AUTHOR CORRECTION 4719 Engel, U., Oezbek, S.,...

Engel, U., Oezbek, S., Engel, R., Petri, B., Lottspeich, F. and Holstein, T. W.(2002). Nowa, a novel protein with minicollagenCys-rich domains, is involved in nematocyst formation in Hydra. 115, 3923-3934.

In both the on-line and print versions of this paper, the second and third authors’ names are incorrect. The correction version isshown below:

Ulrike Engel, Suat Özbek, Ruth Streitwolf-Engel, Barbara Petri, Friedrich Lottspeich and Thomas W. Holstein

In addition, the nucleotide sequence for the Nowa cDNA was shown with an incorrect GenBank accession number. The correctGenBank accession number is AF539862.

AUTHOR CORRECTION4719

IntroductionPattern formation within single cells has been recentlyrecognized as a fundamental but not well understood aspect ofcell biology (Shulman and St Johnston, 1999). A particularlyfascinating example is the morphogenesis of the nematocyst, acomplex structure that develops inside a giant secretory vesiclein the cnidarian nematocyte (Slautterback and Fawcett, 1959;Holstein, 1981). Upon stimulation of the nematocyte, thenematocyst discharges explosively, a process that takes lessthan 3 milliseconds (Holstein and Tardent, 1984).

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

Nematocyst morphogenesis can be subdivided into fivestages (Holstein, 1981). (1) An early growth phase duringwhich the capsule primordium forms and grows by addition ofnew vesicles to the vesicle harboring the capsule. (2) A lategrowth phase during which a tubule forms outside the capsuleby addition of more vesicles; capsule and tubule wall form acontinuous structure. (3) Invagination of the long externaltubule into the capsule. (4) An early maturation phase leadingto the formation of spines by condensation of the proteinspinalin (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 anosmotic pressure of 150 bar that drives discharge (Weber, 1990;Szczepanek et al., 2002).

The extremely high pressure in mature capsules requireshigh tensile strength of the wall. This tensile strength ismediated by minicollagens, a family of very short collagensthat form the capsule’s inner wall (Kurz et al., 1991; Holsteinet al., 1994). In a previous paper (Engel et al., 2001), we haveshown that wall maturation involves polymerization ofminicollagens to an insoluble polymer. This polymerization ismediated by disulfides in the minicollagen cysteine-richdomains (MCCR domains) that undergo a switch from intra-chain to inter-chain disulfide bonds in the late maturationphase. The inner wall layer, formed by minicollagens, iscovered by an outer wall layer, which is more electron-densethan the inner wall in EM sections of nematocysts (Holstein,1981; Watson and Mariscal, 1984) and appeared as a layer ofglobular material in field emission scanning electronmicroscopy (Holstein et al., 1994). The molecular nature of thisouter wall was previously unknown.

In this study we used the monoclonal antibody H22 (mAbH22) (Kurz et al., 1991), which stained the outer wall of Hydranematocysts 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 acompletely novel protein that we call nematocyst outer wallantigen (Nowa). The C-terminal part of the 774-residue protein

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The novel protein Nowa was identified in nematocysts,explosive organelles of Hydra, jellyfish, corals and otherCnidaria. Biogenesis of these organelles is complex andinvolves assembly of proteins inside a post-Golgi vesicle toform a double-layered capsule with a long tubule. Nowa isthe major component of the outer wall, which is formedvery early in morphogenesis. The high molecular weightglycoprotein has a modular structure with an N-terminalsperm coating glycoprotein domain, a central C-type lectin-like domain, and an eightfold repeated cysteine-richdomain at the C-terminus. Interestingly, the cysteine-richdomains are homologous to the cysteine-rich domains ofminicollagens. We have previously shown that the cysteinesof these minicollagen cysteine-rich domains undergo an

isomerization process from intra- to intermoleculardisulfide bonds, which mediates the crosslinking ofminicollagens to networks in the inner wall of the capsule.The minicollagen cysteine-rich domains present in bothproteins provide a potential link between Nowa in the outerwall and minicollagens in the inner wall. We propose amodel for nematocyst formation that integratescytoskeleton rearrangements around the post-Golgi vesicleand protein assembly inside the vesicle to generate acomplex structure that is stabilized by intermoleculardisulfide bonds.

Key words: Minicollagen Cys-rich domain, CTLD, Nematocyst,Microtubules, Assembly

Summary

Nowa, a novel protein with minicollagen Cys-richdomains, is involved in nematocyst formation in HydraUlrike Engel 1,*, Suat Oezbek 2, Ruth Engel 2, Barbara Petri 3, Friedrich Lottspeich 4 and Thomas W. Holstein 1,‡

1Institute of Zoology, Darmstadt University of Technology, 64287 Darmstadt, Germany2Department of Biophysical Chemistry, Biozentrum, University of Basel, 4056 Basel, Switzerland3Institute of Zoology, University of Munich, 80333 Munich, Germany4Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany*Present address: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA‡Author for correspondence (e-mail: [email protected])

Accepted 1 August 2002Journal of Cell Science 115, 3923-3934 © 2002 The Company of Biologists Ltddoi:10.1242/jcs.00084

Research Article

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is characterized by an eightfold repetition of Cys-rich(cysteine-rich) domains homologous to minicollagen Cys-richdomains (MCCR domains). These domains present in Nowaand minicollagens suggest that the two proteins interact duringwall formation. We propose a model that integrates the role ofthe MT (microtubule) cytoskeleton and the interaction of Nowaand minicollagen in forming the nematocyst wall.

Materials and MethodsStrains and culture conditionsHydra 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).

AntibodiesSupernatants from hybridoma cultures of mAb H22 (Kurz et al., 1991)were directly used for immunocytochemistry. A polyclonal antibodydirected against the recombinantly expressed Nowa CTLD wasgenerated in rabbits by Eurogentec (Herstal, Belgium). Immunizationwas carried out following a standard protocol using 100 µg of purifiedand refolded CTLD protein in PBS. Minicollagen antibody (Engel etal., 2001) and spinalin antibody (Koch et al., 1998) are polyclonalrabbit antisera generated against recombinantly expressed Hydraproteins. The mAb directed against β-tubulin (mAb anti-tubulin) fromPhysarium polycephalumwas obtained from Chemicon.

