TY - JOUR
AU1 - Hwang, Jung Shan
AU2 - Takaku, Yasuharu
AU3 - Chapman, Jarrod
AU4 - Ikeo, Kazuho
AU5 - David, Charles N.
AU6 - Gojobori, Takashi
AB - Abstract The cnidocil at the apical end of Hydra nematocytes is a mechanosensory cilium, which acts as a “trigger” for discharge of the nematocyst capsule. The cnidocil protrudes from the center of the cnidocil apparatus and is composed of singlet and doublet microtubules surrounding an electron-dense central filament. In this paper, we identify a novel protein, nematocilin, which is localized in the central filament. Immunofluorescence staining and immunogold electron microscopy show that nematocilin forms filaments in the central core of the cnidocil. Nematocilin represents a new member of the intermediate filament superfamily. Two paralogous sequences of nematocilin are present in the Hydra genome and appear to be the result of recent gene duplication. Comparison of the exon–intron structure suggests that the nematocilin genes evolved from the nuclear lamin gene by conserving exons encoding the coiled-coil domains and replacing the C-terminal lamin domains. Molecular phylogenetic analyses also support the hypothesis of a common ancestor between lamin and nematocilin. Comparison of cnidocil structures in different cnidarians indicates that a central filament is present in the cnidocils of several hydrozoan and a cubozoan species but is absent in the cnidocils of anthozoans. A nematocilin homolog is absent in the recently completed genome of the anthozoan Nematostella. Thus, the evolution of a novel ciliary structure, which provides mechanical rigidity to the sensory cilium during the process of mechanoreception, is associated with the evolution of a novel protein. cnidaria, cnidocil, evolution, Hydra, intermediate filament, nematocyst Introduction Nematocytes, which are also known as “stinging cells,” constitute a cnidarian-specific cell type that function in the capture of prey, in self-defense, and in movement. Hydra has 4 distinct types of nematocytes: stenotele, holotrichous isorhiza, atrichous isorhiza, and desmoneme. All 4 types are derived from interstitial cells of Hydra (David and Murphy 1977). An interstitial stem cell that is committed to nematocyte differentiation undergoes synchronous cell divisions to form nests of 2, 4, 8, 16, or 32 cells (David and Challoner 1974; David and Gierer 1974; Shimizu and Bode 1995). Following a terminal cell division, each nematocyte in a nest differentiates a nematocyst capsule. The nematocyst is a complex intracellular organelle, which is formed during differentiation of nematocytes. After nematocyst formation is completed, the cell cluster falls apart into single nematocytes, which migrate to the tentacles and are mounted in ectodermal epithelial cells (battery cells). At this stage, the cnidocil emerges from the apical surface of the nematocyte. The cnidocil apparatus is the site of mechanoreception in nematocytes, and stimulation of the cnidocil apparatus leads to the explosive evagination of the nematocyst capsule (Holstein and Tardent 1984; Thurm and Lawonn 1990). It is a characteristic feature of nematocytes in all Cnidaria (fig. 1A). It consists of a central cnidocil (modified cilium) surrounded by stereocilia or outer microvilli (Holstein and Hausmann 1988). Significant modifications to this structure occur in different cnidarians. In anthozoans, the cnidocil is short and has the characteristic 9 + 2 arrangement of microtubules (Westfall 1965; Schmidt and Moraw 1982; Watson and Mariscal 1983). By contrast, in Hydra, the cnidocil has a very prominent central filament and only a few singlet microtubules surrounded by 9 doublet microtubules (Slautterback 1967; Golz 1994). By isolating cnidocils and partially dissociating the central filament, Golz (1994) could demonstrate that it consists of densely packed fibers with a diameter of 3–4 nm. FIG. 1.