1 Institute of Zoology, Darmstadt University of Technology, 64287 Darmstadt,
Germany
2 Department of Biophysical Chemistry, Biozentrum, University of Basel, 4056
Basel, Switzerland
3 Institute of Zoology, University of Munich, 80333 Munich, Germany
4 Max-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:
holstein{at}bio.tu-darmstadt.de)
Accepted 1 August 2002
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Summary |
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Key words: Minicollagen Cys-rich domain, CTLD, Nematocyst, Microtubules, Assembly
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Introduction |
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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
).
|
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-
-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.
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Materials and Methods |
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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 against
ß-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 Forskålia
sp. 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 ß-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 ZAP 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 20x20 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.
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Results |
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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.
|
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 in
Fig. 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).
|
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.
|
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.
|
|
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 in Fig. 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 (Figs
7,8,9).
|
|
|
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. 7A
shows 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).
|
![]() |
Discussion |
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Nowa is a novel protein and putative binding partner of the
minicollagens
Nowa was identified as a component of the outer wall of Hydra
nematocyts. 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 (Figs
5,
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 in
Fig. 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).
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References |
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