ImmunofluorescenceAnimals were relaxed in 2% urethane in M solution for 2 minutes andfixed either in Lavdovsky’s fixative (50% ethanol, 3.7%formaldehyde, 4% acetic acid in water) or 4% paraformaldehyde for24 hours. The fixative was removed by several washes in PBS, andmembranes were opened by an incubation of 30 minutes in 0.1%Triton X-100. Animals were incubated in mAb H22 overnight. Afterseveral washes in PBS, the animals were incubated for 5 hours in anti-mouse antibody coupled to Alexa-488 fluorochrome (MolecularProbes) diluted 1:400 in PBS with 1% BSA, and excess antibody wasremoved by washing again. For double-staining, minicollagen-1antibody diluted 1:500 in mAb H22 or spinalin antibody diluted 1:10in mAb H22 were added to animals and subsequently detected bysimultaneous incubation with anti-rabbit antibody coupled to Alexa-568 fluorochrome and anti-mouse antibody coupled to Alexa-488fluorochrome.

For double-staining with the two monoclonal antibodies H22 andanti-tubulin, animals were fixed in 4% paraformaldehyde in PBS with0.1% Trition-X for 10 minutes and an additional hour withoutdetergent. After incubation with mAb H22, binding sites on the FC ofmAb H22 were blocked with a polyclonal anti-mouse IgG antibody(Sigma) diluted 1:100 in PBS with 1% BSA for 5 hours. Detection ofmAb H22 was achieved indirectly with anti-rabbit antibody coupledto Alexa-568 diluted 1:400 in PBS 1% BSA. The subsequent stainingwith mAb anti-tubulin (2 µg/ml in 1% BSA in PBS) followed theprotocol described for mAb H22 described above, but incubationtimes were shortened to limit exchange reactions on the FC of the twomonoclonal 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 threewashes in PBS, an anti-mouse antibody coupled to Alexa-488 wasadded for 4 hours and excess antibody removed by washing.

Isolated capsules (Weber et al., 1987) were stained without fixationin 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 cellsHydra were labeled with [3H]thymidine (50 µCi/ml) as described(Holstein and David, 1990). Macerates (David, 1973) of labeledpolyps were stained with mAb H22 and FITC-conjugated goat anti-mouse antibody, dipped into autoradiographic photo-emulsion (KodakNTB-2), exposed for 10 days at 4°C and developed.

Confocal microscopy and deconvolutionWhole mounts and macerated Hydra were viewed and documentedon a confocal laser scanning microscope (Leica TCS SP). Singlephoton 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 sectionsor as projections through a series of optical planes (indicated in thelegend). Overlay of multiple channels, projections, and 3D renderingof stacks were done using Leica confocal software 2.00 and Imaris3.0 software (Bitplane). Deconvolution of image stacks to removebackground and improve axial and lateral resolution was performedwith Huygens System 2 software (Scientific Volume Imaging).

Electron microscopy Conventional TEM of Hydra vulgaris and Forskålia sp. wasperformed 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 with50 mM phosphate-buffer (pH 7.2). Specimens were dehydrated in di-methyl-formamid (50%, 70%, 90%) and embedded in a series ofincreasing lowicryl resin concentrations (DMF:lowicryl 2:1 for 15minutes, 1:1 for 30 minutes, 1:2 for 2 hours, and 100% lowicryl for12 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 IgGserum (Sigma) diluted 1:20 in PBS for 2 hours at 20°C and washedwith 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 analysisSDS-PAGE of isolated capsules and whole Hydra was performed asdescribed (Engel et al., 2001). For enzymatic deglycosylation, twomillion 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 30minutes at 70°C. The sample was then diluted with digestion bufferto 180 µl and insoluble material removed by centrifugation at 13,000g for 5 minutes. Aprotinin and leupeptin (1 µg/ml) were added to thesupernatant. The sample was split, 10 µl (10 U) N-glycosidase F(Roche) was added to the digestion sample, and 10 µl buffer to themock control, respectively. Both samples were incubated at 37°Covernight, and protein was then precipitated by addition of 30%trichloroacetic acid and analyzed by SDS-PAGE.

2D electrophoresis of capsule proteins was performed using apreviously described method (Görg et al., 1988). In the first dimensionproteins were focussed on IPG-strips with Multiphor II (AmershamPharmacia Biotech) essentially following the manufacturer’sinstructions. Isolated capsules (2 million) were solubilized in 150 µlfirst dimension buffer (8 M urea, 4% CHAPS, 20 mg/ml DTT, 5 µg/mlleupeptin, 5 µg/ml, aprotinin, 1 mM EDTA and 1% IPG-buffer) at

Journal of Cell Science 115 (20)

3925Molecular assembly of nematocysts

35°C for 30 minutes. Insoluble material was removed by 5 minutescentrifugation at 13,000 g and the supernatant supplemented to 350µl with first dimension buffer. IPG-strips (13 cm) with a linear pHgradient from pH 3-10 were rehydrated with the protein sampleovernight and proteins were focussed for 20,000-23,000 Vh. Forsubsequent separation according to molecular weight, strips wereequilibrated in second dimension buffer (50 mM Tris-HCl, pH 6.8,6M urea, 30% glycerol, 2% SDS and a trace of bromphenol blue) for15 minutes with 10 mg/ml DTT, and proteins were separated by SDS-PAGE.

For western analysis of proteins transferred to a nitrocellulosemembrane (Towbin et al., 1979), blots were blocked for 1 hour with1% BSA in TBS with 0.05% Tween (TBST). All further incubationsand washes (3-4 times for 10 minutes after each antibody incubation)were performed in TBST. Blots were incubated with mAb H22 diluted1:10 for 2 hours, washed and incubated with anti-mouse IgG antibodycoupled to alkaline phosphatase (Promega) diluted 1:7000 for 1 hour.The signal was visualized by enyzmatic precipitation of the colorsubstrate 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 ECLchemoluminescence system (Amersham) according to themanufacturer’s instructions.

