Expression and localization of carbonic anhydrase and ATPases in the symbiotic tubeworm Riftia pachyptila
Equipe Ecophysiologie, CNRS-UPMC UMR 7127 CEOBM, Station Biologique, BP 74, F-29682 Roscoff Cedex, France
* Author for correspondence (e-mail: decian{at}sb-roscoff.fr)
Accepted 14 October 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Vestimentifera, carbonic anhydrase, V-H+ATPase, Na+K+-ATPase, immunolocalization, in situ hybridization
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The work described herein focuses on CA and proteins involved in ion
exchange processes in the giant hydrothermal vent tubeworm Riftia
pachyptila Jones. Adults lack both mouth and gut
(Jones, 1981) but, however,
exhibit the highest growth rates among marine animals
(Lutz et al., 1994
). They
actually derive all their nutritional needs from the large population of
sulfur-oxidizing chemolithoautotrophic bacterial symbionts that live in cells
within a specialized organ located in their trunk, the trophosome
(Cavanaugh et al., 1981
;
Felbeck, 1981
). The trophosome
is a soft tissue, composed of highly vascularized lobules, and surrounded by
coelomic fluid (Jones, 1988
).
Because the bacterial symbionts are remote from the external milieu, the worm
must take up sulfide, oxygen, nitrate and carbon dioxide from the environment
and transport these inorganic molecules to the symbionts. All these substances
are taken up from the surrounding water through the large and highly
vascularized plume (Fig. 1)
(Jones, 1988
). They are
transported by circulating fluids to provide energy, carbon and nitrogen
source to the bacterial metabolic cycles, and ultimately transferred to the
worm as organic molecules. As far as carbon dioxide is concerned, a number of
studies have shown that molecular CO2 enters by diffusion
(Goffredi et al., 1997
), and
accumulates at very high concentrations in the body fluids (up to 30
mmoll-1) (Childress et al.,
1993
). Transport of CO2 in body fluids is mainly as
bicarbonate, a limited amount being bound to the extracellular hemoglobins
(Toulmond et al., 1994
).
However, the bacteria per se can only utilize molecular
CO2 (Scott et al.,
1999
). In addition, extracellular pH in the body fluids of
Riftia remains very stable at approximately 7.3, despite these high
amounts of carbon dioxide (Goffredi et
al., 1997
). As suggested by all these authors, carbon dioxide
conversion is needed at various steps of the transport processes and probably
involves high levels of carbonic anhydrase activity.
|
Previous studies biochemically characterized a functional cytoplasmic CA in
the plume and in the trophosome of Riftia
(Kochevar et al., 1993), and
other studies have also quantified V-H+ATPase and
Na+K+-ATPase activities in several tissues of R.
pachyptila (Goffredi and Childress,
2001
; Goffredi et al.,
1999a
). According to Goffredi and Childress
(2001
), Riftia
regulates acidbase balance mainly via high concentrations of
ATPases, 7-55% higher than other deep-sea (Tevnia jerichonana, Calyptogena
magnifica and Coryphaeniodes acrolepis) and shallow-living
(Arenicola marina, Urechis caupo, Chaetopterus veriodepatus and
Themiste pyroides) animals. The authors also emphasized the
importance of the Na+/H+ exchanger and
Na+K+-ATPase in proton elimination and extracellular pH
regulation in the face of the numerous processes acting to acidify the
internal environment in the tubeworm. Moreover, a recent physiological
investigation on isolated bacteriocyte suspensions from the trophosome tissue
of Riftia revealed the complex interaction of carbonic anhydrase with
two important ion-transporting enzymes, the vacuolar-type
V-H+ATPase and the Na+K+-ATPase. Both enzymes
seem to be involved in the transepithelial transport processes for
electrolytes and carbon dioxide (De Cian et al., 2002). These results suggest
that in the trophosome, the proton motive force of the H+ATPase
drives transport processes involved in CO2/HCO3- (and
possibly HS- transport) across the cell membrane by generating
local acidification of the outer layer of the membrane. In this symbiotic
tissue the relative importance of H+ATPase minimizes the role of
Na+K+-ATPase in ion equilibrium. However,
Na+K+-ATPase is likely to play an important role in the
plume.
