Expression and polarity reversal of V-type H+-ATPase during the mineralizationdemineralization cycle in Porcellio scaber sternal epithelial cells
1 Central Facility for Electron Microscopy, University of Ulm,
Albert-Einstein-Allee 11, 89069 Ulm, Germany
2 University of Osnabrück, Department of Animal Physiology,
Barbarastraße 11, 49076 Osnabrück, Germany
3 Mount Desert Island Biological Laboratory, Salsbury Cove ME 04672,
USA
* Author for correspondence (e-mail: andreas.ziegler{at}medizin.uni-ulm.de)
Accepted 18 February 2004
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Summary |
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Key words: biomineralization, calcium carbonate, Crustacea, epithelial H+ transport, epithelium, Isopoda, Porcellio scaber
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Introduction |
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Another interesting aspect of the P. scaber model is the amorphous
character of the sternal deposits (Becker
et al., 2002; Ziegler,
1994
). Because the solubility of amorphous CaCO3 (ACC)
is ten times higher than of its crystalline form
(Brecevic and Nielson, 1989
),
it is ideally suitable as a transient store for Ca2+. Since the
solubility product of ACC depends on the pH, the regulation of the
H+ concentration within the limited space around the deposits by
epithelial H+ transport is of particular significance for avoiding
CaCO3 crystallisation.
Epithelial proton transport may be mediated by several molecular
mechanisms. In crustaceans these include a V-type H+-ATPase (VHA;
Onken and Putzenlechner, 1995;
Weihrauch et al., 2001
,
2002
), a
Na+/H+-exchanger
(Towle et al., 1997
) or
2Na+/H+-exchanger, which may also transport one
Ca2+ in exchange for one H+
(Ahearn et al., 2001
), and a
Cl/
exchanger
(Ahearn et al., 1987
). The
exact mechanisms for H+-transport during mineralization processes
in Crustaceans, however, are unknown. A recent study has shown an upregulation
of the V-type H+-ATPase activity in crustacean gill epithelia from
premolt to the postmolt stage (Zare and
Greenaway, 1998
), raising the possibility that in the hypodermal
epithelium of crustaceans this mechanism contributes to the mineralization and
demineralization processes as well.
In an attempt to test a contribution of the VHA to mineral deposition and resorption we analysed the expression of the VHA in the ASE and PSE of P. scaber by the reverse-transcriptase polymerase chain-reaction (RT-PCR) technique during three different molting stages and used immunocytochemical and ultrastructural techniques to localize the VHA within the epithelial cells.
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Materials and methods |
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Molecular cloning of the V-type H+-ATPase (VHA) cDNA fragments
We extracted total RNA from sternal tissue of about ten P. scaber
under RNAse-free conditions using chemicals obtained from Promega Corporation
(Madison, WI, USA). Reverse transcription of mRNA was done using oligo (dT)
primers and Superscript II (Gibco-BRL, Gaitherberg, MD, USA) reverse
transcriptase. We used the degenerate sense primer HATF2: GCN ATG GGN GTN AAY
ATG GA and the degenerate antisense primer HATR4: TGN GTD ATR TCR TCG TTN GG
(D: A/G/T; N: A/C/G/T; R: A/G; Y: C/T) published previously
(Weihrauch et al., 2001) to
amplify the putative VHA B-subunit protein fragment of P. scaber. PCR
products were separated electrophoretically on 1% agarose gels, extracted from
gel slices (Qiagen Qiaquick, Valencia, CA, USA) and sequenced automatically by
the dideoxynucleotide method (Sanger et
al., 1977
) at the Marine DNA Sequencing Center of Mount Desert
Island Biological Laboratory employing the degenerate PCR primers in the
sequencing reactions. A search of GenBank using the BLAST algorithm
(Altschul et al., 1997
)
revealed close matches with previously published sequences for the VHA. For
multiple alignments we used Gene Doc
(http://www.psc.edu/biomed/genedoc/)
and ClustalW
(http://antheprot-pbil.ibcp.fr/)
software.
Analysis of relative VHE expression
Two independent sets of ASE and PSE were carefully dissected and stored in
RNAlater (Ambion, Austin, TX, USA). For each of the three molting
stages within each set we pooled epithelia from 10 animals. After extraction
of total RNA we determined the RNA concentration photometrically at 260 nm
(Hitachi U2000 ultraviolet-visible spectrophotometer, Tokyo, Japan). Within
each set, equal amounts of total RNA were used in either reaction.