— View largeDownload slide Identification of 2 nematocyte-specific genes in Hydra. (A) Ultrastructure of the cnidocil apparatus of Hydra. cn, cnidocil; f, central filament; s, stereocilia or outer microvilli; im, inner microvilli; and cp, capsule. (B) Schematic drawing of nematocilin A and B genes in Hydra genome. Sequences of nematocilin A and B were aligned with the genomic scaffold assembled by the J. Craig Venter Institute (https://research.venterinstitute.org/files/hydra_magnipapillata_WGS_assembly). The number of each exon is indicated below the boxes. (C) Alignment of amino acid sequences of nematocilin A and B. Both sequences contain an IF domain characterized by the coiled-coil heptad repeat. Below the sequence alignment are the predicted heptad repeats (abcdefg)n where the positions a and d are shaded dark gray. The heptad repeat–containing segments 1A, 1B, 2A, and 2B (solid line) are indicated above the sequence. The linkers L1, L12, and L2 are shown between these segments. The α-helical segment 2B has been extended to amino acid 378 (dashed line) to include the well-conserved motif terminating segment 2B in lamin and invertebrate IF proteins. A stutter (abcd) in segment 2B is indicated by asterisks. FIG. 1.— View largeDownload slide Identification of 2 nematocyte-specific genes in Hydra. (A) Ultrastructure of the cnidocil apparatus of Hydra. cn, cnidocil; f, central filament; s, stereocilia or outer microvilli; im, inner microvilli; and cp, capsule. (B) Schematic drawing of nematocilin A and B genes in Hydra genome. Sequences of nematocilin A and B were aligned with the genomic scaffold assembled by the J. Craig Venter Institute (https://research.venterinstitute.org/files/hydra_magnipapillata_WGS_assembly). The number of each exon is indicated below the boxes. (C) Alignment of amino acid sequences of nematocilin A and B. Both sequences contain an IF domain characterized by the coiled-coil heptad repeat. Below the sequence alignment are the predicted heptad repeats (abcdefg)n where the positions a and d are shaded dark gray. The heptad repeat–containing segments 1A, 1B, 2A, and 2B (solid line) are indicated above the sequence. The linkers L1, L12, and L2 are shown between these segments. The α-helical segment 2B has been extended to amino acid 378 (dashed line) to include the well-conserved motif terminating segment 2B in lamin and invertebrate IF proteins. A stutter (abcd) in segment 2B is indicated by asterisks. We have recently identified 51 nematocyte-specific genes using a cDNA microarray and in situ hybridization. Among these were 2 genes (hmp_08523 and hm_04087), which were strongly expressed in the tentacles after the differentiation of the nematocyst capsule was completed (Hwang et al. 2007). In the present study, we show that these cDNAs are derived from 2 paralogous genes encoding lamin-like proteins of 47 kDa. We have named these proteins nematocilin A and B. Using polyclonal antibodies directed against the recombinant proteins, we show that the proteins are localized in the core of the cnidocil, which was previously described as containing electron-dense, longitudinal filaments by electron microscopy (Slautterback 1967; Golz 1994). Sequence analysis shows that the proteins can form a coiled-coil α-helices consisting of 364 amino acids, which is highly homologous to intermediate filament (IF) proteins. Finally, we compare the structural diversity of cnidocils among different cnidarian lineages and discuss the origin of nematocilin and the function of the central filament. Materials and Methods Strain and Culture Conditions Hydra magnipapillata 105 (Hydrozoa; Cnidaria) was maintained at 18 °C in the “M” medium (1 mM NaCl, 1 mM CaCl2, 0.1 mM KCl, 0.1 mM MgSO4, and 1 mM Tris-(hydroxymethyl) aminoethane pH 7.6). Animals were fed 3 times a week with freshly hatched Artemia and washed with fresh medium after 6–8 h. Animals were starved for more than 24 h before experiments. Antibodies To express nematocilin A, a NotI–SalI fragment (amino acids 268–421) including mostly the coiled-coil segment 2B and partially the C-terminal domain was cloned into a protein expression vector pET28a (Novagen, Madison, WI). Sequence of cloned vector was verified and followed by transformation into Escherichia coli BL21 (DE3). Overexpressed protein in E. coli was purified by Ni-NTA affinity column (Invitrogen, Carlsbad, CA) and subsequently by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The gel fragment isolated from SDS-PAGE was used as an antigen to generate polyclonal antibody in rabbit. This anti-nematocilin polyclonal antibody was prepared by OPERON Biotechnologies, K.K. (Tokyo, Japan) and then purified on protein A-Sepharose column (Pharmacia Biotech, Inc., Tokyo, Japan). Whole Mount In Situ Hybridization In situ hybridization was carried out as described by Grens et al. (1996). The concentration of the riboprobe used for hybridization varied from 50 to 200 ng/ml. Western Blot Cell extracts from whole