The novel protein Nowa was identified in nematocysts, explosive organelles of Hydra, jellyfish, corals and other Cnidaria. Biogenesis of these organelles is complex and involves assembly of proteins inside a post-Golgi vesicle to form a double-layered capsule with a long tubule. Nowa is the major component of the outer wall, which is formed very early in morphogenesis. The high molecular weight glycoprotein has a modular structure with an N-terminal sperm coating glycoprotein domain, a central C-type lectin-like domain, and an eightfold repeated cysteine-rich domain at the C-terminus. Interestingly, the cysteine-rich domains are homologous to the cysteine-rich domains of minicollagens. We have previously shown that the cysteines of these minicollagen cysteine-rich domains undergo an isomerization process from intra- to intermolecular disulfide bonds, which mediates the crosslinking of minicollagens to networks in the inner wall of the capsule. The minicollagen cysteine-rich domains present in both proteins provide a potential link between Nowa in the outer wall and minicollagens in the inner wall. We propose a model for nematocyst formation that integrates cytoskeleton rearrangements around the post-Golgi vesicle and protein assembly inside the vesicle to generate a complex structure that is stabilized by intermolecular disulfide bonds.

Pattern formation within single cells has been recently recognized as a fundamental but not well understood aspect of cell biology(Shulman and St Johnston,1999). A particularly fascinating example is the morphogenesis of the nematocyst, a complex structure that develops inside a giant secretory vesicle in the cnidarian nematocyte(Slautterback and Fawcett,1959; Holstein,1981). Upon stimulation of the nematocyte, the nematocyst discharges explosively, a process that takes less than 3 milliseconds(Holstein and Tardent,1984).

The basic structure of the nematocyst consists of a capsule with a double-layered wall, a matrix with an inverted tubule bearing spines, and an operculum. Based on this structure, a wide diversity of morphological types of nematocysts (Fig. 1A) has evolved that serve different functions such as capture of prey and defense(Mariscal, 1974;Holstein et al., 1990).

Fig. 1.

Localization of mAb H22 antigen in the outer wall of nematocysts. (A)Isolated nematocysts of Hydra. The different capsule types are: d,desmoneme; i, holotrichous isorhiza; s, stenotele. One of the stenoteles has discharged (s*). (A′) Immunofluorescence of mAb H22 viewed by confocal microscopy (maximum projection) in the isolated nematocysts shown in A. (B) Mature, isolated nematocyte of Hydra. Note the size of the nematocyst vesicle, which fills almost the whole cell (N, nucleus). (C) EM immunogold labeling of mAb H22 in the wall of a Hydra nematocyst. Gold particles are exclusively found in the outer wall (ow) and not in the inner wall (iw). (D) SDS-PAGE and western analysis of whole Hydra and isolated capsules. The protein of 500,000 isolated capsules was compared with 1/5 Hydra (1/5H) by silver-staining. In a western blot, protein of 1 Hydra (1H) and 500,000 capsules separated by SDS-PAGE were probed with mAb H22 and minicollagen antibody. A high molecular weight protein is detected by mAb H22 in isolated capsules, but not in whole Hydra. Minicollagens detected by minicollagen-1 antibody represent the major proteins of the capsule. Bars, 5 μm (A,B); 100 nm (C).

Fig. 1.

Localization of mAb H22 antigen in the outer wall of nematocysts. (A)Isolated nematocysts of Hydra. The different capsule types are: d,desmoneme; i, holotrichous isorhiza; s, stenotele. One of the stenoteles has discharged (s*). (A′) Immunofluorescence of mAb H22 viewed by confocal microscopy (maximum projection) in the isolated nematocysts shown in A. (B) Mature, isolated nematocyte of Hydra. Note the size of the nematocyst vesicle, which fills almost the whole cell (N, nucleus). (C) EM immunogold labeling of mAb H22 in the wall of a Hydra nematocyst. Gold particles are exclusively found in the outer wall (ow) and not in the inner wall (iw). (D) SDS-PAGE and western analysis of whole Hydra and isolated capsules. The protein of 500,000 isolated capsules was compared with 1/5 Hydra (1/5H) by silver-staining. In a western blot, protein of 1 Hydra (1H) and 500,000 capsules separated by SDS-PAGE were probed with mAb H22 and minicollagen antibody. A high molecular weight protein is detected by mAb H22 in isolated capsules, but not in whole Hydra. Minicollagens detected by minicollagen-1 antibody represent the major proteins of the capsule. Bars, 5 μm (A,B); 100 nm (C).

Nematocyst morphogenesis can be subdivided into five stages(Holstein, 1981). (1) An early growth phase during which the capsule primordium forms and grows by addition of new vesicles to the vesicle harboring the capsule. (2) A late growth phase during which a tubule forms outside the capsule by addition of more vesicles;capsule and tubule wall form a continuous structure. (3) Invagination of the long external tubule into the capsule. (4) An early maturation phase leading to the formation of spines by condensation of the protein spinalin(Koch et al., 1998) inside the invaginated tubule. (5) A final late maturation step during which poly-γ-glutamate is synthesized in the matrix of the capsule. This generates an osmotic pressure of 150 bar that drives discharge(Weber, 1990;Szczepanek et al., 2002).

The extremely high pressure in mature capsules requires high tensile strength of the wall. This tensile strength is mediated by minicollagens, a family of very short collagens that form the capsule's inner wall(Kurz et al., 1991;Holstein et al., 1994). In a previous paper (Engel et al.,2001), we have shown that wall maturation involves polymerization of minicollagens to an insoluble polymer. This polymerization is mediated by disulfides in the minicollagen cysteine-rich domains (MCCR domains) that undergo a switch from intra-chain to inter-chain disulfide bonds in the late maturation phase. The inner wall layer, formed by minicollagens, is covered by an outer wall layer, which is more electron-dense than the inner wall in EM sections of nematocysts (Holstein,1981; Watson and Mariscal,1984) and appeared as a layer of globular material in field emission scanning electron microscopy(Holstein et al., 1994). The molecular nature of this outer wall was previously unknown.

In this study we used the monoclonal antibody H22 (mAb H22)(Kurz et al., 1991), which stained the outer wall of Hydra nematocysts throughout morphogenesis and in mature ready-to-discharge nematocysts in the tentacles(Engel et al., 2001). Isolation and cloning of the mAb H22 antigen revealed a completely novel protein that we call nematocyst outer wall antigen (Nowa). The C-terminal part of the 774-residue protein is characterized by an eightfold repetition of Cys-rich(cysteine-rich) domains homologous to minicollagen Cys-rich domains (MCCR domains). These domains present in Nowa and minicollagens suggest that the two proteins interact during wall formation. We propose a model that integrates the role of the MT (microtubule) cytoskeleton and the interaction of Nowa and minicollagen in forming the nematocyst wall.

Strains and culture conditions

Hydra magnipapillata (strain 105) was used for all experiments,except for generation of cDNA, where Hydra vulgaris was used. Animals were cultured in M solution under standard conditions(Loomis and Lenhoff,1956).