Semi-quantitative RT-PCR was accomplished by incorporating biotinylated dUTP
in the PCR reaction mixture. Amplification proceeded for 27 cycles of 92°C
(1 min), 45°C (1 min), and 72°C (1 min) using the degenerated primer
pair HATF2 and HATR4 (PCR product size 392 bp). In the logarithmic phase of
amplification the biotinylated products were separated on 1% agarose gels,
transferred to nylon membranes and visualized with the PhotoTope detection
system (New England Biolabs, Beverly, MA, USA). Under the employed conditions
the signal intensity of the PCR product was dependent on the amount of cDNA
template (Fig. 3A).
|
Antibody and western blot analysis of the VHA B-subunit
Sternal epithelia of 24 animals with well-developed sternal
CaCO3 deposits were dissected, frozen in liquid nitrogen and stored
at 30°C. Lysis buffer (200 µl of 10 mmol l1
Tris containing 0.01% SDS, pH 7.4 and protease inhibitor cocktail, Sigma) was
added to the samples, which were then heated to 100°C, homogenized for 3
min using a preheated homogenizer, heated and homogenized for a second round
and centrifuged at 16 000 g for 5 min. The supernatant was
diluted 1:1 in 2x SDS loading buffer
(Laemmli, 1970). SDS page was
done using 10%20% gradient gels (Novex, Invitrogen, Karlsruhe,
Germany). Proteins were electrotransfered overnight onto PVDF-membranes in 10
mmol l1 NaB4O7 at 100 mA. The
membranes were washed in Tris-buffered saline containing Tween 20 (TBST: 25
mmol l1 Tris-HCl, pH 7.5, 150 mmol l1
NaCl, 0,005% thimerosal, 0.1% Tween 20) for 10 min, blocked in 3% skim-milk in
TBST for 1 h, incubated in primary antibody (mouse monoclonal anti yeast VHA
B-subunit; Molecular Probes, Leiden, The Netherlands) diluted in 1% skim-milk
in TBST at 1:200 for a further 1 h, washed 3x in TBST for 10 min,
reacted for 1 h with secondary antibody [horseradish peroxidase (HRP)-coupled
anti-mouse IgG; Amersham, Freiburg, Germany] at a dilution of 1:500 in 1%
skim-milk in TBST and washed 3x in TBST. Bound antibodies were
visualized using a chemiluminescence detection system (ECL, Amersham, Little
Chalfont, UK), followed by exposure to Kodak BioMax MS-film.
Immunofluorescence-labelling on cryosections
For the immunofluorescence experiments we fixed the ASE with 4%
paraformaldehyde in 0.1 mol l1 cacodylate buffer (pH 7.3)
for 1 h. Subsequently, specimens were immersed in 2.3 mol l1
sucrose in 0.1 mol l1 sodium cacodylate buffer for 2 h,
mounted on aluminium rods and frozen in liquid nitrogen. Sections (0.7 µm
thick) were cut with a Leica Ultracut S microtome (Vienna, Austria) equipped
with a FCS cryochamber, glass knives and an antistatic device (Diatome) at
70°C. Sections were transferred to polylysine-coated glass slides
(Polyprep, Sigma, Taufkirchen, Germany) with a droplet of 2.3 mol
l1 sucrose in 0.1 mol l1 sodium cacodylate
buffer. Labelling was done as described previously
(Weihrauch et al., 2001).