animals (42 μg per lane) were separated under reducing conditions by SDS-PAGE on a 12% polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride western blot membrane (Roche Diagnostics K.K., Tokyo, Japan). Following blocking in phosphate-buffered saline (PBS) containing 3% (w/v) bovine serum albumin (BSA) and 0.5% (v/v) Tween 20 overnight at 4 °C, the blot was incubated for 1 h at room temperature (RT) with anti-nematocilin antibody (1.1 mg/ml; 1:5,000) in PBS containing 0.5% gelatin. The blot was washed 3× 10 min with PBS containing 0.5% (v/v) Tween 20 at RT, incubated with goat anti-rabbit lgG conjugated to HRP (Santa Cruz Biotechnology, Santa Cruz, CA), and washed with PBS (as above). The blot was developed using chemiluminescence (Immobilon Western Chemiluminescent HRP substrate, Millipore, Bedford, MA) and exposed to film (Fujifilm, Tokyo, Japan). Confocal Immunofluoresence Microscopy Animals were fixed in Lavdowsky's fixative (50% ethanol, 3.7% formaldehyde, and 4% acetic acid in PBS) overnight at 4 °C. To permeabilize the cell membrane, animals were treated with 0.1% (v/v) Triton X-100 in PBS for 30 min at RT. Before immunostaining, animals were blocked in PBS buffer containing 1% (w/v) BSA and 0.1% (w/v) sodium azide for 1 h at 4 °C. Rabbit polyclonal anti-nematocilin antibody (1.1 mg/ml) was diluted 1:500 in blocking buffer above and incubated with animals overnight at 4 °C. Excess antibody was washed away 3 times with PBS, followed by a 30 min blocking with 0.1% (w/v) BSA in PBS and then a 2-h incubation at RT with Alexa Fluor 488 goat anti-rabbit lgG (1:200, Molecular Probes, Eugene, OR). Finally, animals were washed 3 times with PBS before imaging under a Zeiss confocal laser scanning microscope, LSM510 META (Carl Zeiss Co. Ltd., Jena, Germany) equipped with 488-nm argon-ion laser. Imaging was done using 100× Plan Apochromat NA 1.4 oil immersion objective and digitally captured at a resolution of 512 × 512 pixels. Z-series typically contained 15–20 1.2-μm optical sections for a total stack depth of 18–25 μm. For presentation, some image stacks were converted to maximum intensity projections using the Zeiss LSM510 META 3.0 software. Postembedding Immunogold Electron Microscopy Hydra tentacles were fixed in 0.1 M cacodylate buffer (pH 7.0) containing 4% paraformaldehyde and 0.05% glutaraldehyde overnight at 4 °C, followed by 3 washes of 10 min each in 0.1 M cacodylate buffer (pH 7.0). After dehydration through an increasing series of ethanol solutions, tentacles were infiltrated with medium grade LR White resin (London Resin Co. Ltd., Basingstoke, UK). Polymerization of resin was achieved by an accelerator compound at 4 °C for 2 days. Ultrathin sections (ca. 70 nm) were prepared using a Leica EM UC6 ultramicrotome and collected on a formvar-coated nickel grid. They were then treated with a saturated aqueous sodium metaperiodate (Sigma, St Louis, MO) for 10 min, rinsed with distilled water and finally with 0.1 M HCl for 10 min. After the rinse, nonspecific binding was blocked with BSA (0.1 M cacodylate buffer, pH 7.0; 0.1% Triton X-100; 1% BSA) for 30 min. Sections were then exposed to small drops of polyclonal anti-nematocilin antibody (1:100) diluted in blocking solution for 2 h, washed with 0.1 M cacodylate buffer (pH 7.0), and incubated with 1:100 diluted goat anti-rabbit IgG antibody linked with 1.4-nm colloidal gold particles (Nanoprobes Inc., Stony Brook, NY) for 2 h. Sections were then washed several times with 0.1 M cacodylate buffer (pH 7.0), and the gold particles were silver enhanced by HQ silver enhancement kit (Nanoprobes Inc.) according to the manufacturer's instruction. Finally, sections were stained with 2% aqueous uranyl acetate followed by 0.4% lead citrate for 10 min each and viewed with a JEOL transmission electron microscope (TEM) (JEM-1010) operating at 80 kV. Negative Staining and Immunogold Electron Microscopy of Isolated Cnidocils The isolation, dissociation, and immunogold labeling of cnidocil were performed according to Golz (1994) with a few modifications. Briefly, cnidocils were isolated from 30 to 40 animals by incubation in 7% ethylene glycol in cold M medium for 20 s. Isolated cnidocils were collected by centrifugation at 13,000 rpm for 10 min, and the pellet was resuspended in H2O before absorbing to the formvar- and carbon-coated grid. Although cnidocils were still wet, the grid was placed in a droplet of cold lysis buffer (10 