Antibodies

Supernatants from hybridoma cultures of mAb H22(Kurz et al., 1991) were directly used for immunocytochemistry. A polyclonal antibody directed against the recombinantly expressed Nowa CTLD was generated in rabbits by Eurogentec(Herstal, Belgium). Immunization was carried out following a standard protocol using 100 μg of purified and refolded CTLD protein in PBS. Minicollagen antibody (Engel et al., 2001)and spinalin antibody (Koch et al.,1998) are polyclonal rabbit antisera generated against recombinantly expressed Hydra proteins. The mAb directed againstβ-tubulin (mAb anti-tubulin) from Physarium polycephalum was obtained from Chemicon.

Immunofluorescence

Animals were relaxed in 2% urethane in M solution for 2 minutes and fixed either in Lavdovsky's fixative (50% ethanol, 3.7% formaldehyde, 4% acetic acid in water) or 4% paraformaldehyde for 24 hours. The fixative was removed by several washes in PBS, and membranes were opened by an incubation of 30 minutes in 0.1% Triton X-100. Animals were incubated in mAb H22 overnight. After several washes in PBS, the animals were incubated for 5 hours in antimouse antibody coupled to Alexa-488 fluorochrome (Molecular Probes)diluted 1:400 in PBS with 1% BSA, and excess antibody was removed by washing again. For double-staining, minicollagen-1 antibody diluted 1:500 in mAb H22 or spinalin antibody diluted 1:10 in mAb H22 were added to animals and subsequently detected by simultaneous incubation with anti-rabbit antibody coupled to Alexa-568 fluorochrome and anti-mouse antibody coupled to Alexa-488 fluorochrome.

For double-staining with the two monoclonal antibodies H22 and anti-tubulin, animals were fixed in 4% paraformaldehyde in PBS with 0.1%Triton-X for 10 minutes and an additional hour without detergent. After incubation with mAb H22, binding sites on the FC of mAb H22 were blocked with a polyclonal anti-mouse IgG antibody (Sigma) diluted 1:100 in PBS with 1% BSA for 5 hours. Detection of mAb H22 was achieved indirectly with anti-rabbit antibody coupled to Alexa-568 diluted 1:400 in PBS 1% BSA. The subsequent staining with mAb anti-tubulin (2 μg/ml in 1% BSA in PBS)followed the protocol described for mAb H22 described above, but incubation times were shortened to limit exchange reactions on the FC of the two monoclonal antibodies. Nuclei were stained with 0.5 μg/ml 6′-diamidino-2 phenylindole (DAPI) or Yoyo-1 (Molecular Probes) before mounting the animals on objective slides.

Macerated cells (David,1973) were incubated with mAb anti-tubulin diluted 1:10 in PBS with 1% BSA overnight. After three washes in PBS, an anti-mouse antibody coupled to Alexa-488 was added for 4 hours and excess antibody removed by washing.

Isolated capsules (Weber et al.,1987) were stained without fixation in mAb H22 overnight(4°C), washed three times by centrifugation in PBS 0.003% Triton X-100 (5 minutes, 500 g), and incubated in anti-mouse antibody coupled to Alexa-488 (3 hours) and washed again.

Determination of the labeling-index in mAb-H22-positive cells

Hydra were labeled with [3H]thymidine (50 μCi/ml) as described (Holstein and David,1990). Macerates (David,1973) of labeled polyps were stained with mAb H22 and FITC-conjugated goat anti-mouse antibody, dipped into autoradiographic photo-emulsion (Kodak NTB-2), exposed for 10 days at 4°C and developed.

Confocal microscopy and deconvolution

Whole mounts and macerated Hydra were viewed and documented on a confocal laser scanning microscope (Leica TCS SP). Single photon excitation was generated with an Argon-Crypton laser and 2-photon excitation with a femtosecond pulsed Ti:sapphire laser (Tsunami, Spectra Physics) pumped by a Nd:YVO4 laser (Millenia V, Spectra Physics). The 2-photon laser was used for excitation of DAPI. The confocal micrographs are shown either as single optical sections or as projections through a series of optical planes(indicated in the legend). Overlay of multiple channels, projections, and 3D rendering of stacks were done using Leica confocal software 2.00 and Imaris 3.0 software (Bitplane). Deconvolution of image stacks to remove background and improve axial and lateral resolution was performed with Huygens System 2 software (Scientific Volume Imaging).

Electron microscopy

Conventional TEM of Hydra vulgaris and Forskåliasp. was performed as described (Holstein,1981). For immunogold TEM, Hydra polyps body column pieces were fixed in a mixture of 0.2% glutaraldehyde and freshly prepared 2%formaldehyde buffered with 50 mM phosphate-buffer (pH 7.2). Specimens were dehydrated in dimethyl-formamid (50%, 70%, 90%) and embedded in a series of increasing lowicryl resin concentrations (DMF:lowicryl 2:1 for 15 minutes, 1:1 for 30 minutes, 1:2 for 2 hours, and 100% lowicryl for 12 hours) at 4°C. UV-polymerization occurred at 0°C for three days. Ultrathin sections were transferred to formvar-coated Ni-grids, incubated with mAb H22 (12 hours,20°C), washed with PBS, incubated with 10 nm kolloidal gold-coupled goat anti-mouse IgG serum (Sigma) diluted 1:20 in PBS for 2 hours at 20°C and washed with PBS. Specimens were contrasted with 2% lead citrate (1 minute) and analyzed in a Zeiss EM9-S2 electron microscope.

SDS-PAGE, 2D electrophoresis and western analysis

SDS-PAGE of isolated capsules and whole Hydra was performed as described (Engel et al., 2001). For enzymatic deglycosylation, two million isolated capsules were solubilized in 40 μl digestion buffer (0.5% octoglucoside, 10 mM EDTA, 20 mM Na-phosphate pH 7.2) supplemented with 0.25 M β-mercaptoethanol and 0.5%SDS for 30 minutes at 70°C. The sample was then diluted with digestion buffer to 180 μl and insoluble material removed by centrifugation at 13,000 g for 5 minutes. Aprotinin and leupeptin (1 μg/ml) were added to the supernatant. The sample was split, 10 μl (10 U) N-glycosidase F (Roche) was added to the digestion sample, and 10 μl buffer to the mock control, respectively. Both samples were incubated at 37°C overnight, and protein was then precipitated by addition of 30% trichloroacetic acid and analyzed by SDS-PAGE.

2D electrophoresis of capsule proteins was performed using a previously described method (Görg et al.,1988). In the first dimension proteins were focussed on IPG-strips with Multiphor II (Amersham Pharmacia Biotech) essentially following the manufacturer's instructions. Isolated capsules (2 million) were solubilized in 150 μl first dimension buffer (8 M urea, 4% CHAPS, 20 mg/ml DTT, 5 μg/ml leupeptin, 5 μg/ml, aprotinin, 1 mM EDTA and 1% IPG-buffer) at 35°C for 30 minutes. Insoluble material was removed by 5 minutes centrifugation at 13,000 g and the supernatant supplemented to 350 μl with first dimension buffer. IPG-strips (13 cm) with a linear pH gradient from pH 3-10 were rehydrated with the protein sample overnight and proteins were focussed for 20,000-23,000 Vh. For subsequent separation according to molecular weight, strips were equilibrated in second dimension buffer (50 mM Tris-HCl, pH 6.8, 6M urea, 30% glycerol, 2% SDS and a trace of bromphenol blue) for 15 minutes with 10 mg/ml DTT, and proteins were separated by SDS-PAGE.