Sections were successively incubated with 0.05 mol l1
glycine in phosphate-buffered saline (PBS) for 15 min, 1% SDS in PBS for 5 min
and washed with PBS 3x for 5 min. To block endogenous biotin the
sections were incubated with 0.001% streptavidin in PBS for 15 min and washed
with PBS for 5 min. Subsequently biotin-binding sites of the streptavidin were
blocked by incubation with 0.1% biotin in PBS (15 min). After washing the
sections with PBS twice for 5 min, thesections were blocked with blocking
solution [3% dry milk and 0.1% cold-water fish gelatine (Biotrend, Cologne,
Germany) in PBS] for 15 min. Sections were incubated overnight at 4°C with
primary antibody (anti-yeast V-type H+-ATPase B-subunit) at 2.5
µg ml1 in PBS containing 1% skim-milk. Controls were
incubated in the same buffer without the primary antibody. Then the sections
were washed 3x with high salt PBS (500 mmol l1 NaCl in
PBS) and once with PBS for 5 min each. Subsequently the secondary antiserum,
anti-mouse biotinylated IgG (Amersham) diluted 1:100 in 1% milk in PBS was
added onto the sections for 1 h. After washing 3x for 5 min with high
salt PBS and 5 min with PBS, the sections were treated with blocking solution
(TNB: 0.1 mol l1 Tris-HCL, pH 7.5, 150 mmol
l1 NaCl, 0.5% blocking reagent; NEN, Boston, MA, USA) for 30
min and incubated with HRP-linked streptavidin (NEN) at a dilution of 1:100 in
TNB for 1 h. Sections were washed 3x with high salt PBS and once with
PBS for 5 min each. The sections were then incubated with the fluorescent HRP
substrate Tyramid-Cy3 (NEN) at a dilution of 1:50 in amplification buffer for
8 min in the dark. After washing 3x for 5 min with high salt PBS and 5
min with PBS the sections were mounted in 80% glycine, 20% PBS plus 2%
N-propyl-gallate (to retard fading) and examined with a Zeiss
Axiophot microscope (Jena, Germany). Micrographs were taken using a Spot CCD
camera (Visitron, Puchheim, Germany). The distribution of the basolateral
plasma membrane was labeled using the mouse monoclonal anti
Na+/K-ATPase antibody (
5), and FITC-conjugated anti-mouse
IgG (Sigma) following the procedure described previously
(Ziegler, 1997a
).
Electron microscopy
Anterior and posterior sternites from animals in the intermolt, late
premolt and intramolt stage were high-pressure frozen at
2.3x108 Pa (Leica, EMHPF). Specimens were freeze-substituted
in acetone containing 1% OsO4 and 1% H2O
(Walther and Ziegler, 2002)
using a self-built computer-controlled device, following an exponential 24 h
warming protocol starting from 90 to 0°C. After incubation for a
further 1 h at 0°C in the same solution specimens were washed three times
in acetone at room temperature and embedded in Epon resin. For overviews a few
samples were chemically fixed as described previously
(Ziegler, 1996
). Thin sections
(60 nm) were cut with a Leica Ultracut UCT microtome using a diamond knife
(Diatome), stained with 2% uranyl acetate in H2O and 0.3% lead
citrate, and viewed with a Philips 400 electron microscope at 80 kV.
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Results |
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To test if the expression of a VHA is correlated with epithelial Ca2+-transport we analysed the relative expression in sternal epithelia with high (ASE) and moderate (PSE) transport rates in the non-transporting control stage, and the Ca2+-transporting stages during CaCO3 deposit formation and degradation. In two independent experiments the semiquantitative RT-PCR indicates an increase in VHA expression in both epithelia from the control stage to the Ca2+-transporting stages. In the latter the signal is somewhat larger in the ASE than in the PSE (Fig. 3). The high expression during the formation and resorption of the CaCO3 deposits suggest VHA mediated proton transport in both directions.
Immunocytochemical localization of the VHA
In order to investigate if VHA expression is correlated with a change in
the subcellular distribution of the VHE we used a monoclonal antibody to study
stage dependent localization of the VHA in the ASE. The antibody against the
cytoplasmic B-subunit of the VHA of yeast was successfully used previously to
detect the VHA in crustacean gill epithelium
(Weihrauch et al., 2001). In
western blots of solubilised sternal tissue of P. scaber the antibody
binds to a single band at an apparent molecular mass of 54 kD
(Fig. 4), which is within the
range reported for the B-subunit in various eukaryotic cells (for a review,
see Finbow and Harrison,
1997
), indicating specific binding of the antibody. On
cryosections of the ASE location and intensity of the immunoreaction depends
on the molting stage. Immunofluorescence was generally weak in the early
premolt control stage (Fig.
5A,B). Strong immunofluorescence occurred during the stages of
CaCO3 deposit formation and resorption
(Fig. 5C,D,G,H). Interestingly,
the location of the immunofluorescence changed between the two
Ca2+-transporting stages. During CaCO3 deposit formation
the antibody binds to some extent to the cuticle of the epithelial cells and
to lateral and basal areas (Fig.
5C,D) indicating binding to the basolateral plasma membrane, which
forms a system of ramifying invaginations. The nuclei, the cytoplasm around
the nuclei, and the cytoplasm between the nuclei and the cuticle were
virtually devoid of any signal. The extension of the system of ramifying
invaginations is shown by immunocytochemical localization of the
Na+/K+-ATPase (Fig.