mM cacodylate buffer pH 7.0, 10 mM ethylenediaminetetraacetic acid, 0.1% Triton X-100) containing 5% polyethylene glycol (MW 6,000) for 45 min. The grid was then placed in another droplet of cold lysis buffer containing 1% polyethylene glycol (MW 6,000) and 1 mM dithioerythritol (DTE) for 40 min. Dispersed filaments of cnidocils were fixed with 1% formaldehyde in 10 mM cacodylate buffer for 1 min and followed by staining 1% aqueous uranyl acetate for 20–30 s. After air-drying, negative-stained filaments were ready for TEM observation. For immunogold-labeling experiment, fixed filaments were treated with the blocking solution as above for 30 min and then incubated with anti-nematocilin antibody (1:1,000 dilution) 2 h at 4 °C. After several rinses with 0.1 M cacodylate buffer (pH 7.0), the grids were incubated with goat anti-rabbit IgG conjugated with 1.4-nm colloidal gold particles (Nanoprobes Inc., 1:1,000 dilution) for 2 h. The diameter of gold particles was enlarged by HQ SILVER enhancement kit. Finally, the grid was negatively stained with 1% aqueous uranyl acetate and air-dried before examination by TEM. Results and Discussion Nematocilin Is a Novel Protein Containing a Lamin-Like IF Domain Two nematocyte-specific cDNAs were identified previously by microarray analysis (hmp_08523 and hm_04087 in table 1; Hwang et al. 2007). The complete sequences of the corresponding genes were deduced from expressed tag sequences (ESTs) and the Hydra genome assembly (J. Craig Venter Institute, https://research.venterinstitute.org/files/hydra_magnipapillata_WGS_assembly). We have named the predicted proteins nematocilin A and B (DDBJ/GenBank/EMBL accession numbers BAG48261 and BAG48262, respectively). Nematocilin A and B are encoded by duplicated genes separated by 18.6 kb on one contig in the Hydra genome assembly (fig. 1B). Each nematocilin gene has 6 exons, and the exon structure is highly conserved with respect to number, size, and sequence similarity. By comparison, the introns vary in size and the 5′ and 3′ flanking regions (1,000 bp upstream and downstream) show relatively low sequence conservation (data not shown). Alignment of nematocilin A and B indicates that the amino acid sequences are 93% identical (fig. 1C). Both nematocilin genes appear to be expressed at roughly the same rate based on the frequency of ESTs matching each gene. Because nematocilin A and B encode almost identical proteins, we refer to them hereafter as nematocilin. Blasting nematocilin to the nonredundant protein database yields hits to nuclear lamins in Hydra and other invertebrates. This is due to the presence of an IF domain in nematocilin (fig. 1C). Proteins containing the IF domain are encoded by diverse gene families that only share sequence similarity in the central rod domain. Both N- and C-terminal regions can be variable in length (Parry et al. 2007). The central rod domain of IF proteins contains 4 α-helical heptad repeat motifs, 1A, 1B, 2A, and 2B, separated by short nonhelical sequences. Using the COILS prediction algorithms (version 2.2, Lupas et al. 1991), we could identify 4 heptad repeats in nematocilin. In these heptad repeats, the first and fourth residues were predominantly nonpolar (fig. 1C). The heptad repeats were interrupted by 3 short linker sequences, L1, L12, and L2. Nematocilin also contained a “stutter” in segment 2B, an insertion of 4 amino acids that interrupts the 2B heptad repeat and that is found conserved in all IF domains (fig. 1C) (Brown et al. 1996). Based on the homology to the Hydra nuclear lamin sequence (Erber et al. 1999), we extended segment 2B to include residue 378 that represents the well-conserved end of the 2B helix in this protein. Thus, nematocilin belongs to the IF superfamily. Nematocilin lacks a signal peptide at the N-terminus in contrast to other nematocyte-specific proteins such as minicollagen, spinalin, or nematocyst outer wall antigen (Kurz et al. 1991; Koch et al. 1998; Engel et al. 2002). This suggests that nematocilin is a cytoplasmic protein and not part of the nematocyst capsule. Nematocilin and the Structure of the Central Filament in the Cnidocil To determine the intracellular localization of nematocilin protein, we generated an antibody against the C-terminal half of nematocilin A (amino acids 268–421). The antibody detected a single band of approximately 47 kDa in western blots of cell lysates of Hydra (fig. 2A), corresponding to the expected molecular mass of nematocilin. Immunofluorescence staining of whole animals with the nematocilin antibody yielded a strong signal in the cnidocils of all 4 types of mature nematocytes mounted in the tentacles (fig. 2B–I). No immunofluorescence was present in the stereocilia or the inner microvilli. We also raised an antibody against the heptad repeat motif (amino acids 62–201) of nematocilin B. Western blots indicated that this antibody stained the 47-kDa nematocilin protein (data not shown). Staining whole mounts of Hydra with this antibody revealed an identical pattern to nematocilin A (data not shown). FIG. 2.