Tryptic digestion and peptide sequencingThe 88 kDa protein spot was excised and cleaved directly in gel withtrypsin (Roche, Tutzing) as described (Eckerskorn and Lottspeich,1989). The eluted peptides were separated by reversed phase HPLCon 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 andUV-detection was performed at 206 nm. The peptide fractions werecollected manually and subjected to amino acid sequence analysis onan ABI 472A protein sequencer (Applied Biosystems, Langen) usingthe conditions recommended by the manufacturer. The sequencingresulted in the peptides: T-14, K/R I Y N Q I K; T-15, K/R X X D EI 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 VR; and T-25, K/R X X X Y L R g Q T d L (unequivocal amino acidsare shown in capitals).

PCR-based cloningRapid amplification of cDNA ends (RACE) and synthesis of cDNAlabeled at the 3′ end was performed using the method and primers(QT, Q0 and QI) described (Frohman, 1995). The peptide T-15 wasused 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). Theseoligonucleotides were used as sequence-specific primers for 3′RACEfrom first-strand H. vulgaris cDNA that had been synthesized frompoly A+ RNA with the olig-dT anchor primer QT. PCR was performedwith Taq DNA polymerase (Amersham Pharmacia Biotech) in agradient cycler (Eppendorf) under the following conditions: 5 minutesat 95°C (1 cycle); 1 minute at 95°C, 1 minute at 48.4°C, 1 minute at72°C (35 cycles); and 5 minutes at 72°C (1 cycle). 0.5 µl of theamplification product obtained with H22-1 and Q0 was used foramplification with H22-2 and QI. An amplification product of 650 bpwas ligated into the pGEM-T vector (Promega) according to themanufacturer’s instructions. The presence of another peptidesequence, T-14, within this PCR-fragment, confirmed it to be part ofthe mAb H22 antigen.

Screening of cDNA phage libraryPrimers 5′-GCC TGA TCA TAA TTC AAA ATA TGA-3′ and 5′-

CAA GTT GTT GTG ATT CTC TGC TCC-3′ were used to amplifya sequence from the 650 bp PCR fragment of Nowa and generate aprobe to screen a λZAP cDNA library (Stratagene) consisting of amixture of random and oligo-dT-amplified cDNA from Hydravulgaris. Filter lifts on Biodyne A membrane (Pall) were generatedusing a standard protocol (Sambrook et al., 1989). 80,000 plugs on20×20 cm filters were screened with the probe random-labeled with32P (Prime-It II kit, Stratagene) using the high stringency conditions.Three positive overlapping clones were isolated, which togetherrepresented the complete coding sequence of Nowa. The continuoustranscript 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′-UTRregion. A vector containing the complete coding region of Nowa inpBluescript SK- (Nowa pBluescript) was generated by ligation of twoof the original cDNA clones.

In situ hybridizationWhole mount in situ hybridization was performed as previouslydescribed (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 CTLDThe CTLD-encoding region of the Nowa cDNA comprising residues212-346 was amplified by PCR using the Nowa pBluescript vector asa template. NdeI and BamHI sites were introduced in the 5′- and 3′-primers, respectively, to enable convenient cloning of the amplifiedDNA into the corresponding sites of the prokaryotic expression vectorpet19b (Novagen). Primers used were: 5′-TTT CAT ATG AAA ATAAAA TGT CCA GAT GGC-3′; and 5′-TTT GGA TCC TTA CCTCAT CTT ACA AAC AAA TG-3′. The resulting vector thatintroduces a polyhistidine-tag at the N-terminal end of the proteinsequence was used for transforming E. coli BL21 (DE3) cells.Transformed cells were grown in LB medium at 37°C until the OD600reached 0.6 and then induced by adding IPTG to 0.4 mM. Cells wereharvested 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 bacteriawas achieved by several freeze/thaw steps followed by two cycles ofsonification. Nowa CTLD protein was found exclusively in inclusionbodies, 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. Refoldingwas achieved by solubilizing the inclusion bodies in 6 M GuHCl, 1mM 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 oxidizedglutathione. Precipitates were discarded and the soluble protein wasdialyzed against 50 mM Tris-HCl pH 8.0 and 150 mM NaCl. Finalpurification was achieved using nickel-sepharose chromatographyaccording to the manufacturer’s instructions (Novagen). Folding stateand stability were checked by CD-spectroscopy and trypsin digestion.

Results The mAb H22 antigen is a high molecular weight proteinof the nematocyst outer wall The monoclonal antibody H22 reacted with all nematocysttypes in Hydra, and staining was restricted to the wall of thecapsule (Fig. 1A,A′). In EM cross-sections of maturecapsules, immunogold-coupled mAb H22 labeled adistinctive layer adjacent to the electron-lucent inner wall(Fig. 1C). This outer layer has been previously described asthe nematocyst outer wall by electron microscopy (Holstein,1981; Watson and Mariscal, 1984). When nematocysts wereisolated (Weber et al., 1987) and analyzed by SDS-PAGE, the

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mAb H22 reacted with a high molecular weight protein ofapproximately 90 kDa in capsules, but not in whole Hydra(Fig. 1D). By comparison, minicollagens detected byminicollagen-1 antibody represent the major component ofthe capsule with a molecular weight between 30 and 40 kDa(Engel et al., 2001) (Fig. 1D).

Isolation of the mAb H22 antigen revealed a novelproteinTo purify the mAb H22 antigen, isolated capsules weresubjected to 2D electrophoresis. Capsule proteins, separatedaccording to their pI and molecular weight, were visualized byCoomassie blue (Fig. 2A) and silver-staining (Fig. 2C). Aprotein spot of pI 5.9 and a size of 88 kDa was correlated to animmunoreactive spot in western analysis (Fig. 2B). In gelsstained with Coomassie blue few other proteins were detectedapart from the immunoreactive spot (Fig. 2A), indicating thatthe antigen of mAb H22 represented a major structuralcomponent of capsules. The protein spot was excised from thegel, digested and microsequenced by Edman degradation. Usingdegenerate primers based on one of the resulting peptides (seeMaterials and Methods), a 650 bp fragment was amplified fromHydra vulgariscDNA by PCR-based 3′ rapid amplification ofcDNA ends (3′ RACE). The full length cDNA of 2634 bp wasisolated from a Hydra vulgariscDNA library and contained anORF of 2322 bp. The full nucleotide sequence is available underGenBank accession number AF 559862.