These physiological results prompted us to investigate the respective localization of the various proteins involved, in order to better understand carbon dioxide transport, conversion and incorporation processes that are specific to this symbiosis. The present study describes the expression pattern of cytosolic CA using an RNA probe and its histochemical and immunocytochemical localization in the trophosome and other non-symbiotic tissues of R. pachyptila. Immunolocalization of the two ion transporter enzymes V-H+ATPase and Na+K+-ATPase were also investigated and related to CA localization.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein extraction
Plume, vestimentum, body wall and trophosome tissues were dissected onboard
immediately after recovery of the animals, washed several times with
Riftia saline (Fisher et al.,
1988) and frozen separately into liquid nitrogen until used.
Membrane and crude homogenates were prepared from 1 g of frozen tissue of each
body part. The tissues were homogenized on ice using a Tissuemizer
(Ultra-Turrax, Janke-Kuntel, Ika Labortechnik, Staufen) by three 30 s bursts
in 4 ml extraction buffer. The extraction buffer contained 50
mmoll-1 Tris-HCl, pH 7.5, 200 mmoll-1 sucrose, 150
mmoll-1 KCl containing protease inhibitors including 2
mmoll-1 EDTA, 1 mmoll-1 Pefablock (Boehringer Mannheim),
5 µg ml-1 chymostatin, 10 µg ml-1 leupeptin and 10
µg ml-1 aprotinin. Samples were kept on ice during the whole
procedure and centrifuged at 10 000 g for 20 min at 4°C.
The supernatant (S1) was transferred in a new tube and stored at 4°C. The
pellet was washed twice and then solubilized for 1 h in 2 ml of membrane
buffer containing 1% Triton X-100 (3 vol.), Tris buffer used above (7 vol.)
and 0.2% saponin (1 vol.) (Brion et al.,
1997
). Proteins were solubilized by breaking up the membrane
pellet with a pipette tip, vortexing and sonicating. S1 and the solubilized
pellet (P1) were then ultracentrifuged at 100 000 g for 1.5 h
at 4°C resulting in supernatants S2 (from S1, soluble proteins) and S3
(from P1, membrane-associated proteins). The pellet P3 was solubilized one
more time in 500 µl of the Triton X-100 buffer in the same conditions as
above and ultracentrifuged at 100 000 g for 1.5 h at 4°C,
but the resulting supernatant did not contain significant amount of proteins.
All samples were stored in homogenization buffer with 5x Laemmli sample
buffer at -40°C (Laemmli,
1970
).
Western blots
Protein concentrations were determined by the method of Bradford
(1976). 7µg of protein from
each fraction were loaded on reducing SDS-PAGE gels for silver nitrate
staining and 30 µg were loaded in triplicates for western blotting
(Brion et al., 1997
). Proteins
were transferred to a nitrocellulose (Bio-Rad) membrane using a wet
(Trisglycine20% methanol) transfer unit for 2 h at 4°C and
80 V. All blocking and antiserum incubations were done in TBST (20
mmoll-1 Tris-HCl, pH 7.5, 0.5 moll-1 NaCl, 0.05% Tween
20). After blocking for 2 h in 5% skimmed milk in TBST at room temperature,
the membrane was split up into three identical parts. Each one was incubated
overnight at 4°C with a different primary antibody: (i) rabbit anti-chick
30 kDa CA-II (a generous gift from Dr P. Linser, Whitney Laboratory,
University of Florida, St Augustine, USA) diluted 1:1000, (ii) mouse
monoclonal antibody raised against the 60 kDa ß-subunit of yeast
V-H+ATPase (Molecular Probes) diluted 1:500, and (iii) `
5'
ascites diluted 1:1000. The primary antibody `
5', developed by Douglas
M. Fambrough, was obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the National Institute of Child Health and
Development and maintained by The University of Iowa, Department of Biological
Sciences, Iowa City, IA 52242, USA. This antibody was raised against chicken
Na+K+-ATPase
-subunit, a 1020-amino-acid membrane
protein, with a large spectrum of cross-reactions recorded from mammals to
insect and frog (Lebovitz et al.,
1989
). As positive standard for CA we used bovine CA, present in
the molecular mass marker mix (LMW electrophoresis calibration kit, Amersham
Pharmacia Biotech., France). Pre-immune rabbit serum (Sigma) and omission of
the primary antibody were used as negative controls. The membrane was then
rinsed 3 times in TBST and incubated for 2 h at room temperature with a 1:3000
dilution of pig anti-rabbit horseradish peroxidase-conjugated IgG antibody
(DAKO A/S, Denmark) or sheep anti-mouse HRP-conjugated IgG antibody (Amersham
Pharmacia Biotech.). The antigenantibody complex was revealed by the
chemiluminescence system (ECL, Amersham Pharmacia Biotech., France) according
to the manufacturer's instructions, and by exposing autoradiographic film
X-OMAT AR (Eastman Kodak Co. NY, USA) to the nitrocellulose membrane.
