Cytoplasmic intermediate filament proteins and the nuclear lamins A, B and C share the IFA epitope

The murine monoclonal antibody IFA isolated by Pruss et al. (Cell 27 (1981) 419) reacts with all major proteins of the cytoplasmic intermediate filament family (IF) albeit with different affinities but leaves the nucleus undecorated in standard immunofluorescence microscopy. Here we show that IFA reacts with all three nuclear lamins from rat and man in immunoblotting. This is most easily demonstrated in a cell line in which most cells lack cytoplasmic IFs. Thus the rather minor but ubiquitous 66 kD polypeptides identified by Pruss et al. as IF-associated proteins reflect the lamin triplet. While surprising at first, these results are in agreement with the approximate location of the IFA epitope on IF molecules and the recently discovered sequence homology along the rod domain between lamins A and C and IF proteins. Our results extend this relation to lamin B in spite of its unique behaviour during mitosis.     

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.     

Amino acid sequences and homopolymer-forming ability of the intermediate filament proteins from an invertebrate epithelium.

Intermediate filaments (IF) isolated from the oesophagus epithelium of the snail Helix pomatia contain two polypeptides of mol. wt 66,000 (A) and 52,000 (B), which we have now characterized by in vitro self-assembly studies and by protein sequences. A and B can each form morphologically normal IF and share extended regions of sequence identity. All A-specific sequences seem to locate to an extension of the carboxyl-terminal domain. Although the Helix protein(s) reveal the IF-consensus sequences at the ends of the coiled-coil, the remainder of the rod domain shows conservation of sequence principles rather than extended homology, when compared with any subtype of vertebrate IF proteins. Interestingly, the Helix proteins have the longer coil 1b domain found in nuclear lamins and not in cytoplasmic IF proteins of vertebrates. They lack, however, the karyophilic signal sequence typical for lamins. Obvious implications for IF evolution and structure are discussed.     

Molecular Phylogeny of Metazoan Intermediate Filament Proteins

We have cloned cytoplasmic intermediate filament (IF) proteins from a large number of invertebrate phyla using cDNA probes, the monoclonal antibody IFA, peptide sequence information, and various RT-PCR procedures. Novel IF protein sequences reported here include the urochordata and nine protostomic phyla, i.e., Annelida, Brachiopoda, Chaetognatha, Echiura, Nematomorpha, Nemertea, Platyhelminthes, Phoronida, and Sipuncula. Taken together with the wealth of data on IF proteins of vertebrates and the results on IF proteins of Cephalochordata, Mollusca, Annelida, and Nematoda, two IF prototypes emerge. The L-type, which includes 35 sequences from 11 protostomic phyla, shares with the nuclear lamins the long version of the coil 1b subdomain and, in most cases, a homology segment of some 120 residues in the carboxyterminal tail domain. The S-type, which includes all four subfamilies (types I to IV) of vertebrate IF proteins, lacks 42 residues in the coil 1b subdomain and the carboxyterminal lamin homology segment. Since IF proteins from all three phyla of the chordates have the 42-residue deletion, this deletion arose in a progenitor prior to the divergence of the chordates into the urochordate, cephalochordate, and vertebrate lineages, possibly already at the origin of the deuterostomic branch. Four phyla recently placed into the protostomia on grounds of their 18S rDNA sequences (Brachiopoda, Nemertea, Phoronida, and Platyhelminthes) show IF proteins of the L-type and fit by sequence identity criteria into the lophotrochozoic branch of the protostomia. Received: 2 April 1998 / Accepted: 19 June 1998     

Lamin B shares a number of distinct epitopes with lamins A and C and with intermediate filament proteins

Four monoclonal antibodies raised against rat liver nuclear lamins and an anti-intermediate filament antibody [Pruss, R. M., Mirsky, R., & Raff, M. C. (1981) Cell (Cambridge, Mass.) 27, 419-428] have been used to identify epitopes shared by lamin B with lamins A and C, and with intermediate filament proteins. The antibodies defined two major antigenic regions on the three lamins which were both homologous with mouse epidermal keratins as well as hamster vimentin and desmin. Three distinguishable epitopes shared by lamin B with lamins A and C were identified by competition studies between pairs of antibodies and by reaction against N-chlorosuccinimide and cyanogen bromide cleavage fragments. These results support the hypothesis that lamin B, despite important biochemical differences with lamins A and C, shares with them some of the structural characteristics typical of intermediate filament proteins.     

Nuclear lamin LI of Xenopus laevis: cDNA cloning, amino acid sequence and binding specificity of a member of the lamin B subfamily.

Lamins are karyoskeletal proteins associated with the nuclear envelope which can be divided into two groups, i.e. the type A lamins of near neutral pI and the more acidic lamins, including mammalian lamin B. We have isolated cDNA clones encoding a representative of the type B subfamily from Xenopus laevis, and have deduced its amino acid sequence from the coding portion of the approximately 2.9 kb mRNA. The polypeptide (mol. wt 66,433) is identified as a typical lamin by its homology to Xenopus human type A lamins, but detailed sequence comparison shows that LI is less related to Xenopus lamin A than the latter is to human lamin A. The conformation predicted for LI conforms to the general model of lamins and intermediate filament proteins and is characterized by an extended central alpha-helical coiled coil domain, flanked by non-alpha-helical domains, i.e. a relatively short N-terminal head and a long C-terminal tail. As in lamins A and C, the head of lamin LI is positively charged and the tail presents a similar C-terminal pentapeptide, a putative nuclear accumulation signal, a very negatively charged region and a number of short regions that are highly homologous in all lamins. However, LI differs from the type A lamins by the absence of the oligo-histidine stretch and a di-proline motif in the tail region and by a significantly lower number of identical amino acid positions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence and molecular characterization of murine lamin B as deduced from cDNA clones