5E,F) and transmission electron microscopy
(Fig. 6A). During
CaCO3 resorption we observed a strong signal near and within the
cuticle. Immunoreaction in the cytoplasm, including basolateral areas and the
nuclei, was below the detection limit (Fig.
5G,H). To increase spatial resolution of the strong signal within
the cuticle a few sections were stained without an amplification protocol
using a Cy3-conjugated donkey anti-mouse IgG as a secondary antibody. These
experiments revealed a dot-like distribution of the immunoreaction within the
cuticle (Fig. 5I).
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Ultrastructural detection of portasomes
Since unspecific binding of the primary antibody to the cuticle cannot be
excluded by western blot analysis we confirmed the presence of a VHA within
the apical plasma membrane during the degradation of the CaCO3
deposits using ultrastructural techniques. It is now generally accepted that
the large cytoplasmic V1 domain of the VHA can be visualized by
transmission electron microscopy as 10 nm thick particles, the so-called
portasomes (Wieczorek et al.,
1999b). When the VHA occurs in high abundance portasomes can be
recognized as membrane coats at the cytoplasmic side of the plasma membrane.
We examined the ultrastructure of the ASE and PSE in high-pressure frozen and
freeze-substituted sternal epithelial cells. Regions of the plasma membrane
containing portasome coats were found in the apical membrane of the ASE
(Fig. 6BD) but not the
PSE during the intramolt stage (three animals). No portasome coats were found
at the apical or basolateral plasma membrane of the ASE and PSE during the
control stage (three animals) or during CaCO3 deposit formation
(three animals).
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Discussion |
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Expression of a V-type H+-ATPase
The VHA is a large protein complex composed of two distinct domains: a
cytoplasmic V1 domain consisting of at least eight different
subunits (AH) and the integral membrane V0 domain consisting
of at least five subunits (ad) (for a review, see
Wieczorek et al., 2000). The B
subunit is one of the two ATP binding subunits of the V1 domain.
Sequence alignment of the cDNA-fragment with known sequences and
immunocytochemical experiments indicate the expression of a VHA B-subunit and
hence the expression of the VHA within the sternal epithelium of Porcellio
scaber. The high identity of the deduced amino acid sequence with the
corresponding crustacean and insect protein fragments is in accordance with
the high evolutionary conservation of the VHA B-subunit
(Novak et al., 1992
). Since
cuticle secretion is retarded in the anterior integument during the late
premolt and intramolt stage, the ASE is specialized for the formation and
degradation of the sternal CaCO3 deposits
(Ziegler, 1997b
). Therefore,
the increase in VHA expression within the ASE from the early premolt control
stage to the calcium transporting stages during CaCO3 deposit
formation and CaCO3 deposit resorption suggests a role of the VHA
in the mineralization and demineralization processes, respectively. The PSE
cells are involved in degradation of the old and formation of the new cuticle
of the posterior sternites including demineralization and mineralization
(Glötzner and Ziegler,
2000
; Ziegler,
2002
), but do not form CaCO3 deposits. This implies
that the rate of mineral transport by the PSE is smaller than in the ASE.
Hence, the somewhat smaller expression of the VHA B-subunit within the PSE is
in accordance with a role of the VHA in mineral deposition and resorption.
Subcellular localization of the V-type H+-ATPase
The immunolocalization at the basolateral side of the epithelial cells in
the late premolt stage corresponds well with the spatial distribution of the
basolateral plasma membrane, which increases by an elaborate system of
invaginations (Figs 5E,F and
6A), as shown previously using
lanthanum as an extracellular marker and immunocytochemical localization of
the Na+/K+-ATPase in the basolateral membrane (Ziegler,
1996,
1997a
). Areas within the
cytoplasm that do not include invaginations are virtually devoid of VHA. The
strong increase in immunofluorescence near and within the cuticle of the
epithelium from late premolt to intramolt indicates that the signal is mostly
if not entirely due to a specific reaction of the antibody to the VHA
B-subunit. The ASE has numerous cellular projections of the epithelial cells,
which extend into the new cuticle of the sternites
(Ziegler, 1997b
). The dot-like
signal in specimens without an amplification of the immunoreaction suggests
that the VHA is located within these projections. This is supported by coats
of portasomes lining the apical plasma membranes of the ASE cells in the stage
of CaCO3 deposit resorption. The apparent lack of portasomes within
the basolateral membrane, despite their immunolocalization during
CaCO3 deposit formation, may be explained by the failure of
portasomes to form particle coats due to a lower portasome density. This may
result from the larger surface of the basolateral plasma membrane as compared
to the apical plasma membrane
(Glötzner and Ziegler,
2000
) and by a lower H+ transport rate during the
rather slow CaCO3 deposit formation within 1 or 2 weeks as compared
to their quick resorption within less than 24 h.