For western analysis of proteins transferred to a nitrocellulose membrane(Towbin et al., 1979), blots were blocked for 1 hour with 1% BSA in TBS with 0.05% Tween (TBST). All further incubations and washes (3-4 times for 10 minutes after each antibody incubation) were performed in TBST. Blots were incubated with mAb H22 diluted 1:10 for 2 hours, washed and incubated with anti-mouse IgG antibody coupled to alkaline phosphatase (Promega) diluted 1:7000 for 1 hour. The signal was visualized by enyzmatic precipitation of the color substrate NBT/BCIP (Roche). Detection of minicollagen antibody (1:500) and anti-CTLD antibody (1:200) was performed with anti-rabbit horseradish peroxidase (1:10,000) and the ECL chemoluminescence system (Amersham) according to the manufacturer's instructions.

Tryptic digestion and peptide sequencing

The 88 kDa protein spot was excised and cleaved directly in gel with trypsin (Roche, Tutzing) as described(Eckerskorn and Lottspeich,1989). The eluted peptides were separated by reversed phase HPLC on a Purospher RP18, encapped 5 μm column (Merck, Darmstadt) using a solvent gradient from 0 to 60% acetonitrile in 0.1% trifluoroacetic acid/water(v/v). The flow rate was 60μl/minute and UV-detection was performed at 206 nm. The peptide fractions were collected manually and subjected to amino acid sequence analysis on an ABI 472A protein sequencer (Applied Biosystems,Langen) using the conditions recommended by the manufacturer. The sequencing resulted in the peptides: T-14, K/R I Y N Q I K; T-15, K/R X X D E I A A S G V A K P d h; T-17, K/R F A P D V R; T-18, K/R I L S V R; and T-25, K/R X X X Y L R g Q T d L (unequivocal amino acids are shown in capitals).

PCR-based cloning

Rapid amplification of cDNA ends (RACE) and synthesis of cDNA labeled at the 3′ end was performed using the method and primers (QT,Q0 and QI) described(Frohman, 1995). The peptide T-15 was used to design two overlapping fully degenerate oligonucleotides:5′- GA(CT) GA(AG) AT(ACT) GC(AGCT) GC(AGCT) (AT)(GC)(AGCT) GG-3′(H22-1) and 5′-(AT)(GC) (AGCT) GG(AGCT) GT(AGCT) GC(AGCT) AA(AG)CC-3′ (H22-2). These oligonucleotides were used as sequence-specific primers for 3′RACE from first-strand H. vulgaris cDNA that had been synthesized from poly A+ RNA with the olig-dT anchor primer QT. PCR was performed with Taq DNA polymerase (Amersham Pharmacia Biotech) in a gradient cycler (Eppendorf) under the following conditions: 5 minutes at 95°C (1 cycle); 1 minute at 95°C, 1 minute at 48.4°C, 1 minute at 72°C (35 cycles); and 5 minutes at 72°C (1 cycle). 0.5 μl of the amplification product obtained with H22-1 and Q0 was used for amplification with H22-2 and QI. An amplification product of 650 bp was ligated into the pGEM-T vector (Promega) according to the manufacturer's instructions. The presence of another peptide sequence, T-14,within this PCR-fragment, confirmed it to be part of the mAb H22 antigen.

Screening of cDNA phage library

Primers 5′-GCC TGA TCA TAA TTC AAA ATA TGA-3′ and 5′-CAA GTT GTT GTG ATT CTC TGC TCC-3′ were used to amplify a sequence from the 650 bp PCR fragment of Nowa and generate a probe to screen a λZAP cDNA library (Stratagene) consisting of a mixture of random and oligo-dT-amplified cDNA from Hydra vulgaris. Filter lifts on Biodyne A membrane (Pall)were generated using a standard protocol(Sambrook et al., 1989). 80,000 plugs on 20×20 cm filters were screened with the probe random-labeled with 32P (Prime-It II kit, Stratagene) using the high stringency conditions. Three positive overlapping clones were isolated,which together represented the complete coding sequence of Nowa. The continuous transcript of 2634 bp in length contained a single ORF of 2322 bp,42 bp of the 5′-untranslated region (UTR) and 270 bp of the 3′-UTR region. A vector containing the complete coding region of Nowa in pBluescript SK- (Nowa pBluescript) was generated by ligation of two of the original cDNA clones.

In situ hybridization

Whole mount in situ hybridization was performed as previously described(Technau and Bode, 1999). As probe, a digoxygenine (DIG)-labeled RNA was transcribed from the 650 bp Nowa PCR-fragment using the Sp6 site in the pGEM-T Vector (Promega).

Expression, refolding and purification of Nowa CTLD

The CTLD-encoding region of the Nowa cDNA comprising residues 212-346 was amplified by PCR using the Nowa pBluescript vector as a template. NdeI and BamHI sites were introduced in the 5′- and 3′- primers, respectively, to enable convenient cloning of the amplified DNA into the corresponding sites of the prokaryotic expression vector pet19b(Novagen). Primers used were: 5′-TTT CAT ATG AAA ATA AAA TGT CCA GAT GGC-3′; and 5′-TTT GGA TCC TTA CCT CAT CTT ACA AAC AAA TG-3′. The resulting vector that introduces a polyhistidine-tag at the N-terminal end of the protein sequence was used for transforming E. coli BL21 (DE3) cells. Transformed cells were grown in LB medium at 37°C until the OD600 reached 0.6 and then induced by adding IPTG to 0.4 mM. Cells were harvested after 2 hours and resuspended in 50 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 5 mM EDTA and 5 mM DTT. Complete lysis of bacteria was achieved by several freeze/thaw steps followed by two cycles of sonification. Nowa CTLD protein was found exclusively in inclusion bodies,which were purified by extensive washing with 50 mM Tris-HCl pH 8.0, 0.1 M NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100 and, in a final step in the same buffer without detergent. Refolding was achieved by solubilizing the inclusion bodies in 6 M GuHCl, 1 mM DTT and dialysis against 0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, 400 mM L-arginine, 10 mM reduced glutathione and 1 mM oxidized glutathione. Precipitates were discarded and the soluble protein was dialyzed against 50 mM Tris-HCl pH 8.0 and 150 mM NaCl. Final purification was achieved using nickel-sepharose chromatography according to the manufacturer's instructions (Novagen). Folding state and stability were checked by CD-spectroscopy and trypsin digestion.

The mAb H22 antigen is a high molecular weight protein of the nematocyst outer wall

The monoclonal antibody H22 reacted with all nematocyst types in Hydra, and staining was restricted to the wall of the capsule(Fig. 1A,A′). In EM cross-sections of mature capsules, immunogold-coupled mAb H22 labeled a distinctive layer adjacent to the electron-lucent inner wall(Fig. 1C). This outer layer has been previously described as the nematocyst outer wall by electron microscopy(Holstein, 1981;Watson and Mariscal, 1984). When nematocysts were isolated (Weber et al., 1987) and analyzed by SDS-PAGE, the mAb H22 reacted with a high molecular weight protein of approximately 90 kDa in capsules, but not in whole Hydra (Fig. 1D). By comparison, minicollagens detected by minicollagen-1 antibody represent the major component of the capsule with a molecular weight between 30 and 40 kDa(Engel et al., 2001)(Fig. 1D).