The translated amino acid sequence of 774 residues representsa completely novel protein with no homologues so far in thedatabase. We named the protein Nowa for nematocyst outer wallantigen. As expected for a secreted protein, there is a putativesignal peptide at the N-terminus, comprising the first 18 residues.The computed molecular weight (82.7 kDa) and pI (7.7) of theprotein without signal peptide differed from the values inferredfrom the protein spot in 2D gels (pI 5.9, 88 kDa; Fig. 2). Thisdiscrepancy is due to glycosylation of the protein. There arethree N-glycosylation sites present in the sequence, and N-linkedglycosylation was confirmed by deglycosylation of the proteinwith N-glycosidase F (Fig. 2D). The mAb H22 immunoreactiveband in Western blots disappeared after treatment with theenzyme, suggesting that the antibody reactivity depended on N-linked sugar moieties linked to the peptide backbone. With apolyclonal antibody against a recombinantly expressed domainof Nowa (see below), the molecular weight after deglycosylationwas determined to be 81 kDa (Fig. 2D).

Nowa contains a CTLD and a SCP domainAlthough Nowa has no homologue in the database, a searchfor 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 in Fig. 3A.The SCP domains occur in a variety of eukaryotic extracellularproteins (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 ina wide variety of otherwise unrelated extracellular proteins(Drickamer, 1999). The alignment in Fig. 3B of the NowaCTLD with different representatives of CTLD-containingproteins, shows the conservation of the cysteines (Cys(1-6)) and


Fig. 1.Localization of mAb H22 antigen in the outer wall ofnematocysts. (A) Isolated nematocysts of Hydra. The differentcapsule types are: d, desmoneme; i, holotrichous isorhiza; s,stenotele. One of the stenoteles has discharged (s*).(A′) Immunofluorescence of mAb H22 viewed by confocalmicroscopy (maximum projection) in the isolated nematocysts shownin A. (B) Mature, isolated nematocyte of Hydra.Note the size of thenematocyst vesicle, which fills almost the whole cell (N, nucleus).(C) EM immunogold labeling of mAb H22 in the wall of a Hydranematocyst. Gold particles are exclusively found in the outer wall(ow) and not in the inner wall (iw). (D) SDS-PAGE and westernanalysis of whole Hydraand isolated capsules. The protein of500,000 isolated capsules was compared with 1/5 Hydra (1/5H) bysilver-staining. In a western blot, protein of 1 Hydra (1H) and500,000 capsules separated by SDS-PAGE were probed with mAbH22 and minicollagen antibody. A high molecular weight protein isdetected by mAb H22 in isolated capsules, but not in whole Hydra.Minicollagens detected by minicollagen-1 antibody represent themajor proteins of the capsule. Bars, 5 µm (A,B); 100 nm (C).


most of the conserved aliphatic and aromatic stretches in theNowa CTLD. The conserved carbonyl residues implicated inCa2+-dependent sugar binding in the classical C-type lectins(Weis et al., 1991) are not found in Nowa CTLD, which makesit unlikely that the CTLD functions in sugar binding (seeDiscussion).

The Nowa CTLD was expressed recombinantly (Fig. 4A) ina bacterial expression system. The resulting 18 kDa proteinwas used to generate an antibody that reacted strongly with itsantigen (Fig. 4B). In capsules, it reacted specifically with the88 kDa band recognized by mAb H22, confirming the identityof Nowa and the antigen of mAb H22.

Minicollagen Cys-rich domains are repeated eight timesin the C-terminal part of NowaThe most striking feature of Nowa is a Cys-rich C-terminal partin which six characteristically spaced cysteines are repeatedeight 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 Cys-rich (MCCR) domain, is used in minicollagens to interconnectminicollagen trimers to large polymers (Engel et al., 2001). Itis an intriguing possibility that the multiple MCCR domains inNowa allow Nowa to interact with minicollagens through thematching cysteines and to take part in the crosslinking process(see Discussion).

Nowa transcription occurs only indeveloping nematocytesTo investigate a possible role of Nowa information of the capsule wall, wedetermined its expression in cells of thenematocyte differentiation pathway (Fig.6A). In situ hybridization revealed thatthe mRNA encoding Nowa was notexpressed in mature nematocytes but onlyin differentiating nematocytes ornematoblasts (Fig. 5, arrows). Thetentacles showed no hybridization withthe probe for Nowa mRNA. In the bodycolumn, clusters of small labeled cellswere present (Fig. 5B), representing nestsof nematoblasts or developingnematocytes. Nests with clearly

discernable capsules were negative (Fig. 5B′, arrows withasterisks). Thus, Nowa mRNA was expressed only at thebeginning of nematocyte differentiation.

Nowa protein expression starts concomitant withcapsule formation To determine the onset of Nowa expression in thedifferentiation pathway, Hydrawere continuously labeled with[3H]thymidine to identify proliferating precursors and trace thetransition of proliferating precursors into differentiating cells.Fig. 6E shows that [3H]thymidine-labeled cells at time point 0did not express Nowa detected by mAb H22. However, about5 hours after onset of labeling, some labeled cells becamemAb-H22-positive, indicating expression of Nowa. Completelabeling of differentiating nematocyte nests required 3-4 days,which is in good agreement with previous results (David andGierer, 1974). The rapid appearance of labeled mAb-H22-positive cells indicates that Nowa synthesis starts alreadybefore the terminal mitosis of nematoblasts.

This surprising result was confirmed by analysing dividingnematoblast nests in whole mounts stained with mAb H22. Thepercentage of dividing nests positive for mAb H22 was 84%in 16-cell nests (n=19), 74% of all 8-cell nests (n=101), and17% of all 4-cell nests (n=59). An example of a dividing 8-cellnematoblast nest is shown in Fig. 6B,C. Nuclei in metaphaseare clearly discernable from interphase nuclei in adjacent cells

Fig. 2.Characterization and isolation of themAb H22 antigen. (A-C) 2D electrophoresisof capsule proteins according to pI andmolecular weight. The protein of two millioncapsules was separated and analyzed byCoomassie staining (A), western analysiswith mAb H22 (B), and silver-staining (C).(D) Assay of N-glycosylation. Solubilizedcapsule protein was incubated with N-glycosidase F to remove N-linked sugars. Thedeglycosylated protein and mock control(without enzyme) were separated by SDS-PAGE and probed with mAb H22 (left) andanti-CTLD antibody (right) in westernanalysis.