— View largeDownload slide Immunolocalization of nematocilin using anti-nematocilin antibody. (A) Western blot of Hydra lysate using anti-nematocilin antibody. The band at 47 kDa corresponds to the expected size of nematocilin. (B–I) Confocal images of nematocilin localization using anti-nematocilin antibody. (B and C) Stenotele. (D and E) Holotrichous isorhiza. (F and G) Desmoneme. (H and I) Atrichous isorhiza. Scale bar, 10 μm. FIG. 2.— View largeDownload slide Immunolocalization of nematocilin using anti-nematocilin antibody. (A) Western blot of Hydra lysate using anti-nematocilin antibody. The band at 47 kDa corresponds to the expected size of nematocilin. (B–I) Confocal images of nematocilin localization using anti-nematocilin antibody. (B and C) Stenotele. (D and E) Holotrichous isorhiza. (F and G) Desmoneme. (H and I) Atrichous isorhiza. Scale bar, 10 μm. Electron micrographs of Hydra and Tubularia have shown that the central filament of cnidocils contains longitudinally organized fibers with a characteristic pattern of cross-striations (Slautterback 1967; Golz and Thurm 1991; Golz 1994). To localize nematocilin within the cnidocil, we stained sections with immunogold-labeled antibodies. Figure 3A–C shows thin sections of cnidocils in the tentacles of Hydra. The immunogold staining was clearly localized to the central filament in both longitudinal sections (fig. 3A and B) and cross sections (fig. 3C). The peripheral region containing doublet and singlet microtubules did not stain with the anti-nematocilin antibody. Incubation of isolated cnidocils with the reducing agent DTE causes them to dissociate into individual fibers with a diameter of 3–4 nm (Golz 1994). Figure 3D shows the distal end of such a partially dissociated cnidocil. The central filament is splayed out into hair-like fibers, which stained strongly with the immunogold-labeled nematocilin antibodies (fig. 3E and F). Hence, it seems reasonable to conclude that nematocilin fibers form the central filament. These nematocilin fibers are thinner than 8- to 11-nm IFs formed by IF proteins. However, they could represent protofilaments consisting of coiled-coil tetramers of nematocilin, arranged antiparallel to each other similar to the basic organization of IFs. FIG. 3.— View largeDownload slide Immunogold labeling of central filaments using anti-nematocilin antibody. (A) Immunogold electron microscopy of thin sections of Hydra tentacle. Gold particles (black dots) label the nematocilin in the cnidocil core. (B) Boxed region of (A). Microtubules (arrowhead) surround the central filament of the cnidocil. (C) Cross section near the base of the cnidocil apparatus. Gold particles (black dots) label the central filament (f); surrounding stereocilia (s) are not labeled. (D) Isolated cnidocil partially dissociated with DTE. The distal half of the cnidocil is spread out as single fibers. (E) Negative stain immunogold labeling of the dissociated fibers. (F) High magnification of immunogold-labeled fibers shown in (E). Gold particles (black dots) are associated with the dispersed fibers. Scale bar (A, C, D, E) 1 μm; (B and F) 200 nm. FIG. 3.— View largeDownload slide Immunogold labeling of central filaments using anti-nematocilin antibody. (A) Immunogold electron microscopy of thin sections of Hydra tentacle. Gold particles (black dots) label the nematocilin in the cnidocil core. (B) Boxed region of (A). Microtubules (arrowhead) surround the central filament of the cnidocil. (C) Cross section near the base of the cnidocil apparatus. Gold particles (black dots) label the central filament (f); surrounding stereocilia (s) are not labeled. (D) Isolated cnidocil partially dissociated with DTE. The distal half of the cnidocil is spread out as single fibers. (E) Negative stain immunogold labeling of the dissociated