and the other by anti-lamin AC antibodies. Spisula germinal vesicles were found to contain not only the nuclear envelope-bound lamin (clamin), but also a 65-kDa protein cross-reactive with anti-lamin B antibodies. This protein is present unattached to any structure and is apparently soluble. Our findings provide a possible explanation of the early presence of lamin B in pronuclei of mouse and sea urchin contrary to the late appearance of a lamin B equivalent in amphibian embryos. In Spisula, as in Xenopus, the presence of a lamin B equivalent could not be documented in the nuclear envelopes of early embryos, indicating that a separate lamin B equivalent is not essential for chromatin binding to the envelope in these species during early embryogenesis. The results also indicate that the nuclear complement of structural proteins might vary substantially in the same cell type of different species.     

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.     

The gene structure ofXenopus 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 ofXenopus 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. aras-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.by H. Jäckle     

A new lamin in Xenopus somatic tissues displays strong homology to human lamin A

The nuclear lamina of vertebrates is composed of several major polypeptides that range in mol. wt from 60 to 80 kd. In mammals, the three major lamin proteins are designated A, B and C. Two major lamins have been described in Xenopus somatic tissues; two other lamins are expressed primarily in germ cells. We have analysed a cDNA clone encoding a Xenopus lamin that is highly homologous to human lamins A and C. The predicted protein has the carboxy-terminal domain characteristic of human lamin A and is thus a lamin A homologue. Surprisingly, the lamin encoded by the cDNA clone is not one of the known Xenopus lamins. The encoded protein is distinct in size from the oocyte lamin LIII and the two somatic lamins LI and LII. Monoclonal antibodies specific for LII, LIII and LIV (the lamin of male germ cells) do not recognize the protein encoded by the cDNA clone; conversely, a polyclonal antibody against the encoded protein does not recognize any of the known Xenopus lamins. This lamin is expressed late in embryonic development, and is present in all adult somatic cells examined, except erythrocytes. Thus frogs and mammals are similar in having three major somatic lamins that fall into distinct structural classes.     

A Comparative Study of Drosophila and Human A-Type Lamins

Nuclear intermediate filament proteins, called lamins, form a meshwork that lines the inner surface of the nuclear envelope. Lamins contain three domains: an N-terminal head, a central rod and a C-terminal tail domain possessing an Ig-fold structural motif. Lamins are classified as either A- or B-type based on structure and expression pattern. The Drosophila genome possesses two genes encoding lamins, Lamin C and lamin Dm₀, which have been designated A- and B-type, respectively, based on their expression profile and structural features. In humans, mutations in the gene encoding A-type lamins are associated with a spectrum of predominantly tissue-specific diseases known as laminopathies. Linking the disease phenotypes to cellular functions of lamins has been a major challenge. Drosophila is being used as a model system to identify the roles of lamins in development. Towards this end, we performed a comparative study of Drosophila and human A-type lamins. Analysis of transgenic flies showed that human lamins localize predictably within the Drosophila nucleus. Consistent with this finding, yeast two-hybrid data demonstrated conservation of partner-protein interactions. Drosophila lacking A-type lamin show nuclear envelope defects similar to those observed with human laminopathies. Expression of mutant forms of the A-type Drosophila lamin modeled after human disease-causing amino acid substitutions revealed an essential role for the N-terminal head and the Ig-fold in larval muscle tissue. This tissue-restricted sensitivity suggests a conserved role for lamins in muscle biology. In conclusion, we show that (1) localization of A-type lamins and protein-partner interactions are conserved between Drosophila and humans, (2) loss of the Drosophila A-type lamin causes nuclear defects and (3) muscle tissue is sensitive to the expression of mutant forms of A-type lamin modeled after those causing disease in humans. These studies provide new insights on the role of lamins in nuclear biology and support Drosophila as a model for studies of human laminopathies involving muscle dysfunction.