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.     

Structure of an invertebrate gene encoding cytoplasmic intermediate filament (IF) proteins: implications for the origin and the diversification of IF proteins.

The structure of the single gene encoding the cytoplasmic intermediate filament (IF) proteins in non-neuronal cells of the gastropod Helix aspersa is described. Genomic and cDNA sequences show that the gene is composed of 10 introns and 11 exons, spanning greater than 60 kb of DNA. Alternative RNA processing accounts for two mRNA families which encode two IF proteins differing only in their C-terminal sequence. The intron/exon organization of the Helix rod domain is identical to that of the vertebrate type III IF genes in spite of low overall protein sequence homology and the presence of an additional 42 residues in coil 1b of the invertebrate sequence. Intron position homology extends to the entire coding sequence comprising both the rod and tail domains when the invertebrate IF gene is compared with the nuclear lamin LIII gene of Xenopus laevis presented in the accompanying report of Döring and Stick. In contrast the intron patterns of the tail domains of the invertebrate IF and the lamin genes differ from those of the vertebrate type III genes. The combined data are in line with an evolutionary descent of cytoplasmic IF proteins from a nuclear lamin-like progenitor and suggest a mechanism for this derivation. The unique position of intron 7 in the Helix IF gene indicates that the archetype IF gene arose by the elimination of the nuclear localization sequence due to the recruitment of a novel splice site. The presumptive structural organization of the archetype IF gene allows predictions with respect to the later diversification of metazoan IF genes. Whereas models proposing a direct derivation of neurofilament genes seem unlikely, the earlier speculation of an mRNA transposition mechanism is compatible with current results.     

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.     

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.     

The postanal tail of the enteropneust Saccoglossus kowalevskii is a ciliary creeping organ without distinct similarities to the chordate tail

Stach, T. and Kaul, S. 2011. The postanal tail of the enteropneust Saccoglossus kowalevskii is a ciliary creeping organ without distinct similarities to the chordate tail. —Acta Zoologica (Stockholm) 92 : 150–160. The postanal tail of chordates is one of the key characters in chordate evolution and it has been suggested to be homologous to the postanal tail of harrimaniid enteropneusts. We present electron microscopic data of the ontogeny of the postanal tail in the enteropneust Saccoglossus kowalevskii. The postanal tail develops as a ventral posterior allometric outgrowth with a ventral extension of the telotroch. Transmission electron microscopy of serial sections reveals the epidermal organization of the postanal tail with the exception of short, bilaterally symmetric extensions of the paired metacoels. The epidermis cells are connected by apical junctions, rest basally on the extracellular matrix surrounding the mesoderm, and possess a basiepidermal nerve net. The ventral cells in the postanal tail are multiciliated and used for creeping. Dorsal cells are monociliated with numerous microvilli. Two types of glandular cells are present among the epidermis cells. The mesoderm cells contain myofilaments. We were unable to detect anatomical structures similar to the ones present in the postanal locomotory tail of chordates, such as notochord, neural tube, or endodermal strand. Thus, results of our anatomical study do not support homology of the postanal chordate tail and the postanal tail of harrimaniid enteropneusts.     

The antibacterial activity of the hemichordate Saccoglossus ruber (Enteropneusta)

Antibacterial activity of crude whole body homogenates and mucus from the body surface of the hemichordate Saccoglossus ruber was detected using a turbidometric and plate assay. Activity was found to be strongest against marine bacteria and significantly higher against bacteria isolated from the immediate environment of the animals. The whole body nomogenate was less effective at inhibiting growth of the seven terrestrial bacteria tested. The antibacterial factor from the homogenate retained its activity after boiling and was stable at room temperature for at least 8 weeks, suggesting that it was nonproteinaceous. The loss of all activity during dialysis also indicated that a low molecular weight substance was involved. The antibacterial factor from the mucus and homogenate had similar properties, so that the same molecule may have been involved. The possibility that the antibacterial factor may be related to the halogenated indoles and phenols occurring in other enteropneusts is discussed.     

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.     

Expression profiles of the essential intermediate filament (IF) protein A2 and the IF protein C2 in the nematode Caenorhabditis elegans

The multigene family of intermediate filament (IF) proteins in Caenorhabditis elegans covers 11 members of which four (A1-3, B1) are essential for development. Suppression of a fifth gene (C2) results in a dumpy phenotype. Expression patterns of three essential genes (A1, A3, B1) were already reported. To begin to analyze the two remaining RNAi phenotypes we followed the expression of the A2 and C2 proteins. Expression of A2 mRNA starts in larval stage L1 and continues in the adult. Transgenic A2 promoter/gfp larvae strongly display GFP in the main body hypodermis but not in seam cells. This pattern and the muscle displacement/paralysis induced by RNAi silencing are consistent with the role of this protein in keeping the correct hypodermis/muscle relationship during development. IF protein C2 occurs in the cytoplasm and desmosomes of intestinal cells and in pharynx desmosomes. Expression of C2 starts in the late embryo and persists in all further stages.     

Behavior of the spermatozoon during sperm-blastomere fusion and its significance for fertilization (Saccoglossus kowalevskii: hemichordata)

Summary Association of spermatozoa with blastomeres removed from the egg envelope was studied in order to investigate sperm membrane behavior under circumstances unlike those attending fertilization of normal envelope-enclosed eggs. Under the altered circumstances all parts of the surface of the sperm cell were afforded the opportunity of meeting blastomere plasma membrane. Nevertheless, in each case the spermatozoon first became activated, an acrosomal tubule formed, and it was this organelle alone which fused and established continuity with the blastomere plasma membrane. This behavior of the spermatozoon with respect to the denuded blastomere parallels the behavior of the sperm cell with respect to the envelope-enclosed egg at fertilization. Since the sperm cell behaves so similarly in two situations which are so different with respect to what the spermatozoon encounters, this behavior is considered to reflect the inherent nature of the spermatozoon, and analysis of this behavior in relation to a blastomere is considered to be valid also in relation to an egg during fertilization. It is concluded that membrane fusion, involving the acrosomal tubule of an activated spermatozoon, is the only means by which gametic union is established and that these are obligatory rather than fortuitous features of sperm-egg association in Saccoglossus. It is suggested that these features are probably obligatory also in other species which exhibit this pattern of sperm-egg association.This investigation was supported by Research Grant HD-00007 from the National Institutes of Health, United States Public Health Service.     