Preparation of the probes for in situ hybridization
The plasmid carrying Riftia CA cDNA (EMBL accession number
AJ439711; 356-378 bp fragment) was linearised appropriately for either T7 or
T3 polymerase-directed RNA synthesis. Synthesized RNA was labeled by
incorporating digoxigenin (DIG)-conjugated UTP, as recommended by the
manufacturer (Boehringer-Mannheim). The following solutions were added to 1
µg of linearized template: 2 µl of a 10 mmol l-1 nucleotide
mix (including 3.5 mmol l-1 labeled UTP and 6.5 mmol l-1
unlabeled UTP), 2 µl of 10x transcription reaction buffer, 1 µl of
RNase Inhibitor, 2 µl of the specific polymerase and DEPC (diethyl
pyrocarbonate) water to 20 µl final volume. After 2 h at 37°C, 2 µl
of DNase I RNase-free was added to digest the cDNA template and 2 µl of 0.5
mol l-1 EDTA was added to end the transcription reaction. CA sense
and antisense mRNAs were purified onto Quickspin columns (Boehringer-Mannheim)
and stored in the elution buffer (300-500 ng samples in 5 µl) at
-80°C.
In situ hybridization
Small entire juveniles or tissue pieces 5 mm thick were fixed in 0.1 mol
l-1 phosphate-buffered 4% paraformaldehyde (pH 7.4) for 4 h at
4°C. After rinsing in 100 mmol l-1 phosphate buffer (PB) three
times, the specimens were dehydrated in an ethanol series and embedded in
paraffin wax. Sections (5-7 µm) were collected on Biobond (BBInternational,
Cardiff, UK) coated glass slides and air-dried overnight at 40°C. The
sections were dewaxed in toluene, rehydrated in an ethanol series and washed
in PBS. They were then post-fixed in 4% paraformaldehyde in PBS for 15 min at
room temperature, deproteinized for 10 min with 0.2 mol l-1 HCl,
treated for 10 min in 2x SSC at 70°C, and rinsed in PBS, 0.1 mol
l-1 triethanolamine acid, pH 8, containing 0.5% acetic anhydride
under agitation at room temperature. Slides were then incubated with 50 µg
ml-1 proteinase K for 15 min at 37°C in a wet chamber, rinsed
in PBS, and eventually dehydrated in an ethanol series, air-dried, stored at
-80°C and used within 2 weeks after pre-treatment. All these steps were
carried under RNase-free conditions with RNase-free products, all provided by
Sigma-Aldrich.