fibers. (F) High magnification of immunogold-labeled fibers shown in (E). Gold particles (black dots) are associated with the dispersed fibers. Scale bar (A, C, D, E) 1 μm; (B and F) 200 nm. The situation, however, is more complex. Golz (1994) isolated a monoclonal antibody, CFB43, which bound to the central filament of Hydra cnidocils. This antibody recognized a single protein of 33 kDa in western blots that is clearly different in size from the 47-kDa nematocilin. Hence, nematocilin is not the only protein in the central filament. Based on sequence characteristics, however, it seems likely that nematocilin forms the 3- to 4-nm fibers (see above). These fibers are bundled into a much larger structure, the central filament, and electron micrographs demonstrate that this structure is connected to microtubules surrounding it in the cnidocil (Golz 1994). Thus, a possible function for the CFB43 antigen could be to interact with nematocilin fibers, assembling them into the larger central filament and/or binding them to microtubules in the periphery. Nematocilin Is Expressed at a Late Stage of Nematocyte Differentiation To localize nematocilin transcripts, we carried out in situ hybridization on whole mount Hydra. Nematocilin was strongly expressed in nematocytes in the tentacles of Hydra and in migrating nematocytes in the body column (fig. 4A). Expression was also found in a few clusters of nematocytes containing fully developed capsules at the end of the differentiation process and those that are about to fall apart into single nematocytes (fig. 4B). These results show that expression of nematocilin transcripts begins after the nematocyst capsule is fully assembled. Immunofluoresence staining with nematocilin antibody supported this conclusion. Differentiating nematocytes in nests were not stained. Migrating nematocytes, by comparison, showed diffuse cytoplasmic staining (fig. 4C and D). After single nematocytes were mounted in battery cells, the staining disappeared from the cytoplasm and became localized in the cnidocil (fig. 2B–I). Taken together, these results suggest that nematocilin synthesis begins in migrating nematocytes and that the protein accumulates in the cytoplasm until cnidocil formation starts. When cnidocil formation occurs after the mounting of nematocytes in battery cells, nematocilin disappears from the cytoplasm and is concentrated in the cnidocil. FIG. 4.— View largeDownload slide Expression of nematocilin in nematocytes. (A) Whole mount in situ hybridization with a nematocilin probe showing intense staining in the tentacles and dispersed stained cells in the body column. (B) Enlargement of the body column shown in (A). Arrows indicate clusters of nematocytes at a late stage of differentiation in the body column. (C and D) Immunofluorescence signal shows nematocilin localized in cytoplasm of a migrating stenotele at the body column of Hydra. Arrowheads point to mesoglea. Ec, ectoderm; En, endoderm. Scale bar, 10 μm. FIG. 4.— View largeDownload slide Expression of nematocilin in nematocytes. (A) Whole mount in situ hybridization with a nematocilin probe showing intense staining in the tentacles and dispersed stained cells in the body column. (B) Enlargement of the body column shown in (A). Arrows indicate clusters of nematocytes at a late stage of differentiation in the body column. (C and D) Immunofluorescence signal shows nematocilin localized in cytoplasm of a migrating stenotele at the body column of Hydra. Arrowheads point to mesoglea. Ec, ectoderm; En, endoderm. Scale bar, 10 μm. Evolution of Nematocilin and Its Phylogenetic Relationship with Lamin Nematocilin A and B have highest sequence homology to nuclear lamins and invertebrate IF proteins. The sequence similarity is confined to the amino acids 15–378 that correspond to the IF domain in the homologous proteins. In particular, nematocilin has the long form of the 1B helix domain which is characteristic of all nuclear lamins and invertebrate IF proteins. The C-terminal 90 amino acids of nematocilin show no homology to the C-terminal domains of lamins or IF proteins. Specifically, this means that nematocilin lacks the nuclear localization signal domain, the C-terminal CaaX domain, which is the site of N-farnesylation, and the lamin homology domain, a block of 105 amino acids, which is conserved in lamins (Erber et al. 1998). Hydra has, in addition to nematocilin, a nuclear lamin gene (Erber et al. 1999), which