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

Gene 288 (2002) 187–193 www.elsevier.com/locate/gene

The gene for a cytoplasmic intermediate filament (IF) protein of the hemichordate Saccoglossus kowalevskii; definition of the unique features of chordate IF proteins Alexander Zimek, Klaus Weber* Department of Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Go¨ttingen, Germany Received 13 December 2001; received in revised form 5 February 2002; accepted 8 February 2002 Received by A. Sippel

Abstract 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. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Chordates; Deuterostomes; Hemichordates; Intermediate filament; Metazoa; Phylogeny

1. Introduction In contrast to the F-actin based microfilaments and the microtubules, which are general eukaryotic structures, the cytoplasmic intermediate filaments (IF) seem restricted to animals. Molecular evidence for IF-proteins based on DNA complementary to RNA (cDNA) cloning is only available for metazoa (Erber et al., 1998) and analysis of the complete genomes of the yeast Saccharomyces cerevisiae and the plant Arabidopsis thaliana shows no obvious orthologs. IF stabilise cells against mechanical stress as judged by the various human epidermal fragility syndromes (Fuchs and Cleveland, 1998) and at least in Caenorhabditis elegans cytoplasmic IF proteins play an essential role in nematode development (Karabinos et al., 2001a). There are two prototypes of cytoplasmic IF proteins which seem to parallel metazoan phylogeny. When compared with nuclear lamins the first prototype, originally defined in the vertebrates, lacks 42 residues or six heptads in Abbreviations: bp, base pair(s); cDNA, DNA complementary to RNA; IF, intermediate filament; PCR, polymerase chain reaction; mRNA, messenger RNA * Corresponding author. Tel.: 149-551-201-1486; fax: 149-551-2011578. E-mail address: [email protected] (K. Weber).

the coil 1b domain of the central a-helical rod domain. Most vertebrate IF proteins fall into one of the four subfamilies I– IV. Type I and II account for complementary keratin molecules, which as obligatory heterodimeric coiled coils form the keratin filaments of epithelia. Type III molecules can form homopolymeric IF, while neuronal IF proteins are covered by type IV (Fuchs and Weber, 1994; Parry and Steinert, 1995; Herrmann and Aebi, 2000). All 62 currently known human IF proteins show the short coil 1b domain (Hesse et al., 2001), and this feature also holds for the 13 proteins of the cephalochordate Branchiostoma (Karabinos et al., 2000) and the four IF proteins established in the urochordate Styela (Wang et al., 2000). Some IF proteins of the early chordates can be classified as type I–III proteins on grounds of their obligatory heteropolymeric or homopolymeric IF forming ability. In spite of the strong sequence drift, several type I keratins of the early chordates retain the ability to form IF when mixed with human keratin 8, a type II keratin (Karabinos et al., 2000; Wang et al., 2000). The second prototype of cytoplasmic IF proteins is found in the protostomic invertebrates and has been documented for various members of 11 different phyla (Weber et al., 1989; Erber et al., 1998). Here the coil 1b domain has the same length as in nuclear lamins and many but not all protostomic IF proteins show a further element indicating

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00484-5

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a close relationship with nuclear lamins. They contain a lamin homology segment of some 110 residues in their Cterminal tail domains. The long coil 1b domain holds not only for the eight C. elegans IF proteins established earlier by cDNA cloning (Dodemont et al., 1994) but also for the three additional genes provided by the complete genome sequences. Six of the 11 nematode IF proteins show the lamin homology segment in their tail domain (Karabinos et al., 2001a). Interestingly, not all protostomic phyla display cytoplasmic IF. Earlier electron microscopical work indicated a lack of IF in various arthropods including Drosophila and documented instead a wealth of microtubular bundles in cellular locations which in other phyla are occupied by IF bundles (Mogensen and Tucker, 1988; Bartnik and Weber, 1989). In agreement with this view is the lack of obvious orthologs of cytoplasmic IF proteins deduced from the Drosophila genome (Rubin et al., 2000). The lack of IF in Drosophila does not imply that IF, when present in invertebrates, are a non-essential structure. Recent RNA interference experiments show that four of the 11 C. elegans IF genes are essential for nematode development (Karabinos et al., 2001a). There are two major missing links in our understanding of the evolution of metazoan cytoplasmic IF proteins. One concerns the lack of sequences from primitive animals such as the cnidarians, where so far only nuclear lamins are established (Erber et al., 1999). The other concerns the lower deuterostomes (Turbeville et al., 1994; Cameron et al., 2000) – the hemichordates and echinoderms – which could potentially offer evidence for the phylogenetic transition point between the protostomic and the chordate IF sequence types. Here we report the cloning of a cytoplasmic IF protein from the hemichordate Saccoglossus. 2. Materials and methods 2.1. Animals and cDNA libraries Adult Saccoglossus kowalevskii collected at the Woods Hole Marine Laboratory, MA, USA, was kindly provided by Dr E. Ungewickell. Total messenger RNA (mRNA) was used for cDNA synthesis and construction of a phage library with the Zap Express kit (Stratagene, Heidelberg, Germany) was as described (Erber et al., 1998). Priapulus caudatus was from the marine station in Millport, UK. The resulting Zap phage library was described (Erber et al., 1999). 2.2. Isolation of cDNA clones encoding IF proteins About 250,000 plaques of the cDNA libraries were screened with the murine monoclonal antibody IFA (Pruss et al., 1981) as described (Erber et al., 1998). Inserts from positive single plaques were amplified by polymerase chain reaction (PCR), cloned into the pCR 2.1 vector and characterised by DNA sequencing. Of the 31 IFA positive primary Saccoglossus plaques, only one clone (IF-1) described an IF