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in an optical section (Fig. 6B). Each dividing cell contains onelarge and several very small Nowa-filled structures, as seen inthe surface projection (Fig. 6C). We interpret the largerstructure to be the vesicle containing the capsule primordium.The small structures probably represent TGN vesicles, whichare scattered over the cell due to the disassembly of the Golgiapparatus during mitosis. This pattern of vesicle distribution isalso visible in dividing nematoblasts that were double-stainedwith anti-tubulin antibody to visualize the spindle apparatus(Fig. 6D). The presence of mAb-H22-positive structures inmetaphase nematoblasts demonstrates that nematocystmorphogenesis already starts in nematoblasts before their

terminal division into nematocytes. During this terminalmitosis, one daughter cell inherits the young capsuleprimordium while the second daughter cell forms a newprimordium.

Sorting of capsule proteins into the nematocyst wallThe formation of the wall and tubule structures involves a yetundefined sorting mechanism that leads to the formation of thedouble-layered wall. We used confocal microscopy andimmunogold labeling in EM-sections to follow the subcellulardistribution of Nowa during morphogenesis (Figs 7-9).


Fig. 3.Nowa protein from Hydravulgaris. (A) Nowa domainorganization. CTLD, C-typelectin-like domain (smart00034,E-value of 10–17); SCP, homologyto SCP domains (smart00198, E-value of 10–3); SP, signal peptide.The putative cleavage site for anN-terminal propeptide afterresidues KR is indicated by anarrow. (B) Alignment of CTLDsof Nowa and vertebrate proteins.Sequences most similar to theNowa CTLD were retrieved by aBLAST-search in SWISS-PROT(sp) and TrEMBL (tr) proteindatabases. (sp P16112), aggrecancore precursor (human); (spP55066), neurocan core proteinprecursor (murine); (tr O62623),cartilage proteoglycan (bovine);(tr Q13018), PLA2 RE, secretoryphospholipase A2 receptorprecursor (human); (tr Q64449),mannose receptor, C type 2(murine); (tr Q9IB90), C-typelectin from Cyprinus carpio; (trQ9I840), Aggretin β-chain fromAgkistrodon rhodostoma; (trQ9IAM0), Agkisacutacin β-chainfrom Agkistrodon acutus. Shadingshows conservation of residues ofthe same similarity group withinthe aligned sequences: black,100%; dark grey, 80%; and lightgrey, 60% conservation. Aminoacid similarity groups: DN, EQ,ST, KR, FWY, ILMV.(C) Alignment of cysteines inCys-rich domains of Nowa andminicollagens. The C-terminalCys-rich part of Nowa is aneightfold 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.

Nowa-REP1 464 QITGTCPS---GCSGDCYPECKPGCCGQVNLNAPVQP 497

Nowa-REP2 498 SGYTACSQ-YPNCGLSCQSSCSQSCCQQNPYQPSVMSGTIVIQP 540

Nowa-REP3 541 NEQSVCPQ-HPGCSQHCAPRCSPQCCQQSMNSLYQP 575

Nowa-REP4 576 PQMSACPQ-FPSCSPTCAPQCSQLCCQQSSMPLQM 609

Nowa-REP5 610 PQMPSCPQ-FPSCSASCAPQCSQQCCQQPSMSIQP 643

Nowa-REP6 644 LQISSCPQ-FPSCSPSCAPQCSQQCCQQPSMPIQL 677

Nowa-REP7 678 PLMGSCSQ-MPGCSASCAPLCSQQCCQQQAMLQQSIMQQPMM 718

Nowa-REP8 719 MAQNPCSLQQPGCSSACAPACRLSCCSLGRMNLGR 753

minicollagen-1 28 RDANPCGS---YCPSVCAPACAPVCCYPPPPPPPPPPPPPPP 66

122 PPPPPCPP---VCVAQCVPTCPQYCCPAKRK 149

minicollagen-2 22 RSAQACGY---NCPAICAPACTPICCAPPPPPPPPPPPPPPP 60

116 PPPPPCPP---ICPTQCVPYCPQYCCPLKK 142

minicollagen-Ad 26 REASPCGY---GCPSMCAPACEPTCCAPPPPPPPPP 58

117 PPPPPCPP---ICIQHCIRICPQPCCSPPPPP 145

A

C

BNOWA : 215 CPDGW--KANNGNCYKL- FEEEMAWADAVDHCNVLKSS-- LFSGESVEEGAFLKTMLVG--RSSPSWIGM 277 AGGRECAN : 2205 CEEGW--NK YQGHCYRH-FPDRETWVDAERRCREQQSH--LSSIV TPEEQEFVN-----NNAQDYQ WIGL 2264 NEUROCAN : 1040 CDRGW--HK FQGHCYRY- FAHRRAWEDAERDCRRRAGH--LTSVHSPEEHKFI N-----SFGHENS WIGL 1099 CARTILAGE PG : 509 CEEGW--TK FQGHCYRH-FPDRATWVDAESQCRKQQSH--LSSIV TPEEQEFVN-----NNAQDYQ WIGL 568 PLA2 RE : 380 CEPGW--NP YNRNCYKL-QKEEKTWHEALRSCQADNSA--LIDIT SLAEVEFLVT-LLGDENASETWIGL 443 MANNOSE RE : 234 CETFWDKDQLTDSCYQFNFQSTLSWREAWASCEQQGAD--LLSITEIH EQTYI NG--LLTGYSSTLWIGL 311 C-TYPE LECTIN : 27 CQYGW--TN FGVQCYKF- FSRSTSWIA AERNCIEEHAN-- LASVHNEEENDFLMG--LLPSTTKRCWLGV 89 AGGRETIN : 25 CPSGW--SS YEGHCYKP- FNEPKNWADAERFCKLQPKHSHLVSFQSAEEADFVVKLTRPRLKANLVWMGL 91 AGKISACUTACIN: 25 CPSDW--SS YEGHCYKP- FDEPKTWADAEKFCTQQHKGSHLASFHSSEEADFVVTLTTPSLKTDLVWIGL 91