is homologous to nuclear lamins over its whole length including the C-terminal domains absent in nematocilin. In the anthozoan Nematostella, a cnidarian for which a whole-genome sequence is now available (Putnam et al. 2007), there is a clear homolog of the nuclear lamin gene but no homolog of nematocilin. This observation is consistent with the idea that a nuclear lamin gene was present in the common ancestor of both Hydra and Nematostella and presumably in all cnidarians. Based on the sequence similarity between lamin and nematocilin, it is parsimonious to propose that a primordial lamin gene was duplicated in the cnidarian, lineage giving rise to lamin genes in all Cnidaria and to nematocilin homologs in the lineage leading to Hydra. To support this hypothesis, we have searched available genomic and EST databases from lower metazoans and single-celled eukayotes for lamin and nematocilin homologs. The single-celled eukaryote Monosiga lacks a lamin gene, as do the yeast Saccharomyces cerevisiae and the plant Arabidopsis. This suggests that lamin genes arose in metazoan animals. Trichoplax and the demosponge Amphimedon have lamin genes as do all Cnidaria for which data are available including the anthozoans Nematostella, Tealia, Acropora, and Metridium and the hydrozoans Hydra, Clytia, and Cladonema (supplementary fig. S1, Supplementary Material online). Nematocilin genes, by contrast, are only present in the hydrozoans Hydra, Clytia, and Cladonema (supplementary fig. S1, Supplementary Material online). Molecular phylogenies of the α-helical segments 1A and 1B of cnidarian lamins and nematocilins together with sponge and bilaterian lamin sequences (supplementary figs. S2 and Supplementary Data, Supplementary Material online) support the hypothesis that nematocilin arose from a lamin gene precursor. The Neighbor-Joining (NJ) tree revealed that vertebrate and invertebrate lamins are separated into 2 distinct groups and that vertebrate lamins are further subdivided into 4 groups (A/C, L3, B1, and B2) (supplementary fig. S2, Supplementary Material online). Moreover, the NJ analysis suggests that nematocilins share a common ancestor with cnidarian lamins and emerged after the divergence between the Cnidaria and the Bilateria. The maximum likelihood tree has the same overall topology as the NJ tree although it has relatively low bootstrap values (supplementary fig. S3, Supplementary Material online). The phylogenetic analyses support our hypothesis that nematocilin originated from a lamin gene duplication after Cnidaria separated from the bilaterian lineage. It suggests further that nematocilin gene was lost in the anthozoan lineage, which is consistent with the observation that a central filament is not present in cnidocils of anthozoans (see below). The conservation of exon structure between the lamin and nematocilin genes in Hydra provides additional support for this idea. Exons 2 and 4 are identical in length, and exons 1 and 3 are very similar in length (fig. 5A). Mapping the 4 α-helical domains onto the exons also supports the strong similarity of exons in lamin and nematocilin genes (fig. 5B). In addition, it indicates that exon 5 (221 nt) of lamin corresponds to the first half of exon 5 (370 nt) of nematocilin. The C-terminal exons 6–10 of lamin show no similarity to the nematocilin sequence and were presumably lost during formation of the nematocilin gene. A similar argument has been made previously that the nuclear lamin gene is the ancestor of IFs. Many IF proteins in proteostomes retain the important features of lamin by having a C-terminal “tail” domain and an additional 42 amino acids in the α-helical segment 1B. These are all missing in the vertebrate IF proteins (Erber et al. 1998). Exon–intron structure has also been shown to be conserved between lamins and IF proteins (Dodemont et al. 1990; Döring and Stick 1990). FIG. 5.— View largeDownload slide Evolution of nematocilin. (A) Comparison of exon size between nuclear lamin and nematocilin A. Number of bases of each exon is shown in the box. (B) Sequence alignment of exons of Hydra lamin and nematocilin A. Colored residues indicate the 4 heptad repeat segments found in IF family proteins and lamin (Erber et al. 1999). Red, segment 1A; orange, segment 1B; light blue, segment 2A; and green, segment 2B. Asterisk shows the identical residues. FIG. 5.