protein. The full length cDNA sequence was amplified by PCR from the library, cloned into the pCR 2.1 TOPO vector (Invitrogen, San Diego, CA, USA) and sequenced completely on both strands. Only a single IF clone was obtained from the ten primary IFA positive Priapulus plaques. 2.3. Characterisation of the Saccoglossus IF gene Overlapping genomic fragments were amplified by PCR and sequenced. Intron sequences were identified by comparison with the cDNA. The total length of the genomic sequence including the nine introns was 3165 bp. 2.4. Miscellaneous procedures For recombinant expression of the full-length Saccoglossus IF protein the coding sequence of the cDNA was amplified by PCR and the product was inserted into the pET 23a(1) expression vector (Novagen, Madison, WI, USA) as a Nde1//BamHI fragment. The primers used for this PCR reaction were as follows: forward 5 0 -GTGCACAGCATATGATAAGTAGTGG-3 0 , reverse 5 0 -GTCTTCAACGGGATCCATCAGTTTGG-3 0 . Expression in Escherichia coli BL21 (DE3) pLys cells and purification of the recombinant protein by ion exchange chromatography in 8M urea was as described (Karabinos et al., 2000). Protein purity was monitored by gel electrophoresis and automated Edman degradation.

3. Results 3.1. Cloning of the first hemichordate IF protein Poly(A) 1 RNA from adult Saccoglossus was used to construct a phage library with the ZAP expression kit. This library was screened using the murine monoclonal antibody IFA (Pruss et al., 1981), which detects many, but not all, cytoplasmic IF proteins and nuclear lamins from invertebrates (Erber et al., 1998). The complete cDNA sequence of the isolated clone characterises a protein of 543 amino acid residues with a molecular weight of 63,090 and a calculated isoelectric point of 4.89 (Fig. 1A). The organisation of the corresponding gene was determined by PCR methodology. The position of the nine introns interrupting the coding sequence is shown in Fig. 1A. The protein is clearly related to cytoplasmic IF proteins and nuclear lamins (see below). The lack of a nuclear localisation signal and a terminal CaaX motif identifies the protein as a cytoplasmic IF protein with the typical tripartate domain organisation (Fuchs and Weber, 1994; Parry and Steinert, 1995). The N-terminal head domain, which is rather variable in IF proteins, covers 83 residues and contains a continuous stretch of ten serine residues. Compared with the head domain of other cytoplasmic IF proteins, which are usually rich in basic residues, the IF

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Fig. 1. (A) Nucleotide and predicted protein sequence of the cDNA for the cytoplasmic IF protein from Saccoglossus kowalevskii. The arrowheads mark the position of the nine introns in the corresponding gene. The cDNA sequence is available from EMBL/GenBank under accession number AJ421616. (B) Nucleotide and predicted protein sequence of the cDNA for the cytoplasmic IF protein from Priapulus caudatus. The sequence is available form EMBL/ GenBank under accession number AJ421617.