Isolation of the mAb H22 antigen revealed a novel protein

To purify the mAb H22 antigen, isolated capsules were subjected to 2D electrophoresis. Capsule proteins, separated according to their pI and molecular weight, were visualized by Coomassie blue(Fig. 2A) and silver-staining(Fig. 2C). A protein spot of pI 5.9 and a size of 88 kDa was correlated to an immunoreactive spot in western analysis (Fig. 2B). In gels stained with Coomassie blue few other proteins were detected apart from the immunoreactive spot (Fig. 2A),indicating that the antigen of mAb H22 represented a major structural component of capsules. The protein spot was excised from the gel, digested and microsequenced by Edman degradation. Using degenerate primers based on one of the resulting peptides (see Materials and Methods), a 650 bp fragment was amplified from Hydra vulgaris cDNA by PCR-based 3′ rapid amplification of cDNA ends (3′ RACE). The full length cDNA of 2634 bp was isolated from a Hydra vulgaris cDNA library and contained an ORF of 2322 bp. The full nucleotide sequence is available under GenBank accession number AF 559862.

Fig. 2.

Characterization and isolation of the mAb H22 antigen. (A-C) 2D electrophoresis of capsule proteins according to pI and molecular weight. The protein of two million capsules was separated and analyzed by Coomassie staining (A), western analysis with mAb H22 (B), and silver-staining (C). (D)Assay of N-glycosylation. Solubilized capsule protein was incubated with N-glycosidase F to remove N-linked sugars. The deglycosylated protein and mock control (without enzyme) were separated by SDS-PAGE and probed with mAb H22(left) and anti-CTLD antibody (right) in western analysis.

Fig. 2.

Characterization and isolation of the mAb H22 antigen. (A-C) 2D electrophoresis of capsule proteins according to pI and molecular weight. The protein of two million capsules was separated and analyzed by Coomassie staining (A), western analysis with mAb H22 (B), and silver-staining (C). (D)Assay of N-glycosylation. Solubilized capsule protein was incubated with N-glycosidase F to remove N-linked sugars. The deglycosylated protein and mock control (without enzyme) were separated by SDS-PAGE and probed with mAb H22(left) and anti-CTLD antibody (right) in western analysis.

The translated amino acid sequence of 774 residues represents a completely novel protein with no homologues so far in the database. We named the protein Nowa for nematocyst outer wall antigen. As expected for a secreted protein,there is a putative signal peptide at the N-terminus, comprising the first 18 residues. The computed molecular weight (82.7 kDa) and pI (7.7) of the protein without signal peptide differed from the values inferred from the protein spot in 2D gels (pI 5.9, 88 kDa; Fig. 2). This discrepancy is due to glycosylation of the protein. There are three N-glycosylation sites present in the sequence, and N-linked glycosylation was confirmed by deglycosylation of the protein with N-glycosidase F (Fig. 2D). The mAb H22 immunoreactive band in Western blots disappeared after treatment with the enzyme, suggesting that the antibody reactivity depended on N-linked sugar moieties linked to the peptide backbone. With a polyclonal antibody against a recombinantly expressed domain of Nowa (see below), the molecular weight after deglycosylation was determined to be 81 kDa(Fig. 2D).

Nowa contains a CTLD and a SCP domain

Although Nowa has no homologue in the database, a search for conserved domains revealed a C-type lectin-like domain (CTLD) and a domain belonging to the SCP domains (SCP, rodent sperm-coating glycoprotein), as depicted inFig. 3A. The SCP domains occur in a variety of eukaryotic extracellular proteins (see Discussion). The CTLDs are named after the C-type lectins that mediate Ca2+-dependent sugar binding (Drickamer,1988). The CTLDs occur as modular domains in a wide variety of otherwise unrelated extracellular proteins(Drickamer, 1999). The alignment in Fig. 3B of the Nowa CTLD with different representatives of CTLD-containing proteins, shows the conservation of the cysteines (Cys(1-6)) and most of the conserved aliphatic and aromatic stretches in the Nowa CTLD. The conserved carbonyl residues implicated in Ca2+-dependent sugar binding in the classical C-type lectins (Weis et al.,1991) are not found in Nowa CTLD, which makes it unlikely that the CTLD functions in sugar binding (see Discussion).

Fig. 3.

Nowa protein from Hydra vulgaris. (A) Nowa domain organization. CTLD, C-type lectin-like domain (smart00034, E-value of 10-17);SCP, homology to SCP domains (smart00198, E-value of 10-3); SP,signal peptide. The putative cleavage site for an N-terminal propeptide after residues KR is indicated by an arrow. (B) Alignment of CTLDs of Nowa and vertebrate proteins. Sequences most similar to the Nowa CTLD were retrieved by a BLAST-search in SWISS-PROT (sp) and TrEMBL (tr) protein databases. (sp P16112), aggrecan core precursor (human); (sp P55066), neurocan core protein precursor (murine); (tr O62623), cartilage proteoglycan (bovine); (tr Q13018),PLA2 RE, secretory phospholipase A2 receptor precursor (human); (tr Q64449),mannose receptor, C type 2 (murine); (tr Q91B90), C-type lectin from Cyprinus carpio; (tr Q91840), Aggretin β-chain from Agkistrodon rhodostoma; (tr Q9IAM0), Agkisacutacin β-chain from Agkistrodon acutus. Shading shows conservation of residues of the same similarity group within the aligned sequences: black, 100%; dark grey,80%; and light grey, 60% conservation. Amino acid similarity groups: DN, EQ,ST, KR, FWY, ILMV. (C) Alignment of cysteines in Cys-rich domains of Nowa and minicollagens. The C-terminal Cys-rich part of Nowa is an eightfold repetition(REP1-REP8) of six characteristically spaced cysteines, similar to the MCCR domain in the N- and C-terminal part of minicollagens; shown for minicollagen-1 (tr Q00484) and minicollagen-2 (tr Q00485) both from Hydra magnipapillata, and minicollagen-Ad (tr Q16990) from Acropora donei.

Fig. 3.

Nowa protein from Hydra vulgaris. (A) Nowa domain organization. CTLD, C-type lectin-like domain (smart00034, E-value of 10-17);SCP, homology to SCP domains (smart00198, E-value of 10-3); SP,signal peptide. The putative cleavage site for an N-terminal propeptide after residues KR is indicated by an arrow. (B) Alignment of CTLDs of Nowa and vertebrate proteins. Sequences most similar to the Nowa CTLD were retrieved by a BLAST-search in SWISS-PROT (sp) and TrEMBL (tr) protein databases. (sp P16112), aggrecan core precursor (human); (sp P55066), neurocan core protein precursor (murine); (tr O62623), cartilage proteoglycan (bovine); (tr Q13018),PLA2 RE, secretory phospholipase A2 receptor precursor (human); (tr Q64449),mannose receptor, C type 2 (murine); (tr Q91B90), C-type lectin from Cyprinus carpio; (tr Q91840), Aggretin β-chain from Agkistrodon rhodostoma; (tr Q9IAM0), Agkisacutacin β-chain from Agkistrodon acutus. Shading shows conservation of residues of the same similarity group within the aligned sequences: black, 100%; dark grey,80%; and light grey, 60% conservation. Amino acid similarity groups: DN, EQ,ST, KR, FWY, ILMV. (C) Alignment of cysteines in Cys-rich domains of Nowa and minicollagens. The C-terminal Cys-rich part of Nowa is an eightfold repetition(REP1-REP8) of six characteristically spaced cysteines, similar to the MCCR domain in the N- and C-terminal part of minicollagens; shown for minicollagen-1 (tr Q00484) and minicollagen-2 (tr Q00485) both from Hydra magnipapillata, and minicollagen-Ad (tr Q16990) from Acropora donei.