Hybridization specificity and optimal conditions of the RNA probes were tested on dot blots (data not shown) using total RNA of R. pachyptila tissues as a matrix. Sections were pre-hybridized at 45°C for 1 h in hybridization buffer (50% deionized formamide, 0.9 mol l-1 NaCl, 20 mmol l-1 Tris-HCl, pH 7.5, 0.01% SDS, 2% blocking reagent (Boehringer-Mannheim), and hybridized with DIG-labeled CA riboprobes (100 ng/slide) overnight at 45°C in a wet chamber with a coverslip. Experimental tissue sections were hybridized with the anti-sense CA riboprobe and control sections with the sense riboprobe. After stringent washing (20 mmol l-1 Tis-HCl, pH 7.5, 28 mmol l-1 NaCl, 0.01% SDS, 5 mmol l-1 EDTA) at 47°C for 20 min, immunological detection was performed with DIG Nucleic Acid Detection Kit [sheep anti-DIG-alkaline phosphatase (Fab fragments), and NBT/BCIP as a substrate; Boehringer-Mannheim] according to the manufacturer's instructions.
Immunocytochemistry
Tissue pieces or juveniles (2-10 cm long) were fixed in cold methanol at
-20°C and then cryo-embedded in tissue-teck or embedded in paraffin wax as
described above. Cryosections (7 µm) were cut on a Microm cryotome at
-25°C, collected on glass slides coated with 1% gelatin, air-dried at room
temperature and then stored at -40°C. When used, the sections were first
postfixed in methanol containing 0.3% H2O2 to inhibit
endogenous peroxidase activity, and rinsed in PBS (10 mmol l-1
sodium phosphate buffer, pH 7.4, 150 mmol l-1 NaCl). Sections were
then incubated in blocking solution [10% normal goat serum (NGS), 1% BSA, 0.3%
Tween 20 in PBS] for 1 h prior to application of the primary antibody. The
primary antibodies, i.e. polyclonal anti-CAII, monoclonal anti
V-H+ATPase or monoclonal `5', were diluted 1:50 in PBS
containing 1% NGS, 1% BSA, 0.3% Tween 20 (Carrier Solution, CS). The slides
were incubated for 2 h in a moist chamber at 37°C. The sections were then
rinsed in CS, and incubated with a 1:100 in CS HRP-labeled anti-mouse for
monoclonal antibodies (DAKO), or fluorescein-conjugated goat anti-rabbit IgG
secondary antibody for CA antibody (Sigma), for 1 h at room temperature. After
washing with CS, monoclonal antibody binding was visualized by applying a
solution of 4-chloro-1-naphthol (ImmunoPure, Pierce Biotech., Rockford, IL,
USA) until emergence of the characteristic purple-blue color. Sections treated
with CA antibody were mounted in antifading reagent AF3 (Citifluor Ltd,
London).
Histochemical detection of carbonic anhydrase by fluorescence
The CA enzyme was detected with 1-dimethylamino-naphthalene-5-sulfonamide
(DNSA), a fluorescent inhibitor with a great affinity for the catalytic center
of CA (Chen and Kernohan, 1967;
Dermietzel et al., 1985
). The
emission wavelength of DNSA shifts from 580 to 468 nm when binding to CA. The
trophosome, plume and vestimentum tissue pieces were rinsed in Riftia saline
(Fisher et al., 1988
) and
immediately frozen in liquid nitrogen. Cryostat sections (10 µm in
thickness) were prepared at -27°C, transferred to poly-L-lysine coated
slides (Sigma) and fixed with a drop of cold methanol (-20°C) before
thawing (Just and Walz, 1994
).
Immediately after methanol evaporation, sections were stained with 0.1 mmol
l-1 DNSA (Sigma) in phosphate buffer in a wet chamber at room
temperature for 30 min, then mounted in PB
(Dermietzel et al., 1985
). In
control experiments, non-fluorescent CA inhibitors acetazolamide (AZ) or
ethoxyzolamide (EZ), both provided by Sigma-Aldrich, were added to the
incubation medium at a concentration of 1 mmol l-1.
Microscopy and image acquisition
In situ hybridizations were observed on a Leitz Laborlux D light
microscope. Slides used for immunocytolocalization were observed with an
Olympus BH-2 epifluorescence microscope (Olympus Optical Co. Tokyo, Japan)
equipped with a mercury light source and a x40 UVFL objective
(excitation/emission filters: 490/515 for fluoroisothiocyanate). Micrographs
were taken with Kodak films P160 (tungsten light) or P1600 (fluorescence).