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Fig. 2. Sequence features of the central rod domain (A); and the C-terminal tail domain (B) of the hemichordate IF protein. Residues 84 to 423 of the Saccoglossus (Sac) protein (Fig. 1A) are aligned with the rod domains of human vimentin (hVi) and the Helix aspersa IF-A protein (Hel). The subdomains of the rod (coil 1a, coil 1b and coil 2) are indicated by horizontal arrows. Identical residues in Sac and one or both of the other proteins are given in bold type. Dots indicate gaps introduced to optimise the alignments. Note the hallmark sequence motifs at the beginning and end of the rod domains. Chordate IF proteins, represented by human vimentin, and protostomic IF proteins, represented by the Helix protein, differ in the coil 1b subdomain by a chordate specific deletion of 42 residues (Weber et al., 1989; Erber et al., 1998). This deletion is reduced to 11 residues in the Saccoglossus protein. Filled triangles mark the position of introns in the corresponding genes. Since these are identically placed in the genes for human vimentin and the Helix IF protein (Dodemont et al., 1990), they are indicated only along the Helix sequence. The Saccoglossus gene (Fig. 1A) keeps four of these six intron positions. It has lost the second intron and the third intron position is shifted. The unique three residue insert (QFG) into the coil 2b subdomain of the Saccoglossus protein is marked by asterisks. (B) Alignment of the carboxyterminal 112 residues of the hemichordate cytoplasmic IF protein (Fig. 1A) with the central part of the tail domains of four nuclear lamins. Priapulus caudatus lamin (Pri L), Tealia lamin (Tea L), Xenopus laevis lamin L3 (Xen L3) and the murine lamin B2 (Mus LB2). For references to lamin sequences see (Erber et al., 1999). Identical residues in the sequence of the cytoplasmic IF protein form Saccoglossus (Sac) and one or more of the lamin sequences are given in bold type. Six residues strictly conserved in all sequences are marked by an asterisk. An additional 22 positions highly conserved in all five sequences are marked by a plus sign. The Saccoglossus protein is the first deuterostomic cytoplasmic IF protein with a lamin homology segment in the tail domain. Triangles mark intron positions in the corresponding genes. The three intron pattern indicated along the murine lamin sequence also holds for the Xenopus lamin. In the Priapulus lamin the first intron is absent (Erber et al., 1999). The two introns of the Saccoglossus gene indicated along the sequence are from Fig. 1A. Their position is identical to that of introns 1 and 3 of the vertebrate lamins.

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protein shows an excess of negatively (24) over positively (15) charged residues. Fig. 2A gives an alignment of the central a-helical rod domain of the hemichordate IF protein with the corresponding regions of human vimentin and the Helix aspersa IF-A protein, which serve as representatives of chordate and protostomic IF proteins, respectively. Although there is strong sequence drift, the Saccoglossus protein shows the hallmark sequence motifs of IF proteins early in coil 1a (LNERLAXYXDXV) and at the end of coil 2 (EIAXXRKLLEXEE). In the coil 1b subdomain vimentin and all other chordate IF proteins lack 42 residues present in all protostomic IF proteins (Weber et al., 1988, 1989; Dodemont et al., 1990; Erber et al., 1998). Interestingly, in the

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hemichordate protein the deletion is only 11 residues long. A second unique feature of the Saccoglossus protein is a three-residue insert in the coil 2b subdomain (marked by asterisks in Fig. 2A). This unique insert was confirmed in the genomic sequence. The genes for vimentin and the Helix IF protein show six identically placed introns for the rod domain (Dodemont et al., 1990). The corresponding region of the Saccoglossus IF gene shows two changes. It lacks the second intron and shows a displacement of the third intron. All three genes have an identical organisation over the coil 2 subdomain (Fig. 2A). Fig. 2B shows that the C-terminal 112 residues of the tail domain of the hemichordate IF protein are readily aligned with the central part of the tail domain of nuclear lamins and

