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First published online February 15, 2006
Journal of Experimental Biology 209, 956-964 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02031
An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva
1 Göteborg University, Department of Zoology, Zoophysiology,
Medicinaregatan 18 SE-413 90 Göteborg, Sweden
2 Section on Cell Biology and Signal Transduction, NICHD, NIH, Building 49,
Room 5A-78, 22 Convent Drive, MSC 4480, Bethesda, MD 20892-4480,
USA
* Author for correspondence (e-mail: lena.martensson{at}zool.gu.se)
Accepted 12 December 2005
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Summary |
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Key words: barnacle cyprid, cement gland, cement secretion, exocytosis, dense core granules, granule swelling
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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The life cycle of Balanus improvisus has seven planktonic larval
stages before it metamorphoses into a sessile organism. Between the sixth and
seventh transitions, the larva transforms from a nauplii larva into a cyprid
larva. The cyprid larva searches for a suitable surface to attach itself to
and to metamorphose into a sessile reproducing animal. The prerequisite for
settling and metamorphosis is the ability to produce, store and secrete the
adhesive proteins once the cyprid identifies an appropriate surface to adhere.
The adhesive-secreting cells are located within a pair of cement glands, which
are connected by cement ducts that widen into muscular cement sacs, the
presumed temporary storage location during cement secretion. Cement ducts
connect the sacs to an antenna, which is composed of four segments. The cement
duct extends into the third segment, the adhesive discs. The adhesive is
secreted through the discs, and the cyprid larva is able to attach itself to
the surface and begin metamorphosis
(Harrison and Sandeman, 1999
;
Nott and Foster, 1969).
Our approach is to understand the biology of barnacle cement secretion in
detail so that new techniques could be developed to control their settling on
to man-made marine surfaces. Current methods of control mainly use biocide
doped paints on surfaces, and such biocides leach in significant quantities to
cause serious toxicity to the marine environment. New approaches are necessary
to devise more environmentally benign modes of control of barnacle-induced
biofouling (de Nys and Steinberg,
2002; Omae, 2003).
Understanding the neural control of cement secretion is expected to provide
the lead to develop novel substances that inhibit cement secretion. In the
present study, we have focused on the morphology of the cyprid cement glands
and the physiology of adhesive secretion.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). In
short, adult barnacles were allowed to settle on Plexiglas panels placed in
the sea off the west coast of Sweden in the vicinity of Tjärnö
Biological Laboratory (58°53' N, 11°8' E). The panels were
brought to the laboratory and placed in buckets with running seawater. These
animals were used as brood stock in all laboratory experiments. Adult B.
improvisus will spawn throughout the year when regularly fed with nauplii
of Artemia sp. and the prymnesiophyte Isocrysis galbana.
When kept at 27–28°C, the development to cyprid larvae takes
6–7 days, and the newly moulted cyprid larvae were filtered through a
stack of sieves and washed to remove algae and detritus. The cyprids were then
transported in 25 cm3 flasks to the laboratory, stored at 4°C
and usually used within a week. All experiments were performed in filtered seawater (FSW, 0.22 µm), and drugs used were purchased from Sigma. The drugs were diluted in FSW to desired concentrations.
Preparation for light microscopy and transmission electron microscopy (TEM)
The cyprids were placed in FSW containing no dopamine, or 1 mmol
l–1 dopamine for a time series of 2, 4 and 10 min treatment.
The cyprids were thereafter immediately transferred over to the fixative.
The cyprids were fixed according to Harrison and Sandeman
(Harrison and Sandeman 1999).
Cyprids were chilled to 4°C for 30 min and transferred to fixation
solution containing 2.5% glutaraldehyde and 2% formalin in FSW, pH 8.2. The
cyprids were then microwaved in an ice bath up to 37°C and thereafter
placed in fixative at 4°C overnight. Several washings were then performed
in FSW over 1 h, followed by treatment with 2% osmium tetroxide for 30 min and
then 2% uranyl acetate for 20 min. Dehydration was thereafter performed in an
ethanol series of 50%, 70% and 90%. Finally, propylenoxide and infiltration of
Agar 100 resin (Epon) was performed overnight in 4°C. Samples were
embedded in gelatine capsules in Agar 100 and polymerized at 60°C for 2
days.