— View largeDownload slide Evolution of nematocilin. (A) Comparison of exon size between nuclear lamin and nematocilin A. Number of bases of each exon is shown in the box. (B) Sequence alignment of exons of Hydra lamin and nematocilin A. Colored residues indicate the 4 heptad repeat segments found in IF family proteins and lamin (Erber et al. 1999). Red, segment 1A; orange, segment 1B; light blue, segment 2A; and green, segment 2B. Asterisk shows the identical residues. Evolution of a Novel Structure: The Central Filament in the Hydra Cnidocil Anthozoans have a short 9 + 2 cnidocil, which lacks a central filament. In hydrozoans, the cnidocil is longer and has a central core, which is surrounded by 9 doublet microtubules (fig. 6). In some cases, such as Physalia and Polypodium, this core contains numerous singlet microtubules, which presumably stiffen it (Cormier and Hessinger 1980; Raikova 1990). In other cases, such as Hydra, the core has densely packed filaments in the center surrounded by a few singlet microtubules. This novel structure is found in Hydra, several closely related hydrozoans and the cubozoan Carybdea (Golz and Thurm 1993). Golz and Thurm (1991) have suggested that the function of this central filament is to increase the stiffness of the cnidocil and thus to concentrate the effect of a mechanical stimulus to the base of the cnidocil where it is linked to the stereocilia. They demonstrated the presence of cross bridges linking the membrane of the cnidocil just above its base to the tips of the stereocilia, and they hypothesized that bending of the cnidocil in response to mechanical stimulation stretches these cross bridges leading to transduction of the mechanical stimulus. A similar configuration of cross bridges between a central cilium and stereocilia occurs in the vertebrate hair cell. In this mechanoreceptive organelle, the cross bridges have recently been identified as cadherin, and it has been shown that mutations in cadherin disrupt mechanoreception (Siemens et al. 2004; Söllner et al. 2004). These results provide strong support for the Golz and Thurm hypothesis about the function of cnidocil apparatus in mechanoreception. Hence, the existence of central filament not only gives the structural diversity of cnidocil among the cnidarians but also enriched the functional novelty of nematocytes in response to the mechanical stimuli. In summary, results presented here show that evolution of the central filament was accompanied by the evolution of nematocilin, a novel 47-kDa member of the IF family, which is specifically expressed in differentiating nematocytes and localized in the cnidocil. FIG. 6.— View largeDownload slide Cnidocil structure in different cnidarian groups. Schematic cross sections of cnidocils show 9 doublet microtubules in the peripheral region and either single microtubules and/or filaments in the central core. Groups for which data are not available have been left blank. +, number of structural unit is 10 or less; ++, number of structural unit is more than 10 but less than 100; +++, number of structural unit is 100 or more; and −, not present. FIG. 6.— View largeDownload slide Cnidocil structure in different cnidarian groups. Schematic cross sections of cnidocils show 9 doublet microtubules in the peripheral region and either single microtubules and/or filaments in the central core. Groups for which data are not available have been left blank. +, number of structural unit is 10 or less; ++, number of structural unit is more than 10 but less than 100; +++, number of structural unit is 100 or more; and −, not present. We thank Chie Iwamoto for the construction and purification of recombinant protein. We are grateful to Elsa Denker, Michaël Manuel, and Evelyn Houliston for the Clytia EST sequences and also Daniel Rokhsar for the Amphimedon lamin sequence. The authors also acknowledge all providers of public genome sequences especially The US Department of Energy Joint Genome Institute. This work was generously supported by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan. 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TI - Cilium Evolution: Identification of a Novel Protein, Nematocilin, in the Mechanosensory Cilium of Hydra Nematocytes
JF - Molecular Biology and Evolution
DO - 10.1093/molbev/msn154
DA - 2008-07-17
UR - https://www.deepdyve.com/lp/oxford-university-press/cilium-evolution-identification-of-a-novel-protein-nematocilin-in-the-AiZD2gRGzd
SP - 2009
EP - 2017
VL - 25
IS - 9
DP - DeepDyve
ER -