The nuclear lamina is the karyoskeletal structure, intimately associated with the nuclear envelope, that is widespread among the diverse types of eukaryotic cells. A family of proteins, termed lamins, has been shown to be a prominent component of this lamina, and various members of this family are differentially expressed in different cell types. In mammals, three major lamins (A, B, C) have been identified, and in all cells so far examined lamin B is constitutively expressed while lamins A and C are not, suggesting that lamin B is sufficient to form a functional lamina. Because of this key importance of lamin B, cDNA clones encoding mammalian lamin B were isolated by screening murine cDNA libraries, representing F9 teratocarcinoma cells and fetal liver, with the corresponding cDNA probe of lamin LI of Xenopus laevis. The nucleotide sequence of the murine lamin B mRNA (approximately 2.9 kb) was determined. The deduced amino acid sequence of the encoded polypeptide (587 amino acids; mol. wt. 66760) is highly homologous to X. laevis lamin LI (72.9% identical residues) but displays lower similarity to A-type lamins (53.8% identical amino acid residues with human lamin A). Lamin B also conforms to the general molecular organization principle of the members of the intermediate filament (IF) protein family, i.e., an extended alpha-helical rod domain that is interrupted by two non alpha-helical linkers and flanked by non-alpha-helical head (amino-terminal) and tail (carboxy-terminal) domains. The tail domain, which does not reveal a hydrophobic region of considerable length, contains a typical karyophilic signal sequence and an uninterrupted stretch of eight negatively charged amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The "lamin B-fold". Anti-idiotypic antibodies reveal a structural complementarity between nuclear lamin B and cytoplasmic intermediate filament epitopes.

Previous studies have shown that nuclear lamin B binds specifically to the C-terminal domains of type III intermediate filament (IF) proteins under in vitro conditions. To further explore such site-specific interactions, we have used a two-step anti-idiotypic antibody approach. First, a monoclonal antibody disrupting the cytoplasmic IF network organization of living cells (mAb7A3) (Matteoni, R., and Kreis, T. E. (1987) J. Cell Biol. 105, 1253-1265) was characterized. Epitope mapping demonstrated that this antibody recognized a site located in the C-terminal domains of vimentin and peripherin (type III IF proteins). mAb7A3 was able to inhibit more than 80% of the in vitro binding of nuclear lamin B to PI, a synthetic peptide modeled after the C-terminal domain of peripherin that comprises a lamin B-binding site (Djabali, K., Portier, M. M., Gros, F., Blobel, G., and Georgatos, S. D. (1991) Cell 64, 109-121). In a second step, animals were immunized with mAb7A3 and the resulting anti-idiotypic sera were screened. Two of these antisera reacted specifically with nuclear lamin B but not with type A lamins or cytoplasmic IF proteins. The anti-lamin B activity of one of the antisera was isolated by affinity chromatography using a lamin B-agarose matrix. The reaction of these affinity-purified antibodies with lamin B was inhibited by mAb7A3. Furthermore, the anti-lamin B antibodies reacted with Fab fragments of mAb7A3 and abolished binding of lamin B to PI. From these data we conclude that anti-idiotypic antibodies against the paratope of mAb7A3 recognize specific epitopes of the lamin B molecule that have shapes complementary to the one of the C-terminal domain of type III IF proteins. We speculate that these (regional) conformations, which we term the "lamin B-fold," may also occur in non-lamin proteins that mediate the anchorage of IFs to various membranous organelles.     

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.     

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.     

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.     

Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments

The mechanism by which nuclear and cytoplasmic filaments are sorted in vivo was studied by examining which lamin sequences are required to target an otherwise cytoplasmic IF protein, the small neurofilament subunit (NF-L), to the nuclear lamina. By swapping corresponding domains between NF-L and lamin A, nuclear envelope targeting of NF-L was shown to require the presence of the "head" domain, a 42-amino acid sequence unique to lamin rod domains, a nuclear localization signal and the CAAX motif. Replacement of the entire COOH-terminal tail of lamin A with that of NF-L had no discernible effect on nuclear localization of lamin A, provided the substituted NF-L tail contained a NLS and a CAAX motif. This chimeric protein exhibited characteristics more typical of lamin B than that of the parental lamin A. With regard to cytoplasmic assembly properties, substitution of the head domain of lamin A for that of NF-L did not substantially affect the ability of NF-L to coassemble with vimentin in the cytoplasm. In contrast, insertion of a 42-amino acid sequence unique to lamin rod domains into NF-L profoundly affected NF-L coassembly with vimentin indicating that the 42-amino acid insertion in lamins may be important for sorting lamins from cytoplasmic IF proteins.     

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.     

Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A

The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.     

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.     

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.     

Patterns of nuclear lamins in diverse animal and plant cells and in germ cells as revealed by immunofluorescence microscopy with polyclonal and monoclonal antibodies

Polyclonal and monoclonal antibodies against rat liver nuclear lamins have been used to evaluate the immunological cross-reactivity of lamins with a given antibody in a variety of animal and plant cells. The results indicated that lamins of all vertebrate cells but not invertebrate cells share at least one antigenic determinant, resulting in immunological cross-reaction with polyclonal antisera to lamina from rat liver. The range of cross-reaction with monoclonal antibody to rat lamins includes all mammalian cells tested but we observed no reaction with other vertebrate and invertebrate cells. Thus, by means of immunological cross-reaction a less stringently conserved pattern is observed for lamins than, for example, cytoskeletal proteins. We have also investigated the fate of the nuclear lamins during meiosis in testes and ovaries of the mouse. Lamins are absent from male meiotic cells and during oogenesis in meiotic prophases.     

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