Cilium Evolution: Identification of a Novel Protein, Nematocilin, in the
Mechanosensory Cilium of Hydra Nematocytes
Jung Shan Hwang,* Yasuharu Takaku,* Jarrod Chapman, Kazuho Ikeo,* Charles N. David,à and
Takashi Gojobori*
*Center for Information Biology and DDBJ, National Institute of Genetics, Mishima, Japan; Department of Energy, Joint Genome
Institute, Walnut Creek, CA; and àDepartment Biologie II, Ludwig Maximilians University, Munich, Germany
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.
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 þ 2 arrangement of microtubules (Westfall 1965; Schmidt and Moraw 1982; Watson
Key words: cnidaria, cnidocil, evolution, Hydra, intermediate
filament, nematocyst.
E-mail: [email protected].
Mol. Biol. Evol. 25(9):2009–2017. 2008
doi:10.1093/molbev/msn154
Advance Access publication July 17, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
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.
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 a-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.
2010 Hwang et al.
FIG. 1.—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 a-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.
Antibodies
Whole Mount In Situ Hybridization
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).
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 lg 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/
Modified Cilium in Hydra 2011
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 argonion 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-lm optical sections for a total stack
depth of 18–25 lm. 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 formvarand 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
2012 Hwang et al.
FIG. 2.—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 lm.
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 a-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).
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
Modified Cilium in Hydra 2013
FIG. 3.—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 lm; (B and F) 200 nm.
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.
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.
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
2014 Hwang et al.
FIG. 4.—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 lm.
of lamins or IF proteins. Specifically, this means that nematocilin lacks the nuclear localization signal domain, the Cterminal 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 a-helical segments 1A
and 1B of cnidarian lamins and nematocilins together with
FIG. 5.—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.
Modified Cilium in Hydra 2015
FIG. 6.—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. þ, number of structural
unit is 10 or less; þþ, number of structural unit is more than 10 but less than 100; þþþ, number of structural unit is 100 or more; and , not present.
sponge and bilaterian lamin sequences (supplementary figs.
S2 and S3, 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, Sup-
plementary 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
2016 Hwang et al.
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 ahelical 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 a-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).
elty 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.
Supplementary Material
Supplementary figures S1– S3 are available at
Molecular Biology and Evolution online (http://www.
mbe.oxfordjournals.org/).
Acknowledgments
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.
Evolution of a Novel Structure: The Central Filament in
the Hydra Cnidocil
Literature Cited
Anthozoans have a short 9 þ 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 nov-
Brown JH, Cohen C, Parry DA. 1996. Heptad breaks in alphahelical coiled coils: stutters and stammers. Proteins. 26:134–145.
Carré D, Carré C. 1980. On triggering and control of cnidocyst
discharge. Mar Behav Physiol. 7:109–117.
Clausen C. 1991. Differentiation and ultrastructure of nematocysts in Halammohydra intermedia (Hydrozoa, Cnidaria).
Hydrobiologia. 216/217:623–628.
Cormier SM, Hessinger DA. 1980. Cnidocil apparatus: sensory
receptor of Physalia nematocytes. J Ultrastruct Res. 72:
13–19.
David CN, Challoner D. 1974. Distribution of interstitial cells
and differentiating nematocytes in nests in Hydra attenuata.
Am Zool. 14:537–542.
David CN, Gierer A. 1974. Cell cycle kinetics and development
of Hydra Attenuata III. Nerve and nematocyte differentiation.
J Cell Sci. 16:359–375.
David CN, Murphy S. 1977. Characterization of interstitial stem
cells in hydra by cloning. Dev Biol. 58:372–383.
Dodemont H, Riemer D, Weber K. 1990. Structure of an
invertebrate gene encoding cytoplasmic intermediate filament
(IF) proteins: implications for the origin and the diversification of IF proteins. EMBO J. 9:4083–4094.
Döring V, Stick R. 1990. Gene structure of nuclear lamin LIII of
Xenopus laevis: a model for the evolution of IF proteins from
a lamin-like ancestor. EMBO J. 9:4073–4081.
Engel U, Özbek S, Streitwolf-Engel R, Petri B, Lottspeich F,
Holstein TW. 2002. Nowa, a novel protein with minicollagen
Cys-rich domains, is involved in nematocyst formation in
Hydra. J Cell Sci. 115:3923–3934.
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. Characterization
Modified Cilium in Hydra 2017
of the Hydra lamin and its gene: a molecular phylogeny of
metazoan lamins. J Mol Evol. 49:260–271.
Golz R. 1994. Ultrastructure and molecular composition of the
central filament body in hydrozoan cnidocils. Eur J Cell Biol.
65:39–48.
Golz R. 1995. Cytoskeletal elements in migrating and tentacleintegrated nematocytes of a marine hydrozoan. Biol Cell.
83:49–59.
Golz R, Thurm U. 1991. Cytoskeleton-membrane interactions in
the cnidocil complex of hydrozoan nematocytes. Cell Tissue
Res. 263:573–583.