The Nowa CTLD was expressed recombinantly(Fig. 4A) in a bacterial expression system. The resulting 18 kDa protein was used to generate an antibody that reacted strongly with its antigen(Fig. 4B). In capsules, it reacted specifically with the 88 kDa band recognized by mAb H22, confirming the identity of Nowa and the antigen of mAb H22.

Fig. 4.

Recombinant expression of Nowa CTLD. (A) Nowa CTLD construct used for recombinant expression in E. coli. (B) Purified recombinant Nowa CLTD and capsule proteins (500,000 capsules) analyzed by SDS-PAGE and visualized by silver-staining and western analysis.

Fig. 4.

Recombinant expression of Nowa CTLD. (A) Nowa CTLD construct used for recombinant expression in E. coli. (B) Purified recombinant Nowa CLTD and capsule proteins (500,000 capsules) analyzed by SDS-PAGE and visualized by silver-staining and western analysis.

Minicollagen Cys-rich domains are repeated eight times in the C-terminal part of Nowa

The most striking feature of Nowa is a Cys-rich C-terminal part in which six characteristically spaced cysteines are repeated eight times. This pattern is also found in the short N- and C-terminal Cys-rich domains of minicollagens(alignment Fig. 3C). This domain, which we have named minicollagen Cysrich (MCCR) domain, is used in minicollagens to interconnect minicollagen trimers to large polymers(Engel et al., 2001). It is an intriguing possibility that the multiple MCCR domains in Nowa allow Nowa to interact with minicollagens through the matching cysteines and to take part in the crosslinking process (see Discussion).

Nowa transcription occurs only in developing nematocytes

To investigate a possible role of Nowa in formation of the capsule wall, we determined its expression in cells of the nematocyte differentiation pathway(Fig. 6A). In situ hybridization revealed that the mRNA encoding Nowa was not expressed in mature nematocytes but only in differentiating nematocytes or nematoblasts(Fig. 5, arrows). The tentacles showed no hybridization with the probe for Nowa mRNA. In the body column,clusters of small labeled cells were present(Fig. 5B), representing nests of nematoblasts or developing nematocytes. Nests with clearly discernable capsules were negative (Fig. 5B′, arrows with asterisks). Thus, Nowa mRNA was expressed only at the beginning of nematocyte differentiation.

Fig. 6.

Immunolocalization of Nowa protein in dividing nematoblasts. (A) The nematocyte differentiation pathway in Hydra. Nematocytes originate from interstitial stem cells (I-cells), which divide three- to five-times after commitment (nematoblasts) and remain interconnected by cytoplasmic bridges forming nests of 8-32 cells (David and Challoner, 1974; David and Gierer, 1974). After nematocytes have formed a nematocyst (green),nests brake up into single nematocytes, which migrate to the tentacles. (B-D)Confocal microscopy of mAb H22 (green) in dividing nematoblasts. (B)Nematoblast nest in metaphase (optical section) with nuclei (blue) in metaphase condensation numbered 1-8. One of the mAb-H22-positive capsule primordia is indicated by an arrow. (C) Surface projection of the same nest,the boundary of the nest is indicated by a dotted line. (D) Nematoblast in division with metaphase spindle apparatus visualized by mAb anti-tubulin(red). Projections from different angles show the asymmetrical position of the capsule primordium. (E) Continuous [3H]thymidine labeling of mAb-H22-positive nests. The first labeled nests appeared ∼5 hours after onset of labelling; values represent single measurements from two independent experiments. Bars, 5 μm.

Fig. 6.

Immunolocalization of Nowa protein in dividing nematoblasts. (A) The nematocyte differentiation pathway in Hydra. Nematocytes originate from interstitial stem cells (I-cells), which divide three- to five-times after commitment (nematoblasts) and remain interconnected by cytoplasmic bridges forming nests of 8-32 cells (David and Challoner, 1974; David and Gierer, 1974). After nematocytes have formed a nematocyst (green),nests brake up into single nematocytes, which migrate to the tentacles. (B-D)Confocal microscopy of mAb H22 (green) in dividing nematoblasts. (B)Nematoblast nest in metaphase (optical section) with nuclei (blue) in metaphase condensation numbered 1-8. One of the mAb-H22-positive capsule primordia is indicated by an arrow. (C) Surface projection of the same nest,the boundary of the nest is indicated by a dotted line. (D) Nematoblast in division with metaphase spindle apparatus visualized by mAb anti-tubulin(red). Projections from different angles show the asymmetrical position of the capsule primordium. (E) Continuous [3H]thymidine labeling of mAb-H22-positive nests. The first labeled nests appeared ∼5 hours after onset of labelling; values represent single measurements from two independent experiments. Bars, 5 μm.

Fig. 5.

In-situ hybridization of Nowa transcript in whole mounts of Hydra.(A) Overview of an animal with a small bud showing expression of Nowa in the body column but not in the tentacles. (B) Body column with Nowa-mRNA-positive cells. (B′) Enlargement of B with Nowa-mRNA-positive nematoblast or nematocyte nest indicated by an arrow. An adjacent nest with capsules but no Nowa expression is indicated by an arrow and asterisk. Bars, 100 μm (A); 50μm (B); 10 μm (B′).

Fig. 5.

In-situ hybridization of Nowa transcript in whole mounts of Hydra.(A) Overview of an animal with a small bud showing expression of Nowa in the body column but not in the tentacles. (B) Body column with Nowa-mRNA-positive cells. (B′) Enlargement of B with Nowa-mRNA-positive nematoblast or nematocyte nest indicated by an arrow. An adjacent nest with capsules but no Nowa expression is indicated by an arrow and asterisk. Bars, 100 μm (A); 50μm (B); 10 μm (B′).

Nowa protein expression starts concomitant with capsule formation

To determine the onset of Nowa expression in the differentiation pathway, Hydra were continuously labeled with [3H]thymidine to identify proliferating precursors and trace the transition of proliferating precursors into differentiating cells. Fig. 6E shows that [3H]thymidine-labeled cells at time point 0 did not express Nowa detected by mAb H22. However, about 5 hours after onset of labeling, some labeled cells became mAb-H22-positive, indicating expression of Nowa. Complete labeling of differentiating nematocyte nests required 3-4 days, which is in good agreement with previous results(David and Gierer, 1974). The rapid appearance of labeled mAb-H22-positive cells indicates that Nowa synthesis starts already before the terminal mitosis of nematoblasts.