The increase in the VHA abundance from the control stage to the
Ca2+-transporting stages indicates a contribution of the VHA to the
formation and degradation of CaCO3 within the anterior sternites,
in support of the results of the expression analysis. The results provide the
first example of a function for the VHA in mineral deposition. The formation
of the sternal deposits requires the transport of Ca2+ and
from the basal to the apical side
of the epithelium and the release of protons during the formation of
CaCO3. These protons have to be transported back to the hemolymph
to maintain electro-neutrality during the transport process and to avoid
acidification of the liquid around the growing deposits. The high VHA
expression and abundance within the basolateral membrane suggests a
transcellular route for epithelial H+-transport, with the VHA
functioning in the extrusion of protons from the cytoplasm to the hemolymph.
In most epithelial cells containing a VHA within the plasma membrane the
protein complex is located apically
(Wieczorek et al., 1999a
). The
presence of the VHA in the basolateral plasma membrane, however, is rare. To
our knowledge this has been found only in vertebrate ocular ciliary epithelium
(Wax et al., 1997
) and in
subpopulations of kidney intercalated B-cells. In the latter the VHA is
localized either in the basolateral or apical plasma membranes depending on
its presence in proton or bicarbonate secreting cells, respectively
(Brown and Breton, 2000
;
Brown et al., 1988
).
The most interesting result of the present study is the polarity reversal
of VHA abundance in the ASE from the basolateral to the apical plasma
membrane, correlating with the mineralizationdemineralization cycle of
the sternal CaCO3 deposits. This reversal leads to a reduction of
the VHA within the basolateral plasma membrane to virtually undetectable
values, and to an increase of VHA abundance within the apical membrane. This
polarity reversal correlates with the switch of the ASE cells from
CaCO3 secretion to CaCO3 resorption. During the
resorption of the CaCO3 deposits protons must be transported from
the hemolymph into the sternal exuvial gap to mobilize Ca2+ and
ions, which are then transported
back to the hemolymph. A similar contribution to mineral resorption is known
for a VHA in the apical plasma membrane of osteoclasts during bone resorption
(Blair et al., 1989
) and of the
mantle epithelium of a freshwater clam during acid secretion to the shell
(Hudson, 1993
). The
redirection of the VHA from the basolateral to the apical plasma membrane
compartment within the same cell raises the question of the mechanism by which
the sorting of the VHA switches between the opposite plasma membrane
compartments. Expression analysis and the virtual lack of VHA within the
cytoplasm favours a regulation by gene expression and retargeting to the
appropriate plasma membrane compartment. The 2.8-fold increase of the apical
plasma membrane surface area
(Glötzner and Ziegler,
2000
) from the stage of CaCO3 deposit formation to
CaCO3 resorption is in accordance with a sorting of VHA containing
intracellular vesicles to the target membrane. Such vesicles were reported
from A-type intercalated cells of kidney collecting ducts, in which they
regulate proton pumping by shuttling VHA molecules to and from the plasma
membrane (for a review, see Brown and
Breton, 1996
).
Besides its function in proton transport a VHA functions in the
energization of the plasma membrane by proton motive forces that mediate
secondary transport processes by electric coupling (for a review, see
Harvey and Wieczorek, 1997).
This is the case in epithelia with little activity of the classical energizer
of animal plasma membranes, the Na+/K+-ATPase, as in
lepidopteran midgut (Jungreis and Vaughan,
1977
; Wieczorek et al.,
1999a
) or malpighian tubules
(Weng et al., 2003
). In the
ASE of P. scaber immunocytochemical localization of the
Na+/K+-ATPase indicates a high abundance of the pump
within the basolateral membrane (Ziegler,
1997a
), arguing against an energization by the VHA. However, as
pointed out by Wieczorek et al.
(1999a
), some epithelia
energize the basolateral plasma membrane by the
Na+/K+-ATPase and the apical membrane by a VHA.
Therefore, besides a role of the VHA in H+ extrusion, additional
functions, possibly in facilitating Ca2+ influx or acidbase
regulation, cannot be excluded.
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Acknowledgments |
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