NOWA : 278 S DMAAKGGFQFVDGTPYVYSDWSRESQQLVIDLWNTKKETVKNQCI TAS---YEG WNYKDCFKKLPFVCK 344 AGGRECAN : 2265 N DRTIEGDFRWSDGHPMQFENWRPNQPDNFFAAG--------ED CVVMIWHEKGEWNDVPCNYHLPFTCK 2326 NEUROCAN : 1100 N DRTVERDFQWTDNTGLQYENWREKQPDNFFAGG--------ED CVVMVAHESGRWNDVPCNYNLPYVCK 1161 CARTILAGE PG : 569 N DKTI EGDFRWSDGHSLQFENWRPNQPDNFFATG--------ED CVVMIWHEKGEWNDVPCNYQLPFTCK 630 PLA2 RE : 444 SSNKI PVSFEWSNDSSVI FTNWHTLEPHIFPNRS--------QL CVSAEQS-EGHWKVKNCEERLFYICK 504 MANNOSE RE : 312 N DLDTSGGWQWSDNSPLKYLNWESDQPDNPGE----------EN CGVIRTESSGGWQNHDCSIA LPYVCK 3 71 C-TYPE LECTIN : 90 Q DAVEEGQWLWSDGTPYDYSNWCSNEPNNLNV----------EN CGEINWTSDRCWNDASCSTSMGYVCA 149 AGGRETIN : 92 S NI WHGCNWQWSDGARLNYKDWQEQ--------- --------SE CLAFRGVHT-EWLNMDCSSTCSFVCK 143 AGKISACUTACIN: 92 K NI WNGCYWKWSDGTKLDYKDWREQ-----------------FE CLVSRTVNN-EWLSMDCGTTCSFVCK 143

C(1)

C(6)

C(3)C(2)

C(5)C(4)

SCP CTLD Cys-rich SP

1-18 59 190 215 344 469 744 77434

KR


Already in early stages of capsule growth, Nowa wasenriched in the capsule wall, while minicollagen was foundonly in the capsule matrix and in the ER (Fig. 7A′,B). In anEM-section of such a stage (Fig. 7C), which was stained withimmunogold mAb H22, gold particles were present inside thecapsule vesicle, the Golgi apparatus and large vesiclesclose to the capsule primordium. These protein-filledvesicles most likely correspond to the prominent cap-like structures positive for mAb H22 at the growingapex of the capsule (Fig. 7A′ and D arrows), which weinterpret to be TGN. We previously described similarvesicular structures filled with minicollagen (Engel etal., 2001). Astonishingly, double-labeling experimentsrevealed that Nowa and minicollagen do not colocalizein the same TGN vesicles (Fig. 7E). This suggests asorting mechanism by which a pre-mature interactionof Nowa and minicollagen is prevented. No Nowa wasdetected in the ER, which is in agreement with ourfinding that the mAb H22 reacts with an epitopedependent on N-glycosylation of Nowa (Fig. 2D).

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

At the end of nematocyst morphogenesis, when the outertubule has been invaginated into the capsule, minicollagensundergo a disulfide rearrangement leading to a loss ofminicollagen antibody reactivity (Engel et al., 2001).Minicollagens are still present in mature capsules asdemonstrated by Western blots of isolated capsules (Fig. 1D),but they are not accessible for the antibody byimmunohistochemistry. Fig. 7A shows a nest with two almostmature capsules lacking the minicollagen immunreactivity buthaving a speckled mAb H22 staining pattern, which ischaracteristic of mature capsules (Fig. 1A).

MTs form a dynamic scaffold around differentiatingnematocytesLocalization of the MT cytoskeleton in developingnematocytes by confocal microscopy and in EM-sections,revealed a prominent MT scaffold around the developingnematocyst. This scaffold changed as the nematocyst grew. Inearly 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 outertubule was forming (Fig. 9D-F), MTs formed remarkably long,parallel arrays around the outer tubule. The putative positionof the microtubule-organizing center (MTOC; arrows in Fig.9A-F) was close to the site where Nowa-filled or minicollagen-filled TGN structures were observed (Fig. 9B,C,E,F). A

Fig. 4.Recombinant expression of Nowa CTLD. (A) Nowa CTLDconstruct used for recombinant expression in E. coli. (B) Purifiedrecombinant Nowa CLTD and capsule proteins (500,000 capsules)analyzed by SDS-PAGE and visualized by silver-staining andwestern analysis.

Fig. 5. In-situ hybridization of Nowa transcript in wholemounts of Hydra. (A) Overview of an animal with a smallbud showing expression of Nowa in the body column but notin the tentacles. (B) Body column with Nowa-mRNA-positive cells. (B′) Enlargement of B with Nowa-mRNA-positive nematoblast or nematocyte nest indicated by anarrow. An adjacent nest with capsules but no Nowaexpression is indicated by an arrow and asterisk. Bars, 100µm (A); 50 µm (B); 10 µm (B′).

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longitudinal EM-section through an outer tubule revealed MTsrunning along the outer tubule, except at the very tip (Fig. 9G).The two centrioles marking the position of the MTOC werefound close to the tubule tip, in agreement with theimmunolocalization images (Fig. 9A-F). A cross-section of theouter tubule showed the intimate association of MTs to eachother and to the vesicle membrane around the outer tubule (Fig.9H,H′), demonstrating that the MTs formed a cage-likestructure around the growing part of the nematocyst. Thisarrangement and the localization of the MTOC at the site wheremore protein-filled vesicles are delivered to the nematocystvesicle suggest a regulating function of the MT cytoskeletonin directing nematocyst growth (Fig. 10).

DiscussionThe sorting of proteins to form a complex structure inside an

unstructured vesicle requires self-assembly processes. In aprevious paper, we have shown how minicollagens assembleinto the inner wall and are crosslinked to form a highly stress-resistant network (Engel et al., 2001). Here we expand themodel to consider how shape could be generated inside thenematocyst vesicle. Morphogenesis of the structure ischaracterized by polar fusion of protein vesicles leading topolarized growth of a tubule several cell-diameters in length.Intra-vesicular patterning occurs by assembly of proteins intodistinct layers of the wall. We propose that the cytoskeletonrepresents a force that acts on the vesicle to generate shape.This in concert with the temporally regulated transport of anovel outer wall protein, Nowa, and its interaction withminicollagens inside the nematocyst vesicle results in theformation of the double-layered capsule. Once the structure hasreached its final dimensions, the wall is crosslinked to providehigh mechanical stability.