For light microscopy, the blocks were sectioned into 2 µm-thick sections and stained with 1% Toluidine Blue and Pyronin G for 1 min at 60°C. The sections were then mounted in Pertex and examined in a Nikon Optiphot microscope connected to a Nikon DXM 1200 digital camera. Images were acquired using the ACT-1 software (Nikon Microscopes, Europe BV, Badhoevedorp, The Netherlands). Contrast levels were then adjusted in Adobe Photoshop CS (Adobe Systems, Inc., Mountain View, CA, USA).
Confocal microscopy
Cyprid larvae are transparent animals and thus are easily observed under a
confocal microscope to study the secretory process in situ. We found
that Acridine Orange accumulates within secretory vesicles in the cement
glands of cyprids and could be used as a flourophore to study the exocytotic
secretion. This is similar to secretory granules of pancreatic ß-cells
due to the acidic intragranular environment with respect to the cytosol
(Pace and Sachs, 1982). The
excitation wavelength was 490 nm and fluorescence emission was collected at
519 nm. We incubated cyprid larvae in FSW containing 10 mmol
l–1 of Acridine Orange for 1 h and washed them several times
in FSW prior to microscopy. The cyprids seemed unaffected by the Acridine
Orange loading.
The larvae were mounted on a cover glass using Kwik Sil silicon glue (World Precision Instruments, Herts, UK) at the caudal end, while the anterior end remained free. They were kept in FSW in darkness for 1 h. A perfusion chamber was built up by using Tack ItTM (Faber-Castell, Germany) on a glass slide, and the cover glass with the cyprid was inverted and attached to Tack It, thereby creating a perfusion chamber. Larvae were imaged in a Bio-Rad MRC 1024 laser scanning confocal microscope equipped with a Krypton/Argon laser. Images were acquired using Lasersharp 2000 (Bio-Rad, Hercules, CA, USA) at a magnification of 800x and were analyzed using Adobe Photoshop CS.
Differential interference contrast (DIC) microscopy
Cyprid larvae were immobilized in 1% low gelling temperature agarose, type
VII (Sigma A-9045, gelling temperature below 25°C) dissolved in FSW. Agar
was poured over the larvae and they were placed inside a refrigerator for 5
min for polymerization. Animals were then examined under an inverted
microscope and imaged. Images were acquired using a CCD camera (Pulnix Corp.,
San Jose, CA, USA), and the microscope and camera systems were controlled by
Synapse (Synergy Research Inc., Silver Spring, MD, USA) as described earlier
(Simpson et al., 1997).
Cyprids immobilized in agarose appeared unharmed and tried to move within the
gel during microscopic observation.
Estimation of secretory granule number and size
In order to measure the changes in secretory granule type and number during
the secretory event, we stimulated cement secretion in intact larvae using
dopamine (1 mmol l–1). Larvae were exposed to dopamine for 0,
2, 4 or 10 min and were immediately fixed as described earlier. Sections were
cut and were stained with Toluidine Blue for light microscopic observation and
granule counting. Statistical comparison was carried out using analysis of
variance (ANOVA, P<0.0001). Newman–Keuls multiple comparison
test was used as a post-hoc test with an 
-value of 0.05.
For secretory vesicle size measurements, we used Easy Image Measurement
2000 (Bergström Instrument AB, Solna, Sweden), and the cross-sectional
area of the granules in six different sections showing all four types of
granules was measured. All granules of type 2, 3 and 4 within a section were
measured. Due to the presence of large numbers of type 1 granules, the
cross-sectional area of all granules within 2–3 cells was measured. The
result was evaluated by Kruskall–Wallis test (P<0.0001) and
Dunn's multiple comparison as post-hoc test with an -value of
0.05. ANOVA evaluations were done using Graph Pad Software (GraphPad Software,
Inc., San Diego, CA, USA).
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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) and live for up to a month. The cement gland is easy to
recognize under a microscope, and Fig.
1 shows the gland and the cement duct, which widens into a
muscular sac. The muscular sac is connected by another cement duct to the
antennae. The individual cement granules within the gland are large enough to
be seen as individual bumps on the gland surface.
Characterization of cement granules
Light microscopic observation of Toluidine Blue-stained sections of the
gland revealed that control untreated glands contained columnar cells filled
with secretory vesicles stained blue. When stimulated with dopamine (1 mmol
l–1), however, four different types of secretory granules can
be recognized within the glands on the basis of morphological appearance.