Fig. 3. Phylogenetic distribution of the short (2) and long (1) coil 1b subdomain and the presence of a lamin homology segment in the tail domain (T) of cytoplasmic IF proteins. The schematic phylogeny with two protostomic branches (Ecdysozoa and Lophotrochozoa) and the separation of the deuterostomes into chordates versus hemichordates plus echinoderms is based on 18S rDNA sequences (Aguinaldo et al., 1997; Cameron et al., 2000). Numbers in parentheses give the total number of sequences of cytoplasmic IF proteins known per phylum. Numbers without parentheses give the number of sequences established for a single species of the phylum. Earlier references have been summarised (Erber et al., 1998). The figure incorporates the following additional information. The number of IF sequences for the annelid Hirudo medicinalis has increased from 1 to 3 (Xu et al., 1999). The nematode C. elegans genome shows 11 cytoplasmic IF genes (Karabinos et al., 2001a). The number of sequences for the cephalochordate Branchiostoma (Karabinos et al., 2000) and the urochordate Styela clava (Wang et al., 2000) have increased as shown. The value of 62 for man (vertebrates) is based on the recent analysis of the draft sequence of the human genome (Hesse et al., 2001). The presence of the IF subfamilies I–IV is indicated for the chordates. The vertical line in front of the arthropods and their closest relatives indicates the lack of cytoplasmic IF based on electron microscopical results (Bartnik and Weber, 1989). Analysis of the Drosophila genome proves the absence of genes for cytoplasmic IF proteins (Rubin et al., 2000). The results on the hemichordate Saccoglossus are from this study (see text). The asterisk in parenthesis indicates the presence of a small deletion in coil 1b (see Fig. 2). All protostomic cytoplasmic IF proteins have the long coil 1b (1) and most display a lamin homology segment in their tail domains. All chordate IF proteins have the 42 residue deletion in coil 1b (2) and never display a lamin homology segment in their tail domain. The hemichordate sequence (this study) is the first deuterostomic IF sequence with a lamin homology segment in the tail domain. This molecular difference versus the chordates is also seen in coil 1b. It has only a deletion of 11 residues (asterisk) versus the protostomic cytoplasmic IF protein and not the 42 residue deletion characteristic of the chordates.

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thus form the lamin tail homology segment (Weber et al., 1989; Erber et al., 1998). The four nuclear lamin sequences used in Fig. 2B are from vertebrates (mouse lamin B2 and Xenopus laevis LIII), P. caudatus (Priapulida), a protostomic animal, and the cnidarian Tealia. Although the sequence identity levels of the hemichordate cytoplasmic IF protein and the nuclear lamins reach only 23%, there are six strictly conserved and 22 highly conserved residues. In addition, the corresponding part of the gene for the cytoplamsic IF protein of Saccoglossus shows two of the two to three intron positions found in the lamin genes (Fig. 2B). The hemichordate IF protein was expressed in E. coli and the recombinant protein was purified in 8M urea. Removal of the urea by dialysis provided only aggregated material without the formation of IF. The failure of single IF proteins to form IF in vitro can be due to the presence of an obligatory heteropolymer assembly system (Karabinos et al., 2000). Currently we do not know whether this explanation also holds for the Saccoglossus protein. Immunoblotting experiments showed that the recombinant protein was recognised by the monoclonal antibody IFA (data not shown). 3.2. Cloning of a priapulid IF protein Using the monoclonal antibody IFA we also isolated a complete cDNA for a cytoplasmic IF protein from P. caudatus, a member of the Priapulida (Fig. 1B). The predicted protein sequence of 547 residues has a calculated molecular weight of 64,436 and a calculated isoelectric point of 5.71. As expected from the phylogenetic position of the priapulida (Fig. 3), the sequence shows the features of protostomic cytoplasmic IF proteins (Erber et al., 1998). It has the long coil 1b version and shows a lamin homology segment in the tail domain. 4. Discussion Previous studies have documented that metazoan cytoplasmic IF proteins come in two prototypes, which seem to parallel metazoan phylogeny (for references see Section 1). The first prototype has the long lamin-like coil 1b domain and is often accompanied by a lamin tail homology segment in the carboxyterminal tail domain. It has been documented for 11 protostomic phyla and our results on P. caudatus (see Section 3) extend this analysis to a further phylum. The second prototype has the short coil 1b domain due to a deletion of 42 residues and never shows a lamin tail homology segment. It is recognised in all chordate IF sequences (for a summary see Fig. 3). The cDNA cloning of a cytoplasmic protein from the hemichordate Saccoglossus provides the first IF sequence (Fig. 2) from the lower deuterostomes, which cover hemichordates and echinoderms and form the sister branch of the chordates (Turbeville et al., 1994; Cameron et al., 2000). Strikingly, the domain organisation of the hemichordate