This surprising result was confirmed by analysing dividing nematoblast nests in whole mounts stained with mAb H22. The percentage of dividing nests positive for mAb H22 was 84% in 16-cell nests (n=19), 74% of all 8-cell nests (n=101), and 17% of all 4-cell nests (n=59). An example of a dividing 8-cell nematoblast nest is shown inFig. 6B,C. Nuclei in metaphase are clearly discernable from interphase nuclei in adjacent cells in an optical section (Fig. 6B). Each dividing cell contains one large and several very small Nowa-filled structures, as seen in the surface projection(Fig. 6C). We interpret the larger structure to be the vesicle containing the capsule primordium. The small structures probably represent TGN vesicles, which are scattered over the cell due to the disassembly of the Golgi apparatus during mitosis. This pattern of vesicle distribution is also visible in dividing nematoblasts that were double-stained with anti-tubulin antibody to visualize the spindle apparatus (Fig. 6D). The presence of mAb-H22-positive structures in metaphase nematoblasts demonstrates that nematocyst morphogenesis already starts in nematoblasts before their terminal division into nematocytes. During this terminal mitosis, one daughter cell inherits the young capsule primoridum while the second daughter cell forms a new primordium.

Sorting of capsule proteins into the nematocyst wall

The formation of the wall and tubule structures involves a yet undefined sorting mechanism that leads to the formation of the double-layered wall. We used confocal microscopy and immunogold labeling in EM-sections to follow the subcellular distribution of Nowa during morphogenesis (Figs7,8,9).

Fig. 7.

Immunolocalization of Nowa protein in the early growth phase of nematocyst morphogenesis. (A,B) Confocal microscopy of mAb H22 (green) and minicollagen antibody (red) in early stages of nematocyte differentiation. Nuclei are stained with DAPI (blue). (A) Early and late stages of capsule development in an optical section. (A′) Enlargment of one of the early stage nematocytes with nematocyst primordium. Staining of mAb H22 is restricted to the wall and tubular-vesicular structure at the apex (arrow), while minicollagen is detected in the ER and matrix of the capsule, as depicted in the drawing in B. (C) EM-section of nematocyte with nematocyst primordium. Immunogold mAb H22 labeling is found in the capsule matrix (cm), the outer layer of the capsule wall (cw), the Golgi apparatus (g), as indicated by an arrow, and membrane compartments associated with the primordium but not in the ER (n, nucleus). (D,D′) Formation of the inner wall layer by minicollagen. Minicollagen is no longer found in the matrix of the capsule but forms the inner wall (iw) adjacent to the mAb-H22-positive outer wall (ow).(E) Isorhizatype nematocyst with tubular-vesicular structures at the growing apex (arrow) in an optical section. Bars, 5 μm (A,A′,B,D); 1 μm(C).

Fig. 7.

Immunolocalization of Nowa protein in the early growth phase of nematocyst morphogenesis. (A,B) Confocal microscopy of mAb H22 (green) and minicollagen antibody (red) in early stages of nematocyte differentiation. Nuclei are stained with DAPI (blue). (A) Early and late stages of capsule development in an optical section. (A′) Enlargment of one of the early stage nematocytes with nematocyst primordium. Staining of mAb H22 is restricted to the wall and tubular-vesicular structure at the apex (arrow), while minicollagen is detected in the ER and matrix of the capsule, as depicted in the drawing in B. (C) EM-section of nematocyte with nematocyst primordium. Immunogold mAb H22 labeling is found in the capsule matrix (cm), the outer layer of the capsule wall (cw), the Golgi apparatus (g), as indicated by an arrow, and membrane compartments associated with the primordium but not in the ER (n, nucleus). (D,D′) Formation of the inner wall layer by minicollagen. Minicollagen is no longer found in the matrix of the capsule but forms the inner wall (iw) adjacent to the mAb-H22-positive outer wall (ow).(E) Isorhizatype nematocyst with tubular-vesicular structures at the growing apex (arrow) in an optical section. Bars, 5 μm (A,A′,B,D); 1 μm(C).

Fig. 8.

Immunolocalization of Nowa protein in the late growth phase of nematocyst morphogenesis. Confocal microscopy of three nematocysts with an outer tubule.(A) Surface projection of mAb H22 (green) and spinalin antibody staining(red). A′ shows mAb H22 staining only to visualize staining of the outer tubule. (B) Schematic representation of a nematocyst with an outer tubule. Bars, 5 μm.

Fig. 8.

Immunolocalization of Nowa protein in the late growth phase of nematocyst morphogenesis. Confocal microscopy of three nematocysts with an outer tubule.(A) Surface projection of mAb H22 (green) and spinalin antibody staining(red). A′ shows mAb H22 staining only to visualize staining of the outer tubule. (B) Schematic representation of a nematocyst with an outer tubule. Bars, 5 μm.

Fig. 9.

MT scaffold around growing nematocysts in the early (A-C) and late(D-H′) growth phase of nematocyst morphogenesis. (A,D) Localization of tubulin in Hydra macerates. (B,C,E,F) Confocal microscopy of nematocyst nests in the body of Hydra whole mounts stained by mAb H22 and mAb anti-tubulin or minicollagen and anti-tubulin (all shown as projections). The putative position of the MTOC is indicated by an arrow.(G-H′) EM sections through the outer tubule (ot). (G) Tangential section of the outer tubule (see white box in E). The pair of centrioles (ce) are found at the tip of the tubule. MTs are running parallel to the tubule (yellow arrows). (H) Cross-section of the tubule, the enlargement in H′ shows the intimate association of MTs with the membrane around the outer tubule. Bars, 5 μm (A-F); 0.5 μm (G); 2 μm (H); 100 nm (E′).

Fig. 9.

MT scaffold around growing nematocysts in the early (A-C) and late(D-H′) growth phase of nematocyst morphogenesis. (A,D) Localization of tubulin in Hydra macerates. (B,C,E,F) Confocal microscopy of nematocyst nests in the body of Hydra whole mounts stained by mAb H22 and mAb anti-tubulin or minicollagen and anti-tubulin (all shown as projections). The putative position of the MTOC is indicated by an arrow.(G-H′) EM sections through the outer tubule (ot). (G) Tangential section of the outer tubule (see white box in E). The pair of centrioles (ce) are found at the tip of the tubule. MTs are running parallel to the tubule (yellow arrows). (H) Cross-section of the tubule, the enlargement in H′ shows the intimate association of MTs with the membrane around the outer tubule. Bars, 5 μm (A-F); 0.5 μm (G); 2 μm (H); 100 nm (E′).

Already in early stages of capsule growth, Nowa was enriched in the capsule wall, while minicollagen was found only in the capsule matrix and in the ER(Fig. 7A′,B). In an EM-section of such a stage (Fig. 7C), which was stained with immunogold mAb H22, gold particles were present inside the capsule vesicle, the Golgi apparatus and large vesicles close to the capsule primordium. These protein-filled vesicles most likely correspond to the prominent caplike structures positive for mAb H22 at the growing apex of the capsule (Fig. 7A′ and D arrows), which we interpret to be TGN. We previously described similar vesicular structures filled with minicollagen(Engel et al., 2001). Astonishingly, double-labeling experiments revealed that Nowa and minicollagen do not colocalize in the same TGN vesicles(Fig. 7E). This suggests a sorting mechanism by which a pre-mature interaction of Nowa and minicollagen is prevented. No Nowa was detected in the ER, which is in agreement with our finding that the mAb H22 reacts with an epitope dependent on N-glycosylation of Nowa (Fig. 2D).