Nowa is a novel protein and putative binding partner ofthe minicollagens Nowa was identified as a component of the outer wall of Hydranematocyts. It is a novel protein of 88 kDa that is modified byN-glycosylation (Fig. 2D). The sequence starts with a putativesignal peptide of 18 residues. The presence of the residues KRat position 33/34 might suggest cleavage of a propeptide asdemonstrated for minicollagens (Engel et al., 2001) and manyother capsule proteins (Anderluh et al., 2000).

By sequence comparison, we were able to identify threedomains with homology to domains of other extracellularproteins (Fig. 3A). The C-terminal Cys-rich domain proved tobe an eightfold repetition of the MCCR domain (Fig. 3C).These domains, which also flank the collagenous part of theminicollagens (Kurz et al., 1991), are defined by sixcharacteristically spaced Cys-residues. Previous work hasshown that this Cys-rich domain functions in the crosslinkingof minicollagens to an oligomeric structure. Crosslinkingoccurs at a late stage of morphogenesis when the minicollagenshave assembled into the wall. Minicollagens are first producedas soluble trimers that display intra-chain disulfide bondswithin a single MCCR domain. By isomerization of the intra-


Fig. 6. Immunolocalization of Nowa protein in dividingnematoblasts. (A) The nematocyte differentiation pathway in Hydra.Nematocytes originate from interstitial stem cells (I-cells), whichdivide three- to five-times after commitment (nematoblasts) andremain interconnected by cytoplasmic bridges forming nests of 8-32cells (David and Challoner, 1974; David and Gierer, 1974). Afternematocytes have formed a nematocyst (green), nests brake up intosingle nematocytes, which migrate to the tentacles. (B-D) Confocalmicroscopy 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) Surfaceprojection of the same nest, the boundary of the nest is indicated by adotted line. (D) Nematoblast in division with metaphase spindleapparatus visualized by mAb anti-tubulin (red). Projections fromdifferent angles show the asymmetrical position of the capsuleprimordium. (E) Continuous [3H]thymidine labeling of mAb-H22-positive nests. The first labeled nests appeared ~5 hours after onset oflabelling; values represent single measurements from twoindependent experiments. Bars, 5 µm.


chain to inter-chain disulfide bonds in a late stage ofmorphogenesis, the minicollagens are crosslinked and thecapsule wall achieves its high tensile strength (Engel et al.,2001). Similarly to the minicollagens, Nowa protein could notbe solubilized from mature capsules without a reducing agent(data not shown), which indicates that it is crosslinked bydisulfide bonds. We propose that the cysteines of the NowaMCCR domains undergo a similar isomerization process andthat Nowa could be covalently linked to minicollagens by itsmatching cysteines. Since the eightfold repetition of MCCRdomains in Nowa could possibly crosslink up to eightminicollagen molecules, Nowa may function as acrystallization center for minicollagen assembly. We therefore

speculate that minicollagen and Nowa molecules at theinterface of the outer and inner wall are crosslinked by hetero-oligomers, while the majority of minicollagen and Nowa in thetwo wall layers form homo-oligomers (Fig. 7D).

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

The CTLD, which is detected in Nowa is a conserved proteinmodule, which was initially identified in a group of C-type(Ca2+-dependent) animal lectins (Drickamer, 1988). The Nowa

Fig. 7.Immunolocalization of Nowaprotein in the early growth phase ofnematocyst morphogenesis.(A,B) Confocal microscopy of mAbH22 (green) and minicollagen antibody(red) in early stages of nematocytedifferentiation. Nuclei are stained withDAPI (blue). (A) Early and late stages ofcapsule development in an opticalsection. (A′) Enlargment of one of theearly stage nematocytes with nematocystprimordium. Staining of mAb H22 isrestricted to the wall and tubular-vesicular structure at the apex (arrow),while minicollagen is detected in the ERand matrix of the capsule, as depicted inthe drawing in B. (C) EM-section ofnematocyte with nematocystprimordium. Immunogold mAb H22labeling is found in the capsule matrix(cm), the outer layer of the capsule wall(cw), the Golgi apparatus (g), asindicated by an arrow, and membranecompartments associated with theprimordium but not in the ER (n,nucleus). (D,D′) Formation of the innerwall layer by minicollagen.Minicollagen is no longer found in thematrix of the capsule but forms the innerwall (iw) adjacent to the mAb-H22-positive outer wall (ow). (E) Isorhiza-type nematocyst with tubular-vesicularstructures at the growing apex (arrow) inan optical section. Bars, 5 µm(A,A ′,B,D); 1 µm (C).

Fig. 8. Immunolocalization ofNowa protein in the late growthphase of nematocystmorphogenesis. Confocalmicroscopy of threenematocysts with an outertubule. (A) Surface projection ofmAb H22 (green) and spinalinantibody staining (red). A′ shows mAb H22 staining only to visualize staining of the outer tubule. (B) Schematic representation of a nematocystwith an outer tubule. Bars, 5 µm.

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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 fourextracellular CTLDs (26% identity) (Reidling et al., 2000),indicating that the CLTDs are divergent already in Hydra.

The Nowa CTLD belongs to the intron-positive subfamiliyof CTLDs, which possess six Cys-residues (Drickamer, 1989),with disulfide bonds between Cys(1)-Cys(2), Cys(3)-Cys(6), andCys(4)-Cys(6) demonstrated in many of these CTLDs (Llera etal., 2001; Usami et al., 1993). The CTLD of Nowa lacks theresidues with carbonyl side chains implicated in Ca2+-dependent sugar binding (Weis et al., 1991). However, it hasbeen shown for other CTLDs that lack the Ca2+-coordinatingresidues, that these CTLDs function in highly specific proteinbinding independent of sugarmoieties (Llera et al., 2001). Thus,the Nowa CTLD may function innon-covalent binding ofminicollagens, prior to disulfidecrosslinking.

