Gene structure of nuclear lamin LIII of Xenopus laevis; a model for the evolution of IF proteins from a lamin-like ancestor.

The lamin LIII gene of Xenopus laevis has been characterized. The gene is duplicated in the Xenopus genome. The transcribed region spreads over 22 kb of genomic DNA encoding 12 exons. Two alternatively spliced mRNAs are observed which encode LIII isoforms that differ only by the 12 C-terminal amino acids which, however, both contain the CaaX motif known to be the target of post-translational modifications. The intron pattern of the lamin LIII gene is strikingly similar to that of an invertebrate intermediate filament (IF) gene over the entire protein coding sequence. The similarity in gene structure is restricted to the rod domain when compared with vertebrate types I-III IF genes. Our data suggest a model of how IF proteins evolved from a lamin-like ancestor by deletion of two signal sequences; the nuclear localization signal and the C-terminal ras-related CaaX motif. The data rule out the previously proposed hypothesis that IF proteins evolved from an intronless ancestor with an early divergence of neuronal and non-neuronal IF proteins. Together with the data presented in the accompanying paper by Dodemond et al. it can be concluded that the tail domains of lamins and invertebrate IF proteins, but not those of vertebrate IF proteins, are homologous. Thus, the different vertebrate IF proteins probably evolved by combination of the central rod domain with different tail domains by exon shuffling.     

Analysis of the cDNA and gene encoding a cytoplasmic intermediate filament (IF) protein from the cephalochordate Branchiostoma lanceolatum; implications for the evolution of the IF protein family.

We report the molecular cloning of a full-length cDNA encoding a non-neuronal cytoplasmic intermediate filament (IF) protein of the cephalochordate Branchiostoma lanceolatum. Sequence and structural characteristics of IF-1 reveal a close relation to vertebrate IF proteins: they all lack the extended coil 1b version and the lamin tail homology found in protostomic IF proteins. This implies that divergence of type I to IV IF genes from a common ancestor either coincided with the origin of chordates or occurred at an earlier stage in the evolution of deuterostomes. The structural organization of the cephalochordate gene shows a closer relation to vertebrate type III genes than to type I or II genes. The single gene (approximately 19 kb) is composed of 7 exons and 6 introns which are all located within the sequence encoding the rod domain. The positions and phases of the introns show perfect homology to vertebrate type III genes. In line with the absence of protein sequence similarity of the tail domain, the Branchiostoma gene does not possess the introns interrupting this region in type III genes of vertebrates.     

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.     

Rapidly evolving lamins in a chordate, Oikopleura dioica, with unusual nuclear architecture

Metazoan lamins are implicated in the organization of numerous critical nuclear processes. Among chordates, the appendicularian, Oikopleura dioica, has an unusually short life cycle involving rapid growth through extensive recourse to endoreduplication, a characteristic more associated with some invertebrates. In some tissues, this is accompanied by the formation of elaborate, bilaterally symmetric nuclear morphologies associated with specific gene expression patterns. Lamin composition can mediate nuclear shape in spermiogenesis as well as during pathological and normal aging and we have analyzed the O. dioica lamin and intermediate filament (IF) complement, comparing it to that present in other deuterostomes. O. dioica has one lamin gene coding two splice variants. Both variants share with the sister class ascidians a highly reduced C-terminal tail region lacking the immunoglobulin fold, indicating this derivation occurred at the base of the urochordate lineage. The OdLamin2 variant has a unique insertion of 63 amino acids in the normally short N-terminal region and has a developmental expression profile corresponding to the occurrence of endocycling. O. dioica has 4 cytoplasmic IF proteins, IF-A, C, Dalpha, and Dbeta. No homologues to the ascidian IF-B or F proteins were identified, reinforcing the suggestion that these proteins are unique to ascidians. The degree of sequence evolution in the rod domains of O. dioica cytoplasmic IF proteins and their closest ascidian and vertebrate homologues was similar. In contrast, whereas the rate of lamin B rod domain sequence evolution has also been similar in vertebrates, cephalochordates and the sea urchin, faster rates have occurred among the urochordates, with the O. dioica lamin displaying a far greater rate than any other lamin.     

The gene for a cytoplasmic intermediate filament (IF) protein of the hemichordate Saccoglossus kowalevskii; definition of the unique features of chordate IF proteins

We have isolated full length cDNAs encoding a cytoplasmic intermediate filament (IF) protein of a priapulid (Priapulus caudatus) and of a hemichordate (Saccoglossus kowalevskii) and determined the organisation of the hemichordate gene. As expected, the priapulid protein shows the long coil 1b subdomain and the lamin tail homology segment already known for cytoplasmic IF proteins from 11 other protostomic phyla. Surprisingly, the hemichordate protein follows in domain organisation much more closely the protostomic IF proteins than the chordate IF proteins. Thus, the lack of a lamin tail homology segment together with a deletion of 42 residues in the coil 1b domain is a molecular feature restricted to the chordates. We propose that the known IF subfamilies I to IV developed by gene duplications and sequence drift after the deletion in coil 1b arose at the base of the chordate branch.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.     

Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

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.     

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.     

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.     

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     

The role of the head and tail domain in lamin structure and assembly: analysis of bacterially expressed chicken lamin A and truncated B2 lamins.