protein is closer to the protostomic proteins than to the chordate proteins. It has the lamin tail homology segment, which is never found in chordate IF proteins, and lacks the 42 residue deletion in coil 1b characteristic for chordate IF proteins. Interestingly, the coil 1b of the Saccoglossus proteins has not the canonical lamin-like length of protostomic IF proteins, but is shortened due to a unique 11 residue deletion. If we take the Saccoglossus sequence as a true representative of the lower deuterostomes, then the second IF prototype (see above) can be clearly defined as a molecular property of the chordate phyla. IF proteins with a coil 1b shortened by 42 residues and lacking a lamin tail homology segment are restricted to the chordate branch. Only after the 42 residue deletion occurred at the origin of the chordates subsequent gene duplications and sequence drift established the subfamilies I–III. Originally defined in the vertebrate sequences (Fuchs and Weber, 1994; Parry and Steinert, 1995; Herrmann and Aebi, 2000) the corresponding orthologs of the cephalochordates and urochordates are meanwhile clearly documented (Riemer and Weber, 1998; Karabinos et al., 2000; Wang et al., 2000). The neurofilament IV subfamily may be a later and vertebrate specific acquisition (Karabinos et al., 2001b). The definition of the chordate branch by the unique domain organisation of their cytoplasmic IF proteins is not without precedence. An amino terminal sequence signature of muscle actins separates the chordates from the lower deuterostomes and all other metazoa (Vandekerckhove and Weber, 1984; Kovilur et al., 1993; Bovenschulte and Weber, 1997; Kusakabe et al., 1997). Fig. 3 summarises the sequence information on metazoan cytoplasmic proteins currently available and also resolves an earlier problem. In line with the absence of IF in electron microscopic studies of various arthropods (Bartnik and Weber, 1989) the complete genome of Drosophila lacks cytoplasmic IF genes (Rubin et al., 2000). Thus it seems that arthropods use special microtubular bundles in cellular locations which in other phyla are occupied by IF bundles (Karabinos et al., 2001a). Fig. 3 also indicates the few questions on IF phylogeny still to be resolved. Thus there is a need for sequences from the primitive animals such as for instance Hydra, a member of the cnidarians. In addition it would be useful to have a broader data base for the lower deuterostomes by obtaining additional information on echinoderms. This information should be forthcoming from large-scale projects involving expressed sequence tags (ESTs). Such an analysis from an enriched population of mid-gastrula stage primary mesenchyme cells of the sea urchin Strongylocentrotus purpuratus covering 8293 ESTs was recently reported (Zhu et al., 2001). Interestingly this collection contains no obvious ortholog of a cytoplasmic IF protein.

Acknowledgements We thank Drs A. Karabinos and J. Wang for helpful

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discussions. This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (We-338/7) to K.W. The sequences reported above are available from the EMBL/GenBank under the accession numbers AJ421616 and AJ421617. References Aguinaldo, A.M.A., Turbeville, J.M., Linford, L.S., Rivera, M.C., Garey, J.R., Raff, R.A., Lake, J.A., 1997. Evidence for a clade of nematodes, arthropods and other moulting animals. Nature 387, 489–492. Bartnik, E., Weber, K., 1989. Widespread occurrence of intermediate filaments in invertebrates; common principles and aspects of diversion. Eur. J. Cell Biol. 50, 17–33. Bovenschulte, M., Weber, K., 1997. Deuterostomic actin genes and the definition of the chordates: cDNA cloning and gene organisation for cephalochordates and hemichordates. J. Mol. Evol. 45, 653–660. Cameron, C.B., Garey, J.R., Swalla, B.J., 2000. Evolution of the chordate body plan: New insights from phylogenetic analyses of deuterostome phyla. Proc. Natl. Acad. Sci. USA 97, 4469–4474. Dodemont, H., Riemer, D., Weber, K., 1990. Structure of an invertebrate gene encoding cytoplasmic intermediate filament proteins: implications for the origin and the diversification of IF proteins. EMBO J. 9, 4083– 4094. Dodemont, H., Riemer, D., Ledger, N., Weber, K., 1994. Eight genes and alternative RNA processing pathways generate an unexpectedly large diversity of cytoplasmic intermediate filament proteins in the nematode Caenorhabditis elegans. EMBO J. 13, 2625–2638. Erber, A., Riemer, D., Bovenschulte, M., Weber, K., 1998. Molecular phylogeny of metazoan intermediate filament proteins. J. Mol. Evol. 47, 751–762. Erber, A., Riemer, D., Hofemeister, H., Bovenschulte, M., Stick, R., Panopoulou, G., Lehrach, H., Weber, K., 1999. Characterisation of the Hydra lamin and its gene; a molecular phylogeny of metazoan lamins. J. Mol. Evol. 49, 260–271. Fuchs, E., Weber, K., 1994. Intermediate filaments: structure, dynamics, function and disease. Ann. Rev. Biochem. 63, 345–382. Fuchs, E., Cleveland, D.W., 1998. A structural scaffolding of intermediate filaments in health and disease. Science 279, 514–519. Herrmann, H., Aebi, U., 2000. Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr. Opin. Cell Biol. 12, 79–90. Hesse, M., Magin, T.M., Weber, K., 2001. Genes for intermediate filament proteins and the draft sequence of the human genome; novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. J. Cell Sci. 114, 2569–2575. Karabinos, A., Riemer, D., Panopoulou, G., Lehrach, H., Weber, K., 2000. Characterisation and tissue specific expression of the two keratin subfamilies of intermediate filament proteins in the cephalochordate Branchiostoma. Eur. J. Cell Biol. 79, 1–10. Karabinos, A., Schmidt, H., Harborth, J., Schnabel, R., Weber, K., 2001a. Essential roles for four cytoplasmic intermediate filament proteins in