Slides used for histolocalization of CA were observed with a Confocal Laser
Scanning Biological Microscope (CLSM, Fluoview, Olympus Optical Co. Ltd,
Tokyo, Japan). Optical sections (0.7 µm) were taken through bacteriocytes
with the CLSM equipped with a pulsed laser (Mira 900, Coherent, Santa Clara,
CA, USA) to obtain an equivalent biphoton excitation at 380 nm and at 470 nm.
The 470 nm barrier filter (bandwidth 10 nm) allows the detection of the blue
fluorescence of the CA-bound DNSA (maximum emission at 468 nm), but shuts down
the yellow-green fluorescence of unbound DNSA (maximum emission at 580 nm;
Chen and Kernohan, 1967).
Micrographs were processed with Adobe Photoshop 5.5 (Adobe Systems Inc., San José, USA) and NIH Image (http://rsb.info.nih.gov/nih-image) where appropriate.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of CA and ATPases
Western blot analyses were performed on total soluble and
membrane-associated protein extracts from the four main tissues of
Riftia for CA (Fig. 2)
and from branchial plume and trophosome for the ion transporters
(Fig. 3).
Fig. 2A shows the specific
protein patterns of each tissue as revealed by silver nitrate staining. The
vestimentum and the body wall exhibited very similar patterns for both soluble
and membrane-associated protein fractions, and differed from the plume protein
profile by several major bands. Meanwhile, the proteins constituting the
symbiotic tissue appeared different from the other three tissues. Polyclonal
rabbit antibody raised against chick 30 kDa CA cross-reacted with a 27 kDa
cytosolic protein in the plume and a 28 kDa cytosolic protein in the
trophosome (Fig. 2B). This
antibody did not cross-react with any other protein in the four tissues
tested. The 5 monoclonal antibody raised against a chicken
1020-amino-acid Na+K+-ATPase cross-reacted with
membrane-associated proteins only, labeling two bands in the trophosome and
three bands in the plume, all of them with a molecular mass of 115 kDa
(Fig. 3A). The monoclonal
antibody raised against yeast 60 kDa ß-subunit of V-H+ATPase
cross-reacted with proteins from the membrane fraction (S3), producing two
distinct bands of 59 and 57 kDa in the plume and one band of 57 kDa in the
trophosome (Fig. 3B). A lighter
band appears in the cytosolic fraction (S2) in both tissues, at 59 kDa in the
plume and at 57 kDa in the trophosome.
|
|
CA localization
To identify the expression of CA genes in the tissues of R.
pachyptila, degenerate oligonucleotide primers
(Table 1) were designed on the
basis of conserved regions in published cDNA sequences coding for cyotosolic,
membrane-associated, mitochondrial and secreted CA in a wide range of species.
A cDNA sequence of 225 nucleotides was amplified, clones and identified
via BLAST search of GenBank as a cytosolic-like CA fragment. This
cloned fragment was checked for specificity by northern blot (not shown), and
then used for in situ hybridization experiments. Hybridization
experiments with the CA antisense probe and immunolocalization on transverse
sections showed an intense label in the trophosome
(Fig. 4) and the branchial
filaments (Fig. 5).