Nowa is localized in the outerwall in a globular layerNowa localized by mAb H22 wasfound exclusively in the outer wallof mature nematocysts (Fig. 1). Thestaining pattern, which looked likespeckles evenly distributed on thecapsule surface, was reminiscent ofthe structure observed by fieldemission scanning electronmicroscopy on the surface ofisolated nematocysts (Holstein et al.,1994), where the outer wall wasshown to consist of globules. Thespeckled pattern of mAb H22staining was found in all nematocysttypes of Hydra (Fig. 1A′), innematocysts of the anthozoanNematostella vectensis, thecubomedusan Carybdea marsupialisand the hydrozoanHydractiniaechinata (U.E. and T.W.H.,unpublished). This strongly suggeststhat the outer wall formed by Nowais common to the nematocysts ofthese cnidarian classes andpresumably is an indispensableconstituent. To date, no function ofthe outer wall has been proposed innematocyst discharge, but theputative interaction of Nowa withminicollagen through the MCCRdomains implies a function forNowa in morphogenesis. The factthat Nowa expression and proteinsynthesis start prior to the finaldivision of nematoblasts (Figs 5, 6)indicates that it is one of the veryfirst proteins to occur in the

nematocyst primordium, concomitant with the formation of thenematocyst wall.

A model for sorting of proteins into the nematocyst wallImmunostaining of MTs visualized by confocal microscopyallowed us to follow the MT rearrangements around thegrowing nematocyst (Fig. 9). In EM sections, centriolesmarking the position of the MTOC are visible at the growingapex of the vesicle (Holstein, 1981; Watson and Mariscal,1984). One intriguing function of MTs radiating from theMTOC could be the correct positioning of the Golgi apparatusand TGN relative to the site where the capsule vesicle grows.Accordingly, MTs could provide tracks for transport of


Fig. 9. MT scaffold around growing nematocysts in the early (A-C) and late (D-H′) growth phaseof nematocyst morphogenesis. (A,D) Localization of tubulin in Hydramacerates.(B,C,E,F) Confocal microscopy of nematocyst nests in the body of Hydrawhole mounts stained bymAb H22 and mAb anti-tubulin or minicollagen and anti-tubulin (all shown as projections). Theputative position of the MTOC is indicated by an arrow. (G-H′) EM sections through the outertubule (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 withthe membrane around the outer tubule. Bars, 5 µm (A-F); 0.5 µm (G); 2 µm (H); 100 nm (E′).


protein-filled vesicles to the site where new material isdeposited (Fig. 10A). However, there seems to be an additionalfunction of the MT scaffold: as shown in the EM cross-sectionof an outer tubule (Fig. 9H), the MTs are intimately linked tothe nematocyst vesicle membrane. By their tight spacing toeach other and to the membrane (14 nm and 12 nm,respectively) (Holstein, 1981), the MTs form a cage that canaccount 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 thenematocyst part under construction (capsule or outer tubule,Fig. 9) and might regulate its diameter. The dense packing ofMTs efficiently prevents fusion of vesicles along the outertubule 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 thenematocyst vesicle may contribute to the polar growth of thenematocyst vesicle and determine its shape.

The question arises how the shape enforced onto the vesicleby the MT cytoskeleton is passed on to the developingstructures in the interior of the vesicle. Based on electronmicroscopy of nematocyst development, Watson proposed thatwall 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 layerlining the membrane already in very early stages would makeit an ideal candidate for such a function. Thus, Nowa could

serve as positional information for minicollagen assembly asschematically depicted in a cross-section of the nematocystvesicle in Fig. 10B. Minicollagens transported into the capsuleswould bind to Nowa and aggregate to form the inner wall. Inagreement with this model, Nowa is also found to line theforming outer tubule (Fig. 8) and could fulfil a similar functionin patterning of this part of the nematocyst.

An obvious question that arises from the model describedabove is, how a pre-mature interaction of Nowa andminicollagen in the ER, Golgi apparatus and TGN is prevented.Remarkably, we observed separate transport compartments forminicollagen and Nowa, which we interpret as TGN (Fig. 7E).In the ER, where both proteins are synthesized, we can onlyspeculate that the two proteins at this stage are not yetcompetent for interaction, as they have not undergone theirpost-translational modifications (e.g. glycosylation). Weenvisage that the first steps of ordering the wall structures inthe nematocyst vesicle occur by noncovalent interactions.Early covalent crosslinking between minicollagens and Nowathrough disulfide bonds would lead to disordered assembly ofthe proteins and not to the distinct layers observed.Furthermore, the isomerization of disulfide bonds inminicollagens 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 stepsof Nowa assembly, such as sorting of the protein to themembrane, the self-aggregation to form a distinct outer wall

Fig. 10. Formation of the capsule wall formedfrom Nowa (green) and minicollagen (red).(A) Protein sorting and transport as detected byminicollagen antibody and mAb H22.Minicollagen and Nowa synthesized in the ERare transported in separate vesicles to thenematocyst vesicle. Nowa is detected by mAbH22 only modification by glycosylation in theGolgi apparatus and forms the outer wall. MTs(yellow) are organized in a scaffold around thegrowing part of the nematocyst, the MTOC islocalized between the Golgi apparatus and thegrowing apex of the nematocyst. Minicollagenfirst accumulates in the capsule matrix and is thensorted to the wall to form the inner layer of thewall (3). By further transport of protein-filledvesicles, the outer tubule forms (4). It issubsequently invaginated into the cyst, and spines(s) are formed in the tubule lumen (5). Finally,minicollagen crosslinkage leads to a compactionof the wall structure (6). (B) Model of nematocystpatterning by the MT cytoskeleton. The growingpart of the nematocyst vesicle is shown in aschematic cross-section. MTs form a cage aroundthe vesicle and determine its shape (1). The outerwall formed by Nowa on the membrane (2) isused as template for minicollagen assembly.Soluble minicollagen trimers aggregate on theouter wall to form the inner wall (3-5). Finally,Nowa and minicollagen are crosslinked bydisulfide bond isomerization to stabilize thestructure (6).

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are achieved. The multi-domain nature of Nowa opens theintriguing possibility that each of the domains serves a differentfunction in this process. The best understood example so far isthe MCCR domain present in Nowa and minicollagens, whichwas shown to mediate the transition of a state of solubility toan oligomeric network of extremely high stability (Engel et al.,2001).

Many thanks to Hans Bode and Michael P. Sarras for providing theHydra vulgariscDNA library, and to Charles David and Jürgen Engelfor discussions and critical reading of the manuscript. Supported bythe DFG (SFB 269 and GK 361).

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