Nuclear lamins like cytoplasmic intermediate filament proteins exhibit a characteristic tripartite domain structure with a segmented alpha-helical rod domain flanked by an N-terminal head and a C-terminal tail domain. To examine the influence of the head and tail domains on the structure and assembly properties of nuclear lamins, we have engineered "headless," "tailless," and "rod" chicken lamin B2 cDNAs and expressed them in Escherichia coli. A full-length chicken lamin A cDNA was also expressed in E. coli, and the recombinant protein compared with the structure and assembly properties of full-length chicken lamin B2 (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495). As with lamin B2, at their first level of structural organization, lamin A and the headless lamin B2 formed myosin-like dimers consisting of a 51- to 52-nm-long tail flanked by two globular heads at one end. Similarly, the tailless and rod lamin B2 fragments formed tropomyosin-like dimers consisting of a 51 to 52-nm-long rod. In contrast to the lateral mode of association of cytoplasmic IF dimers into four-chain tetramers, at their second level of structural organization, lamin A dimers, just as lamin B2 dimers (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495), associated longitudinally to form polar head-to-tail polymers. Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head-to-tail association. Finally, at their third level of structural organization, rather than assembling into stable 10-nm filaments, both lamin A and the three truncated B2 lamins formed paracrystalline arrays exhibiting distinct transverse banding patterns with axial repeats of either 24 or 48-49 nm depending on the species.     

The cephalochordate Branchiostoma genome contains 26 intermediate filament (IF) genes: Implications for evolution of chordate IF proteins

We analyzed the draft genome of the cephalochordate Branchiostoma floridae (B. floridae) for genes encoding intermediate filament (IF) proteins. From 26 identified IF genes 13 were not reported before. Four of the new IF genes belong to the previously established Branchiostoma IF group A, four to the Branchiostoma IF group B, one is homologous to the type II keratin E2 while the remaining four new IF sequences N1 to N4 could not be readily classified in any of the previously established Branchiostoma IF groups. All eleven identified A and B2-type IF genes are located on the same genomic scaffold and arose due to multiple cephalochordate-specific duplications. Another IF gene cluster, identified in the B. floridae genome, contains three keratins (E1, Y1, D1), two keratin-like IF genes (C2, X1), one new IF gene (N1) and one IF unrelated gene, but does not show any similarities to the well defined vertebrate type I or type II keratin gene clusters. In addition, some type III sequence features were documented in the new IF protein N2, which, however, seems to share a common ancestry with the Branchiostoma keratins D1 and two keratin-related genes C. Thus, a few type I and type II keratin genes existed in a common ancestor of cephalochordates and vertebrates, which after separation of these two lineages gave rise to the known complexities of the vertebrate cytoplasmic type I–IV IF proteins, as well as to the multiple keratin and related IF genes in cephalochordates, due to multiple gene duplications, deletions and sequence divergences.     

The sequence of a cytoplasmic intermediate filament (IF) protein from theannelid Lumbricus terrestris emphasizes a distinctive feature of protostomic IF proteins

The complete cDNA clone for a cytoplasmic intermidiatefilament (IF) protein from the annelid Lumbricus terrestris reported here, shows an extra 42 residues in the coil 1b subdomain of the central rod, as do the IF proteins from nematodes and moluscs. These extra six heptads are also present in all nuclear lamins but not in any known vertebrate cytoplasmic IF protein. Thus, it seems that protostomic metazoa conserve a lamin-like structural element in their cytoplasmic IF proteins, which was lost in the deuterostomic metazoan branch leading to the vertebrates.     

Tunicates have unusual nuclear lamins with a large deletion in the carboxyterminal tail domain

Lamins are essential proteins of metazoa. They give rise to the nuclear lamina lining the nucleoplasmic face of the inner nuclear membrane. Here we report the isolation of complete lamin cDNA clones from three urochordate (tunicate) libraries - adult Ciona intestinalis, the tailbud stage of Styela clava and the gastrula stage of Molgula oculata. Lamins L1 and L2 of adult Ciona are derived from two distinct genes. The sequence of the 3' part of the Ciona lamin L1 gene shows that the alpha and beta variants of lamin L1 in Ciona and Styela arise by alternative choice of the 5' splice site at the last intron. Strikingly, all urochordate sequences reveal a 90 residue deletion which removes nearly the entire 105-box. This region is the only long sequence homology segment in the carboxyterminal tail domain of lamins from animals as diverse as Hydra, Drosophila, Priapulus, Caenorhabditis elegans, several echinoderms, the cephalochordate Branchiostoma and various vertebrates. We discuss this unexpected plasticity of lamin sequences as a urochordate specific marker. To increase the database for the chordates we completed the partial sequence of the Branchiostoma lamin by the N-terminal head and central rod domains. The molecular phylogenetic analysis of the metazoan lamin sequences emphasises the monophyletic nature of the chordates in line with the morphological evidence.     

Intermediate filament proteins in echinoderm coelomocytes

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droebachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single M_r 68 000 sea urchin lamin, as well as two putative lamin isoforms of approximately M_r 70 000 and 68 000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of M_r 46 000 and 58 000 polypeptides, while M_r 215 000 and 185 000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.