|
|
|
The trophosome consists of numerous lobules, each of these being a functional unit (Fig. 4A). Each lobule is provided with an axial efferent blood vessel, several afferent vessels situated at the periphery of the lobule, and many capillaries joining them. Four cell types make up the trophosome lobules: non-bacteriocyte cells, either (i) unspecialized cells or (ii) muscle cells in the external layer immediately adjacent to the axial vessel; (iii) bacteriocytes filling the whole central part of the lobule, and (iv) a layer of peritoneal cells in the outermost region. The bacteriocytes are the specialized cells containing the chemosynthetic bacteria within vacuoles. No label was found in the controls incubated with sense riboprobe (Fig. 4A). Cytosolic CA mRNA was consistently found in all lobules, except for blood vessels (Fig. 4B). Close-ups show that both the bacteriocytes and the peritoneal cells are labelled (Fig. 4C,D). The intensity of the label was often weaker in the bacteriocytes compared to the peritoneal cells (Fig. 4D). Protein immunoreactivity was also apparent in all trophosome lobules from the inner to the outer part (Fig. 4E). The central efferent vessel and the peripheral afferent vessels were all free of staining (Fig. 4E). The intensity of the green label was maximum around the intracellular bacteria, which appeared in red (Fig. 4F). Using DNSA, a fluorescent, specific inhibitor of CA, the cytoplasm of the peritoneal cells showed a bright fluorescence, owing to the formation of the enzymesubstrate complex between CA and DNSA (Fig. 4G). When ethoxyzolamide, the non-fluorescent competitor of DNSA, was added to the incubation medium, a dose-and time-dependent disappearance of the fluorescence was observed, until the fluorescence was completely abolished (Fig. 4H).
The same study was performed on the non-symbiotic tissues of the worm: the trunk body wall, vestimentum and plume. CA mRNA hybridization experiments in the body wall and in the vestimentum only gave weak signals, thus we chose to focus our results on CA expression and immunolocalization in the branchial plume. It is a conspicuous organ, held rigid by the axial collagenous obturaculum, and composed of many rows of lamellae formed by contiguous filaments (Fig. 5A). The free distal tip of each filament bears, on its posterior side, two close rows of pinnules (Fig. 5B). Each filament contains an afferent and an efferent blood vessel bathing in coelomic fluid (Fig. 5D,E). Cytosolic CA mRNA hybridization was labeled in the epidermis of the branchial filaments, where the cells are in contact with the coelomic cavity, but also more apically (Fig. 5B,D). The connective tissue between joined lamellae was always free of staining (Fig. 5C), and only a weak signal could be detected directly around the afferent and efferent vessels (Fig. 5D). The enzyme itself appeared more specifically localized in the apical region of the epithelial cells, at the interface between environmental water and plume epidermis, as shown by immunolocalization with anti-chick CA-II (Fig. 5F). Indeed the expression of CA mRNA appeared to be enhanced in the pinnules of each filament (Fig. 5B), and the same intense staining was observed in immunolocalization experiments with anti-chick CA-II (data not shown). A similar pattern resulted from the fluorescence signal of CA-bound DNSA (Fig. 5G), which emphasized the apical side of the branchial epidermis. Controls performed with sense riboprobe (Fig. 5A), pre-immune serum (Fig. 5E) and competition with EZ (Fig. 5H) did not result in any aspecific background staining.
ATPase localization
Immunolocalizations of V-H+ATPase and
Na+K+-ATPase were also investigated on trophosome
lobules (Fig. 6) and branchial
filaments (Fig. 7), to explore
any possible colocalization of these two ion transporters with the cytosolic
carbonic anhydrase visualized within the same tissues.
Fig. 6A shows a cross-section
of a trophosome lobule from the inner part, the efferent vessel, to the
periphery, stained with DAPI. Dense blue-colored dots marked nuclei of the
non-symbiotic cells, around the efferent vessel and in the outer part of the
lobule, while bacteriocytes exhibited a more diffuse signal due to the
staining of prokaryotic DNA. DAPI staining also distinguished pink-colored
host cell membranes on the trophosome section, which were barely visible by
light microscopy. Fig. 6B
illustrates the corresponding immunolabeling with the V-H+ATPase
antibody, showing bright label at the level of the bacteriocytes
(Fig. 6B), but much less around
peritoneal cells. Higher magnification of the bacteriocytes revealed that the
label surrounds the bacteria (Fig.