Fig. 2 shows a typical
micrograph depicting all the four types of secretory granules following
stimulation of a cyprid for 10 min with dopamine. The most abundant are the
dense, dark granules stained blue (type 1). The granules that appear light
blue represent type 2 vesicles. Granules that appear to be `moth eaten' are
type 3 granules. The fourth type of granules are partially or completely empty
and appear as vacuoles. Fig. 3
summarizes the differences between the size of the different granule types.
Overall, type 1 granules were the smallest, and dopamine stimulation seemed to
induce granule swelling followed by varying degrees of emptying of granule
contents, finally resulting in completely empty vesicles that appear as
vacuoles. Control, unstimulated glands contained mostly type 1 granules, and
only occasionally were type 2 and 3 vesicles found in some cells. This
observation supports our view that the loss of granule contents and vacuole
formation were not experimental artefacts caused by fixatives.
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). Fig. 5B
shows type 2 granules that appear swollen, filled with smooth, amorphous
contents that differ in appearance from the organized regular pattern seen in
dense-core type 1 granules. Type 3 granules appeared partially empty,
containing empty holes that gave a moth-eaten appearance
(Fig. 5C) reflecting their
appearance under the light microscope. Closer examination shows that the empty
holes contain disorganized proteinaceous material with a filamentous structure
interspersed within the smooth amorphous material similar to type 2 granules.
Finally, we classified the empty vacuole-like granules as post-exocytotic type
4 granules devoid of contents. Fig.
5D shows an example of a vacuole, and in this example it is seen
together with type 1 granules. Thus, different types of granules occasionally
occur within the same cell. Type 4 granules (vacuoles) are membrane-bound
areas where the cement proteins have been almost completely lost, and only
some remnants are retained. These structures appear similar to degranulation
sacs seen in histamine-secreting mast cells following maximal stimulation
(Cho et al., 2002b;
Crivellato et al., 2002a).
Overall, these morphological characteristics of secretory granules in the
cement gland of cyprids appear to be similar to secretory vesicles in other
secretory systems during exocytosis such as the entero-endocrine cells
(Crivellato et al., 2002b), the
different cells of the immune system
(Crivellato et al., 2003) and
the pancreatic acinar cells (Raraty et
al., 2000) (for a review, see
Jena, 2005).
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). We tested a number of biogenic amines using this
technique, namely serotonin, histamine, octopamine, tyramine and melatonin,
all at a concentration of 1 mmol l–1 (data not shown). Unlike
dopamine and noradrenaline, none of the other amines tested stimulated cement
secretion (vacuole formation). In control experiments, we tested spontaneous
vacuole formation in the absence of stimulation. Ten animals were incubated in
filtered seawater for 2 h and none of them showed any signs of vacuole
formation during that time. Thus, vacuoles do not form spontaneously in the
absence of stimulation in cement glands of cyprids kept in FSW. The loss of
fluorescence and vacuole formation is probably due to a decrease in
concentration of Acridine Orange within vesicles as they swell and a loss of
dye as secretion of vesicle contents proceeds.
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In another set of experiments, we investigated dopamine-induced cement secretion in intact live cyprids immobilized in low-melting-point agarose using DIC microscopy. Similar to our observation of loss of Acridine Orange fluorescence, we were able to monitor changes in interference contrast followed by vacuole formation within cement glands in all the animals that we examined (Fig. 7). We counted the appearance of the different types of granules at various times during dopamine (1 mmol l–1) stimulation. Fig. 8 shows the change in number of different types of granules during dopamine stimulation over time. Similar to the results from Toluidine Blue-stained sections, dense-core, type 1 granules reduced in number as stimulation proceeded, and the number of vacuoles increased. Thus, a number of different microscopy techniques – light and electron microscopy of fixed and stained sections, confocal microscopy of Acridine Orange-loaded cyprids and DIC microscopy of live cyprids – all show stimulation-dependent changes in granule morphology and granule emptying. While the loss of intragranular material is easily observed, the mechanism through which such loss occurs is not revealed by these experiments. The morphological data, however, suggest that sequential exocytosis might account for the loss of granule contents (see Figs 2, 9). The emergence of vacuoles is seen as early as 2 min after onset of stimulation, and significant loss of granules is observed after 10 min, when approximately half of the dense-core granules have been secreted. Throughout the duration of stimulation, there is no difference in the number of type 2 and type 3 granules. This observation might suggest that the granules undergo swelling upon stimulation and sequentially undergo exocytosis.