Golz R, Thurm U. 1993. Ultrastructural evidence for the
occurrence of three types of mechanosensitive cells in the
tentacles of the cubozoan polyp Carybdea marsupialis.
Protoplasma. 173:13–22.
Grens A, Gee L, Fisher DA, Bode HR. 1996. CnNK-2, an NK-2
homeobox gene, has a role in patterning the basal end of the
axis in Hydra. Dev Biol. 180:473–488.
Hausmann K, Holstein T. 1985. Bilateral symmetry in the
cnidocil-nematocyst complex of the freshwater medusa
Craspedacusta sowerbii Lankester (Hydrozoa, Limnomedusae). J Ultra Res. 90:89–104.
Holstein T, Hausmann K. 1988. The cnidocil apparatus of
hydrozoans: A progenitor of higher metazoan mechanoreceptors. In: Hessinger DA, Lenhoff HM, editors. The biology
of nematocysts. New York: Academic Press. p. 53–73.
Holstein T, Tardent P. 1984. An ultrahigh-speed analysis of
exocytosis: nematocyst discharge. Science. 223:830–833.
Holtmann M, Thurm U. 2001. Variations of concentric hair cells
in a cnidarian sensory epithelium (Coryne tubulosa). J Comp
Neurol. 432:550–563.
Hwang JS, Ohyanagi H, Hayakawa S, Osato N, NishimiyaFujisawa C, Ikeo K, David CN, Fujisawa T, Gojobori T.
2007. The evolutionary emergence of cell type specific genes
inferred from the gene expression analysis of Hydra. Proc
Natl Acad Sci USA. 104:14735–14740.
Koch AW, Holstein TW, Mala C, Kurz E, Engel J, David CN.
1998. Spinalin, a new glycine- and histidine-rich protein in
spines of Hydra nematocysts. J Cell Sci. 111:1545–1554.
Kurz EM, Holstein TW, Petri BM, Engel J, David CN. 1991. Minicollagens in hydra nematocytes. J Cell Biol. 115:1159–1169.
Lupas A, Van Dyke M, Stock J. 1991. Predicting coiled coils
from protein sequences. Science. 252:1162–1164.
Parry DAD, Strelkov SV, Burkhard P, Aebi U, Herrmann H. 2007.
Towards a molecular description of intermediate filament
structure and assembly. Exp Cell Res. 313:2204–2216.
Putnam NH, Srivastava M, Hellsten U, et al. (19 co-authors).
2007. Sea anemone genome reveals ancestral eumetazoan
gene repertoire and genomic organization. Science. 317:
86–94.
Raikova EV. 1990. Fine structure of the nematocytes of
Polypodium hydriforme Ussov (Cnidaria). Zool Scr. 19:
1–11.
Schmidt H, Moraw B. 1982. Die Cnidogenese der Octocorallia
(Anthozoa, Cnidaria): II. Reifung, Wanderung und Zerfall
von Cnidoblast und Nesselkapsel. Helgoland Marine Research. 35:97–118.
Shimizu H, Bode HR. 1995. Nematocyte differentiation in
Hydra: Commitment to nematocyte type occurs at the
beginning of the pathway. Dev Biol. 169:136–150.
Siemens J, Lillo C, Dumont RA, Reynolds A, Williams DS,
Gillespie PG, Müller U. 2004. Cadherin 23 is a component of
the tip link in hair-cell stereocilia. Nature. 428:950–955.
Slautterback DB. 1967. The cnidoblast-musculoepithelial
cell complex in the tentacles of Hydra. Z Zellforsc.
79:296–318.
Söllner C, Rauch GJ, Siemens J, Geisler R, Schuster SC. The
Tübingen 2000 Screen Consortium, 2004. Müller U,
Nicolson T. 2004. Mutations in cadherin 23 affect tip links
in zebrafish sensory hair cells. Nature. 428:955–959.
Thurm U, Lawonn P. 1990. The sensory properties of the
cnidocil-apparatus as a basis for prey capture in Hydra
attenuata. Verh Dtsch Zool Ges. 83:431–432.
Watson GM, Mariscal RN. 1983. Comparative ultrastructure of
catch tentacles and feeding tentacles in the sea anemone
Haliplanella. Tissue Cell. 15:939–953.
Weis VM, Keene DR, Buss LW. 1985. Biology of Hydractiniid
hydroids. 4. Ultrastructure of the planula of Hydractinia
echinata. Biol Bull. 168:403–418.
Westfall JA. 1965. Nematocysts of the sea anemone Metridium.
Am Zool. 5:377–393.
Westfall JA. 1970. The nematocyte complex in a hydromedusan,
Gonionemus vertens. Cell Tissue Res. 110:457–470.
Westfall JA, Sayyar KL, Elliott CF. 1998. Cellular origins of
kinocilia, stereocilia, and microvilli on tentacles of sea
anemones of the genus Calliactis (Cnidaria: Anthozoa).
Invertebr Biol. 117:186–193.
Sudhir Kumar, Associate Editor
Accepted June 30, 2008