6C). The use of Na+K+-ATPase antibody led to
a signal that was mostly localized in bacteriocytes at the periphery of the
lobules (Fig. 6D). The linear
appearance of the label suggests its localization on membranes
(Fig. 6E), although these were
not clearly discernible.
|
|
In transverse sections of branchial filaments, the epithelial cells appeared to possess a V-H+ATPase with localization restricted to the apical membrane and intracellular vesicles (Fig. 7A,B), and a densification of the vacuoles close to the apical area (Fig. 7B). Na+K+-ATPase immunostaining on plume filament cross sections was intense and restricted to the basolateral membrane of the cells surrounding the coelomic cavity containing the afferent and efferent vessels (Fig. 7C).
No staining was detected when pre-immune mouse or rabbit serums were used instead of primary antibody, either on trophosome lobules or on branchial filaments.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The expression pattern of CA mRNA was analyzed in the branchial plume and
trophosome of Riftia pachyptila, as well as the localization of the
corresponding proteins. Evidence was also found for two ion pumps, type
V-H+ATPase and Na+K+-ATPase, which are
functionally associated to CA (Goffredi
and Childress, 2001; De Cian et al., 2002). How do the present
results help our understanding of the overall transport processes of
CO2 in Riftia? Some of the major implications and
hypotheses are discussed below.
CO2 transport across the branchial plume epithelium
The branchial plume plays a key role in the physiology of Riftia,
since it is the organ where almost all ion and metabolite exchanges take
place, especially CO2 incorporation and internal concentration
processes. High activity levels of CA and ATPase have been reported in R.
pachyptila plume tissue: 254 µmol CO2 min-1
g-1 wet mass for CA (Goffredi
et al., 1999b) and 646 µmol Pi h-1
g-1 wet mass for total ATPase, 13% of which was attributed to
Na+K+-ATPase, and 14% to V-H+ATPase
(Goffredi and Childress,
2001
). Physiological studies have also demonstrated the importance
of proton exclusion mechanisms as well as CA activity in maintaining an inward
CO2 flux through Riftia gill (Goffredi et al.,
1997
,
1999b
). It is therefore
interesting that in the branchial epidermis, CA messenger RNA as well as CA
protein are mainly localized in the pinnules and in the apical part of the
epidermis, in close contact with surrounding seawater.
V-H+ATPase generates both electrochemical and pH gradient across
the endo- and exomembranes of eukaryote cells
(Nelson, 1992), and is thus
involved not only in acidifying endosomes but also the cell environment. In
Riftia plume, the epidermal cells appeared to possess a
V-H+ATPase only at their apical membrane and in intracellular
vesicles located mainly in the apical part of the cell cytosol. This
repartition could induce local decreases of pH mediated by massive proton
excretion, and shift the boundary layer equilibrium such that
PCO2 would increase, driving CO2
across the membrane by diffusion. Such a proton elimination process could be
greatly facilitated by the high concentrations of CA found in the pinnules.
Indeed, CA efficiently contributes to internal pH regulation and regulation of
the net entering CO2 gradient in the circulating fluids by
reversible interconversion of CO2 into bicarbonate and protons
(Goffredi et al., 1997
). Thus,
the dual role of cytoplasmic CA and V-H+-ATPase, essential to
sustain CO2 influx while regulating intracellular pH, is fully
compatible with the localization of both proteins revealed in this study.
CO2 transport in the trophosome lobule
Physiological studies have suggested a role for carbonic anhydrase in the
bacteriocytes: the final consumers of CO2 are the bacteria that use
only molecular CO2 (Scott et
al., 1999), but the main form of inorganic carbon in the blood is
bicarbonate (Toulmond et al.,
1994
), hence the need for conversion. CA activity in trophosome
extracts is high (109 µmol CO2 g-1 min-1;
Goffredi et al., 1999b
) and
recent studies on isolated bacteriocytes have shown that inhibition of CA by
acetazolamide induces both extra- and intracellular pH variations (De Cian et
al., 2002).