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), and ß-cells, based on histological criteria
and the morphological appearance of the cement granules. In our hands, within
B. improvisus cyprid cement glands, different types of granules could
be observed within the same cell (Figs
2,
4B). While the possibility
exists that different types of cells might exist within the cement gland, it
appears that different types of granules occur within the same cell. We
conclude that the difference in appearance of the granules is more likely
linked to the secretory process. |
Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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The observation that secretory granules undergo a series of morphological
changes upon stimulation of secretion is significant and may be somewhat
similar to two previous reports on barnacle cement secretion
(Okano et al., 1996;
Walker, 1971). Walker
described cement gland morphology in both free-swimming as well as settled
Balanus amphitrite cyprids. He discovered that the cement gland in
non-settled animals contains a large number of dense granules with a diameter
of approximately 3–5 µm, inside columnar cells, and called these
cells -cells. He also described another type of cell, ß-cells,
with granules that had a reticulated appearance. In addition, Walker observed
vacuoles in the apical part of -cells, where material had been
discharged within glands from settled cyprids. We found that in unstimulated
cyprids, the cement glands contain small, dense-core secretory granules, which
begin to swell within minutes of stimulation with dopamine. Swollen granules
gradually lose their contents, possibly due to secretion, to finally become
empty vacuole-like organelles. The appearance of vacuoles indeed occurred near
the median central groove of the cement glands, similar to the finding by
Walker. In addition, like Walker, we occasionally found granules with type 2
or type 3 appearance in control, unstimulated cyprid cement glands. In cement
glands from dopamine-stimulated cyprids, we always found the three different
types of granules as well as vacuoles within the same cells (Figs
2,
4B,
9B,C).
This finding might question the previous assertion that different types of
cells exist in cyprid cement glands containing different types of granules
possibly differing in their protein compositions
(Walker, 1971). The fact that
Walker found vacuoles and reticulated granules in unsettled cyprids might
indicate that the cyprid larvae, as well as the recently settled cyprids,
might have undergone partial secretion of cement, associated with larval
settlement. Vacuole formation suggests secretion has occurred, possibly in an
attempt at settlement by the cyprid (see later). Similar vacuole-like
structures have been seen in other secretory systems where secretory cells
contain dense-core secretory vesicles, e.g. pancreatic acinar cells
(Cho et al., 2002b;
Raraty et al., 2000),
gastrointestinal epithelia (Crivellato et
al., 2002b; Kuver et al.,
2000) and mast cells
(Crivellato et al., 2002a).
Similar observations of exocytosis and membrane retrieval in invertebrate
neurosecretion were made in the crab sinus gland over two decades ago
(May and Golding, 1983;
Morris and Nordmann,
1980).
The current dogma in the case of cyprid settling is that cement secretion
occurs in an all-or-none fashion. The observation of granules with partial
loss of contents in normal unstimulated cyprid cement glands might suggest
that some secretion can occur in the absence of settling in a form of
piecemeal degranulation (Aravanis et al.,
2003; Crivellato et al.,
2003). Secondly, it is possible that only some of the
cement-secreting cells participate in secretion during a settling attempt,
sparing other cells in order to provide the cyprid with the possibility of
multiple attempts to settle. Finally, the conclusion that the different types
of granules found in stimulated cement glands indeed represent granules that
have undergone varying degrees of loss of contents calls into question the
idea that vesicles with different intragranular composition might exist in
order to support cement curing. While it is entirely possible that several
different proteins make up the secreted cement, it is not clear if they are
derived from multiple types of secretory granules. Not enough is known of the
composition of the vesicle contents, nor the cement, to conclude if the
barnacle adhesive is a single- or multi-component glue. Dense-core secretory
vesicles in general are known to contain multiple protein and other components
that form a complex mixture that is held at extremely high concentrations
within the vesicles (Lagercrantz,
1976; Uvnäs et al.,
1970; Winkler,
1976). It is quite likely that all the necessary components that
make up the barnacle adhesive are stored within the vesicles such that, upon
secretion, interaction with seawater or solid surfaces with the right chemical
features results in glue hardening. More detailed information of the chemistry
of the cement or the granule contents is necessary in order to precisely
understand the adhesive curing mechanism.