The intermediate filament protein keratin 8 is a novel cytoplasmic substrate for c-Jun N-terminal kinase.

Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. A number of keratin in vitro and in vivo phosphorylation sites have been identified. One example is K8 Ser-73, which has been implicated as an important phosphorylation site during mitosis, cell stress, and apoptosis. We show that K8 is strongly phosphorylated on Ser-73 upon stimulation of the pro-apoptotic cytokine receptor Fas/CD95/Apo-1 in HT-29 cells. Kinase assays showed that c-Jun N-terminal kinase (JNK) was also activated with activation kinetics corresponding to that of K8 phosphorylation. Furthermore, K8 was also phosphorylated on Ser-73 by JNK in vitro, yielding similar phosphopeptide maps as the in vivo phosphorylated material. In addition, co-immunoprecipitation studies revealed that part of JNK is associated with K8 in vivo, correlating with decreased ability of JNK to phosphorylate the endogenous c-Jun. Taken together, K8 is a new cytoplasmic target for JNK in Fas receptor-mediated signaling. The functional significance of this phosphorylation could relate to regulation of JNK signaling and/or regulation of keratin dynamics.     

Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum.

Treponema pallidum and other members of the genera Treponema, Spirochaeta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane. These cytoplasmic filaments have a ribbon-like profile and consist of a major cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of approximately 80,000. Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid sequences of T. pallidum subsp. pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA). A 6.8-kb EcoRI fragment containing all but the 5' end of cfpA was identified by hybridization with the resulting PCR product and cloned into Lambda ZAP II. The 5' region was obtained by inverse PCR, and the complete gene sequence was determined. The cfpA sequence contained a 2,034-nucleotide coding region, a putative promoter with consensus sequences (5'-TTTACA-3' for -35 and 5'-TACAAT-3' for -10) similar to the sigma70 recognition sequence of Escherichia coli and other organisms, and a putative ribosome-binding site (5'-AGGAG-3'). The deduced amino acid sequence of CfpA indicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15. No significant homology to known proteins or structural motifs was found among known prokaryotic or eukaryotic sequences. Expression of a LacZ-CfpA fusion protein in E. coli was detrimental to survival and growth of the host strain and resulted in the formation of short, irregular filaments suggestive of partial self-assembly of CfpA. The cytoplasmic filaments of T. pallidum and other spirochetes appear to represent a unique form of prokaryotic intracytoplasmic inclusions.     

mua-6, a gene required for tissue integrity in Caenorhabditis elegans, encodes a cytoplasmic intermediate filament

Locomotion in Caenorhabditis elegans requires force transmission through a network of proteins linking the skeletal muscle, via an intervening basal lamina and epidermis (hypodermis), to the cuticle. Mutations in mua-6 result in hypodermal rupture, muscle detachment from the bodywall, and progressive paralysis. It is shown that mua-6 encodes the cytoplasmic intermediate filament (cIF) A2 protein and that a MUA-6/IFA-2::GFP fusion protein that rescues the presumptive mua-6 null allele localizes to hypodermal hemidesmosomes. This result is consistent with what is known about the function of cIFs in vertebrates. Although MUA-6/IFA-2 is expressed embryonically, and plays an essential postembryonic role in tissue integrity, it is not required for embryonic development of muscle-cuticle linkages nor for the localization of other cIFs or hemidesmosome-associated proteins in the embryo. Finally, the molecular lesion in the mua-6(rh85) allele suggests that the head domain of the MUA-6/IFA-2 is dispensable for its function.     

The intermediate filament protein peripherin is a marker for cerebellar climbing fibres

Immunocytochemical staining with antibodies to the class III intermediate filament protein peripherin reveals discrete subpopulations of neurons and nerve fibres throughout the rat central nervous system. Some of these fibres enter the cerebellar granular and molecular layers. Here we use light and electron microscopic immunocytochemistry and confocal fluorescence microscopy to identify the peripherin positive fibres in the molecular layer of the cerebella of various mammals. 1) The peripherin positive fibres in the molecular layer have morphological attributes of climbing fibres, and peripherin positive fibres are also detected in the olivo-cerebellar tract. Furthermore peripherin positive neurons can be seen in the inferior olive, from which climbing fibres originate. (2 ) The peripherin positive molecular layer fibres rapidly degenerate in rats treated with 3-acetylpyridine (3-AP), a reagent which destroys neurons in the inferior olive, and the time course of degeneration of these mirrors that previously described for 3-AP induced destruction of climbing fibres. (3) Cerebella of other mammal species tested (mouse, rabbit, pig, cow and human) revealed a similar peripherin staining pattern in the cerebellum, including fibres in the molecular layer with the morphology of climbing fibres. (4) We also noted peripherin positive spinocerebellar and vestibulocerebellar mossy fibres in the cerebellar granular layer of folia known to receive these inputs. (5) A subset of perivascular nerve fibres are also peripherin positive. These results show that peripherin is a useful marker for mammalian cerebellar climbing fibres, and that a subset of morphologically distinct cerebellar mossy fibres are also peripherin positive.