At a later stage of capsule growth, minicollagen staining disappeared from the matrix and minicollagen was now found to form the inner wall adjacent to the H22-positive outer wall; this is most clearly seen in the nest of nematocysts in Fig. 7D and 7D′. After formation of the double-layered wall of the nematocyst, fusion of vesicles at the apex led to outgrowth of the outer tubule. Nowa formed a continuos thin layer on the capsule and the outer tubule, as visible from its relative position to spinalin in the capsule matrix (Fig. 8).

At the end of nematocyst morphogenesis, when the outer tubule has been invaginated into the capsule, minicollagens undergo a disulfide rearrangement leading to a loss of minicollagen antibody reactivity(Engel et al., 2001). Minicollagens are still present in mature capsules as demonstrated by Western blots of isolated capsules (Fig. 1D), but they are not accessible for the antibody by immunohistochemistry. Fig. 7Ashows a nest with two almost mature capsules lacking the minicollagen immunreactivity but having a speckled mAb H22 staining pattern, which is characteristic of mature capsules (Fig. 1A).

MTs form a dynamic scaffold around differentiating nematocytes

Localization of the MT cytoskeleton in developing nematocytes by confocal microscopy and in Em-sections, revealed a prominent MT scaffold around the developing nematocyst. This scaffold changed as the nematocyst grew. In early stages, MTs assumed an umbrella-like arrangement(Fig. 9A) that covered the growing apex of the capsule (Fig. 9B,C). In the next stage of nematocyst morphogenesis, when the outer tubule was forming (Fig. 9D-F), MTs formed remarkably long, parallel arrays around the outer tubule. The putative position of the microtubule-organizing center(MTOC; arrows in Fig. 9A-F) was close to the site where Nowa-filled or minicollagenfilled TGN structures were observed (Fig. 9B,C,E,F). A longitudinal EM-section through an outer tubule revealed MTs running along the outer tubule, except at the very tip (Fig. 9G). The two centrioles marking the position of the MTOC were found close to the tubule tip, in agreement with the immunolocalization images(Fig. 9A-F). A cross-section of the outer tubule showed the intimate association of MTs to each other and to the vesicle membrane around the outer tubule(Fig. 9H,H′),demonstrating that the MTs formed a cage-like structure around the growing part of the nematocyst. This arrangement and the localization of the MTOC at the site where more protein-filled vesicles are delivered to the nematocyst vesicle suggest a regulating function of the MT cytoskeleton in directing nematocyst growth (Fig. 10).

Fig. 10.

Formation of the capsule wall formed from Nowa (green) and minicollagen(red). (A) Protein sorting and transport as detected by minicollagen antibody and mAb H22. Minicollagen and Nowa synthesized in the ER are transported in separate vesicles to the nematocyst vesicle. Nowa is detected by mAb H22 only modification by glycosylation in the Golgi apparatus and forms the outer wall. MTs (yellow) are organized in a scaffold around the growing part of the nematocyst, the MTOC is localized between the Golgi apparatus and the growing apex of the nematocyst. Minicollagen first accumulates in the capsule matrix and is then sorted to the wall to form the inner layer of the wall (3). By further transport of protein-filled vesicles, the outer tubule forms (4). It is subsequently invaginated into the cyst, and spines (s) are formed in the tubule lumen (5). Finally, minicollagen crosslinkage leads to a compaction of the wall structure (6). (B) Model of nematocyst patterning by the MT cytoskeleton. The growing part of the nematocyst vesicle is shown in a schematic cross-section. MTs form a cage around the vesicle and determine its shape (1). The outer wall formed by Nowa on the membrane (2) is used as template for minicollagen assembly. Soluble minicollagen trimers aggregate on the outer wall to form the inner wall (3-5). Finally, Nowa and minicollagen are crosslinked by disulfide bond isomerization to stabilize the structure(6).

Fig. 10.

Formation of the capsule wall formed from Nowa (green) and minicollagen(red). (A) Protein sorting and transport as detected by minicollagen antibody and mAb H22. Minicollagen and Nowa synthesized in the ER are transported in separate vesicles to the nematocyst vesicle. Nowa is detected by mAb H22 only modification by glycosylation in the Golgi apparatus and forms the outer wall. MTs (yellow) are organized in a scaffold around the growing part of the nematocyst, the MTOC is localized between the Golgi apparatus and the growing apex of the nematocyst. Minicollagen first accumulates in the capsule matrix and is then sorted to the wall to form the inner layer of the wall (3). By further transport of protein-filled vesicles, the outer tubule forms (4). It is subsequently invaginated into the cyst, and spines (s) are formed in the tubule lumen (5). Finally, minicollagen crosslinkage leads to a compaction of the wall structure (6). (B) Model of nematocyst patterning by the MT cytoskeleton. The growing part of the nematocyst vesicle is shown in a schematic cross-section. MTs form a cage around the vesicle and determine its shape (1). The outer wall formed by Nowa on the membrane (2) is used as template for minicollagen assembly. Soluble minicollagen trimers aggregate on the outer wall to form the inner wall (3-5). Finally, Nowa and minicollagen are crosslinked by disulfide bond isomerization to stabilize the structure(6).

The sorting of proteins to form a complex structure inside an unstructured vesicle requires self-assembly processes. In a previous paper, we have shown how minicollagens assemble into the inner wall and are crosslinked to form a highly stress-resistant network (Engel et al., 2001). Here we expand the model to consider how shape could be generated inside the nematocyst vesicle. Morphogenesis of the structure is characterized by polar fusion of protein vesicles leading to polarized growth of a tubule several cell-diameters in length. Intra-vesicular patterning occurs by assembly of proteins into distinct layers of the wall. We propose that the cytoskeleton represents a force that acts on the vesicle to generate shape. This in concert with the temporally regulated transport of a novel outer wall protein, Nowa, and its interaction with minicollagens inside the nematocyst vesicle results in the formation of the double-layered capsule. Once the structure has reached its final dimensions, the wall is crosslinked to provide high mechanical stability.

Nowa is a novel protein and putative binding partner of the minicollagens

Nowa was identified as a component of the outer wall of Hydranematocyts. It is a novel protein of 88 kDa that is modified by N-glycosylation (Fig. 2D). The sequence starts with a putative signal peptide of 18 residues. The presence of the residues KR at position 33/34 might suggest cleavage of a propeptide as demonstrated for minicollagens (Engel et al., 2001) and many other capsule proteins(Anderluh et al., 2000).