We confirm previously published observations that dopamine and, to a lesser
extent, noradrenaline stimulate cement secretion from cyprid larvae
(Okano et al., 1996). The
major difference between the previously published work and the present study
is that Okano's experiments were carried out using isolated cement gland
preparations in vitro while our study is performed in intact cyprids
in vivo. Perhaps, for that reason, our experiments required a higher
concentration of dopamine to achieve stimulation. The higher concentration of
dopamine might induce a massive cement secretion and, consequently, more
granule swelling and vacuole formation
(Kelly et al., 2004). Another
difference could be that the extracellular medium used in Okano's studies did
not support granule swelling like the extracellular fluid in the living cyprid
kept in seawater. Granule swelling is known to be dependent on medium pH,
Ca2+ (Espinosa et al.,
2002) and ionic strength
(Finkelstein et al., 1986;
Nanavati and Fernandez, 1993),
which may differ in the two experimental conditions. In zymogen granules of
the pancreas, the mechanism of granule swelling during exocytosis was found to
be regulated by a GTP-mediated process involving Gi3 and
aquaporin (Cho et al.,
2002a).
As in most secretory systems, the cement proteins are stored within the
vesicles at extremely high concentrations in a colloidal complex with numerous
components such that the osmotic pressure inside vesicles is low
(Kreuger et al., 1989). The
hydration forces on the granule will be expected to be controlled by the
colloid osmotic pressure within the vesicles and would be modulated by changes
in the osmolality of the surrounding medium
(Whitaker and Zimmerberg,
1987). The intragranular complex needs to dissociate during
exocytosis for secretion to occur. In some granules, a phase boundary between
compact electron-dense material and less-compact amorphous material within
vesicles has been observed, together with loss of material (see
Walker, 1971 for comparison).
Granule swelling following hydration of the granule contents might therefore
be essential for secretion to occur. The structure within the type 3 granules,
appearing as hydration channels, might be due to such changes, and similar
structures have been observed in other dense-core granules such as the sea
urchin egg cortical granules and mast cell granules
(Dvorak and Morgan, 2000;
Whalley et al., 2000).
Exocytosis is an ubiquitous event in biology and several hypotheses have
been suggested as possible molecular mechanisms. It seems likely that the
detailed molecular mechanisms of exocytosis might differ in different
secretory systems depending upon the physiologically required speed of the
exocytotic secretory event. It seems possible that in some cellular systems a
single granule might undergo multiple fusion–secretion cycles (for
reviews, see Burgoyne and Morgan,
2003; Lindau and Alvarez de
Toledo, 2003), leading to partial secretion of vesicle contents or
a pulsatile form of secretion. Similar `kiss-and-run' type partial exocytosis
has been observed in synaptic transmitter release
(Aravanis et al., 2003). During
exocytosis, the granule membrane transiently becomes part of the cell membrane
and then is selectively retrieved, and it is possible that rapid cycling
between a fusion state and a non-fusion state may occur
(Burgoyne and Morgan, 2003;
Schneider, 2001). The duration
and diameter of the fusion pore opening will regulate how much granular
material is secreted for each secretion cycle
(Tsuboi and Rutter, 2003;
Tabares et al., 2003). Thus,
it is possible that the empty vacuoles, like the type 4 granules we observe,
could result from multiple rounds of partial exocytosis, or complete
exocytosis and total emptying.
In conclusion, the data we present here support the suggestion that the dense-core secretory vesicles within cement glands in cyprid larvae are secreted through a process of exocytosis. Exocytotic secretion of barnacle cyprid cement resembles the secretory event observed in many mammalian cell systems, including pulsatile or partial secretion of granule contents. In addition, we observed four different types of cement granules in dopamine-stimulated cyprids. With the exception of type 1 granules, all others appear swollen with partial or complete loss of granule contents and might represent vesicles that have undergone partial or complete loss of contents. Thus, the cement gland in barnacles appears to be a precisely regulated exocrine organ that is more complex in its organization and regulation than previously thought.
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Acknowledgments |
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