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Caenorhabditis elegans development. Proc. Natl. Acad. Sci. USA 98, 7863–7868. Karabinos, A., Wang, J., Wenzel, D., Panopoulou, G., Lehrach, H., Weber, K., 2001b. Developmentally controlled expression patterns of intermediate filament proteins in the cephalochordate Branchiostoma. Mech. Dev. 101, 283–288. Kovilur, S., Jacobson, J.W., Beach, R.L., Jeffery, W.R., Tomlinson, C.R., 1993. Evolution of the chordate muscle actin gene. J. Mol. Evol. 36, 361–368. Kusakabe, T., Araki, I., Satoh, N., Jeffery, W.R., 1997. Evolution of chordate actin genes: evidence from genomic organisation and amino acid sequences. J. Mol. Evol. 44, 289–298. Mogensen, M.M., Tucker, J.B., 1988. Intermicrotubular actin filaments in the transalar cytoskeletal arrays of Drosophila. J. Cell Sci. 91, 431–438. Parry, D.A.D., Steinert, P.M., 1995. Intermediate Filament Structure, Springer Verlag, New York. Pruss, R.M., Mirsky, R., Raff, M.C., Thorpe, R., Dowding, A.J., Anderton, B.H., 1981. All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell 27, 419–428. Riemer, D., Weber, K., 1998. Common and variant properties of intermediate filament proteins from lower chordates and vertebrates; two proteins from the tunicate Styela and the identification of a type III homologue. J. Cell Sci. 111, 2967–2975. Rubin, G.M., Yandell, M.D., Wortman, J.R., Gabor Miklos, G.L., Nelson, C.R., Hariharan, I.K., Fortini, M.E., Li, P.W., Apweiler, R., Fleischmann, W., Cherry, J.M., et al., 2000. Comparative genomics of the eukaryotes. Science 287, 2204–2215. Turbeville, J.M., Schulz, J.R., Raff, R.A., 1994. Deuterostome phylogeny and the sister group of the chordates: evidence from molecules and morphology. Mol. Biol. Evol. 11, 648–655. Vandekerckhove, J., Weber, K., 1984. Chordate muscle actins differ distinctly from invertebrate muscle actins. The evolution of the different vertebrate muscle actins. J. Mol. Biol. 179, 391–413. Wang, J., Karabinos, A., Schu¨ nemann, J., Riemer, D., Weber, K., 2000. The epidermal intermediate filament proteins of tunicates are distant keratins; a polymerisation competent hetero coiled coil of the Styela D protein and Xenopus keratin 8. Eur. J. Cell Biol. 79, 478–487. Weber, K., Plessmann, U., Dodemont, H., Kossmagk-Stephan, K., 1988. Amino acid sequences and homopolymer-forming ability of the intermediate filament proteins from an invertebrate epithelium. EMBO J. 7, 2995–3001. Weber, K., Plessmann, U., Ulrich, W., 1989. Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterisation of two muscle proteins of a nematode. EMBO J. 8, 3221–3227. Xu, Y., Bolton, B., Zipser, B., Jellies, J., Johansen, K.M., Johansen, J., 1999. Gliarin and macrolin, two novel intermediate filament proteins specifically expressed in sets and subsets of glial cells in leech central nervous system. J. Neurobiol. 40, 244–253. Zhu, X., Mahairas, G., Illies, M., Cameron, R.A., Davidson, E.H., Ettensohn, C.A., 2001. A large scale analysis of mRNAs expressed by primary mesenchyme cells of the sea urchin embryo. Development 128, 2615–2627.