By sequence comparison, we were able to identify three domains with homology to domains of other extracellular proteins(Fig. 3A). The C-terminal Cys-rich domain proved to be an eightfold repetition of the MCCR domain(Fig. 3C). These domains, which also flank the collagenous part of the minicollagens(Kurz et al., 1991), are defined by six characteristically spaced Cys-residues. Previous work has shown that this Cys-rich domain functions in the crosslinking of minicollagens to an oligomeric structure. Crosslinking occurs at a late stage of morphogenesis when the minicollagens have assembled into the wall. Minicollagens are first produced as soluble trimers that display intra-chain disulfide bonds within a single MCCR domain. By isomerization of the intrachain to inter-chain disulfide bonds in a late stage of morphogenesis, the minicollagens are crosslinked and the capsule wall achieves its high tensile strength(Engel et al., 2001). Similarly to the minicollagens, Nowa protein could not be solubilized from mature capsules without a reducing agent (data not shown), which indicates that it is crosslinked by disulfide bonds. We propose that the cysteines of the Nowa MCCR domains undergo a similar isomerization process and that Nowa could be covalently linked to minicollagens by its matching cysteines. Since the eightfold repetition of MCCR domains in Nowa could possibly crosslink up to eight minicollagen molecules, Nowa may function as a crystallization center for minicollagen assembly. We therefore speculate that minicollagen and Nowa molecules at the interface of the outer and inner wall are crosslinked by hetero-oligomers, while the majority of minicollagen and Nowa in the two wall layers form homo-oligomers (Fig. 7D).

The two other domains identified in Nowa have not been found in any other capsule protein. The SCP domains, which have been identified in a wide variety of extracellular proteins, such as the allergen from vespid wasps,pathogenesis-related proteins of plants and mammals(Szyperski et al., 1998), have not yet been assigned a common function.

The CTLD, which is detected in Nowa is a conserved protein module, which was initially identified in a group of C-type (Ca2+-dependent)animal lectins (Drickamer,1988). The Nowa CTLD showed higher homology to vertebrate CTLDs(e.g. 30% identity with the CTLD of human mannose receptor;Fig. 3B) than to a recently identified Hydra protein with four extracellular CTLDs (26% identity)(Reidling et al., 2000),indicating that the CLTDs are divergent already in Hydra.

The Nowa CTLD belongs to the intron-positive subfamiliy of CTLDs, which possess six Cys-residues (Drickamer,1989), with disulfide bonds between Cys(1)-Cys(2), Cys(3)-Cys(6), and Cys(4)-Cys(6) demonstrated in many of these CTLDs(Llera et al., 2001;Usami et al., 1993). The CTLD of Nowa lacks the residues with carbonyl side chains implicated in Ca2+-dependent sugar binding(Weis et al., 1991). However,it has been shown for other CTLDs that lack the Ca2+-coordinating residues, that these CTLDs function in highly specific protein binding independent of sugar moieties (Llera et al., 2001). Thus, the Nowa CTLD may function in non-covalent binding of minicollagens, prior to disulfide crosslinking.

Nowa is localized in the outer wall in a globular layer

Nowa localized by mAb H22 was found exclusively in the outer wall of mature nematocysts (Fig. 1). The staining pattern, which looked like speckles evenly distributed on the capsule surface, was reminiscent of the structure observed by field emission scanning electron microscopy on the surface of isolated nematocysts(Holstein et al., 1994), where the outer wall was shown to consist of globules. The speckled pattern of mAb H22 staining was found in all nematocyst types of Hydra(Fig. 1A′), in nematocysts of the anthozoan Nematostella vectensis, the cubomedusan Carybdea marsupialis and the hydrozoan Hydractinia echinata(U.E. and T.W.H., unpublished). This strongly suggests that the outer wall formed by Nowa is common to the nematocysts of these cnidarian classes and presumably is an indispensable constituent. To date, no function of the outer wall has been proposed in nematocyst discharge, but the putative interaction of Nowa with minicollagen through the MCCR domains implies a function for Nowa in morphogenesis. The fact that Nowa expression and protein synthesis start prior to the final division of nematoblasts (Figs5,6) indicates that it is one of the very first proteins to occur in the nematocyst primordium, concomitant with the formation of the nematocyst wall.

A model for sorting of proteins into the nematocyst wall

Immunostaining of MTs visualized by confocal microscopy allowed us to follow the MT rearrangements around the growing nematocyst(Fig. 9). In EM sections,centrioles marking the position of the MTOC are visible at the growing apex of the vesicle (Holstein, 1981;Watson and Mariscal, 1984). One intriguing function of MTs radiating from the MTOC could be the correct positioning of the Golgi apparatus and TGN relative to the site where the capsule vesicle grows. Accordingly, MTs could provide tracks for transport of protein-filled vesicles to the site where new material is deposited(Fig. 10A). However, there seems to be an additional function of the MT scaffold: as shown in the EM cross-section of an outer tubule (Fig. 9H), the MTs are intimately linked to the nematocyst vesicle membrane. By their tight spacing to each other and to the membrane (14 nm and 12 nm, respectively) (Holstein,1981), the MTs form a cage that can account for the stabilization of the nematocyst shape (Holstein,1981; Watson and Mariscal,1984). The diameter of the cage-like scaffold of MTs correlates with the diameter of the nematocyst part under construction (capsule or outer tubule, Fig. 9) and might regulate its diameter. The dense packing of MTs efficiently prevents fusion of vesicles along the outer tubule except at the very tip, which is free of MTs(Fig. 9G). Here, Nowa- and minicollagen-filled vesicles were observed(Fig. 9B,C,E,F). In summary,the MT scaffold around the nematocyst vesicle may contribute to the polar growth of the nematocyst vesicle and determine its shape.

The question arises how the shape enforced onto the vesicle by the MT cytoskeleton is passed on to the developing structures in the interior of the vesicle. Based on electron microscopy of nematocyst development, Watson proposed that wall precursors assemble on the membrane stabilized by MTs, to form a template of material in shape of the mature capsule(Watson, 1988). The localization of Nowa forming a layer lining the membrane already in very early stages would make it an ideal candidate for such a function. Thus, Nowa could serve as positional information for minicollagen assembly as schematically depicted in a cross-section of the nematocyst vesicle inFig. 10B. Minicollagens transported into the capsules would bind to Nowa and aggregate to form the inner wall. In agreement with this model, Nowa is also found to line the forming outer tubule (Fig. 8)and could fulfil a similar function in patterning of this part of the nematocyst.

An obvious question that arises from the model described above is, how a pre-mature interaction of Nowa and minicollagen in the ER, Golgi apparatus and TGN is prevented. Remarkably, we observed separate transport compartments for minicollagen and Nowa, which we interpret as TGN(Fig. 7E). In the ER, where both proteins are synthesized, we can only speculate that the two proteins at this stage are not yet competent for interaction, as they have not undergone their post-translational modifications (e.g. glycosylation). We envisage that the first steps of ordering the wall structures in the nematocyst vesicle occur by noncovalent interactions. Early covalent crosslinking between minicollagens and Nowa through disulfide bonds would lead to disordered assembly of the proteins and not to the distinct layers observed. Furthermore,the isomerization of disulfide bonds in minicollagens was shown to occur in the late maturation phase (Engel et al.,2001) and not during nematocyst growth.

It is not yet completely understood how the different steps of Nowa assembly, such as sorting of the protein to the membrane, the self-aggregation to form a distinct outer wall are achieved. The multi-domain nature of Nowa opens the intriguing possibility that each of the domains serves a different function in this process. The best understood example so far is the MCCR domain present in Nowa and minicollagens, which was shown to mediate the transition of a state of solubility to an oligomeric network of extremely high stability (Engel et al.,2001).

Many thanks to Hans Bode and Michael P. Sarras for providing the Hydra vulgaris cDNA library, and to Charles David and Jürgen Engel for discussions and critical reading of the manuscript. Supported by the DFG (SFB 269 and GK 361).

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