The in situ hybridization experiments on whole Riftia
sections showed that CA mRNA is intensely transcribed throughout the
trophosome lobules. Bacteriocytes and peritoneal cells were labeled for CA
mRNA and CA protein, the intensity of the labeling appearing higher in
peritoneal cells than in bacteriocytes. But the differences in organelle
content between the two cell types revealed by ultrastructural observations
(Gardiner and Jones, 1993) may
account for the difference in staining. The cytoplasm of the bacteriocytes is
filled with bacteria and does not contain as much endoplasmic reticulum (RER)
as the peritoneal cells, so transcriptional activity may be higher in the
latter. They constitute a cell layer directly surrounding the multiple
peripheral afferent vessels that bring CO2 and other solutes to the
symbionts, but the inner part of the lobule is also filled with capillaries
inbetween the bacteriocytes. Our results with CA labeling, however, do not
allow us to distinguish any variation of the staining between the peripheral
bacteriocytes and the central ones. Further investigations should be done
using more sensitive techniques allowing quantitative analysis of the
labeling, through the use of confocal microscopy and fluorescence
quantification analysis software.
Immunohistological localizations of the ion pumps showed that, in trophosome sections, V-H+ATPase was colocalized with CA immunolabeling at the cellular level in the bacteriocytes, but the peritoneal cells were free of staining. Na+K+-ATPase immunostaining was also limited to bacteriocytes.
The branchial epidermis and the bacteriocytes of Riftia appear to
highly express vacuolar-type proton-pumping V-H+ATPase both on
intracellular vesicles and on specific domains of their plasma membrane. They
also contain high levels of cytosolic carbonic anhydrase (CA). In addition to
the Na+K+-ATPase localized herein, future investigation
should focus on HCO3- exchange processes present in
Riftia. HCO3- generated intracellularly in the
branchial cells has to be actively transported across the plume epithelium to
the circulating fluids, and then from the circulating fluids to the
bacteriocyte cytosol. Attempts to identify a
Cl-/HCO3- anion exchanger analog to Band-3 in
the tissues of Riftia through histological and molecular biology
approaches have failed (M.-C. De Cian and A. C. Andersen, unpublished
results), which is not surprising since Band-3 is absent from red blood cells
of lower vertebrates such as lamprey
(Bogdanova et al., 1998;
Hagerstrand et al., 1999
;
Cameron et al., 2000
) and
hagfish (Peters et al., 2000
),
and exhibits a different structure from mammals when present in kidney, liver
and muscles of these species. However, evidence for different types of anion
exhangers has been obtained in invertebrates, such as the
Na+-coupled Cl-/HCO3- exchanger,
newly described in Drosophila
(Romero et al., 2000
). This
transporter exchanges Na+ and HCO3- (or
another anion) against Cl- and H+ and requires a
sustained gradient for Na+, so may constitute an interesting
alternative and should be investigated in Riftia.
Conclusion
The present study has enhanced the importance of CA in two tissues of
Riftia pachyptila involved in ion exchange, the branchial plume,
particularly the symbiotic tissue, the trophosome, in carbon transport and
conversion, pH regulation and ionic balance. Interestingly, as described in
the cnidarian/algal association (Weis et
al., 1989), the cytosolic CA localized in Riftia belongs
to the
-CA gene family, according to the nucleotide sequence obtained,
and not the bacterial
-CA family. Reynolds et al.
(2000
) showed that the
upregulation of CA gene is a function of symbiosis, since CA expression is
enhanced in the presence of symbiotic algae in the host tissue. Since
Riftia acquires symbionts at each generation after metamorphosis, it
would be of interest to investigate the regulation of CA expression in
Riftia as a function of larval development, and acquisition of the
symbiosis by the worm through in situ hybridization and
immunolocalization. The study of the establishment and regulation of the
expression of the related ATPases in the plume and trophosome cells under a
variety of physiological stimuli which modify proton transport and related
ion-transport processes would also open new perspectives.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersen, A. C., Jolivet, S., Claudinot, S. and Lallier, F. H. (2002). Biometry of the branchial plume in the hydrothermal vent tubeworm Riftia pachyptila (Vestimentifera; Annelida). Can. J. Zool. 80,320 -332.[CrossRef]
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Bogdanova, A. Y., Sherstobitov, A. O. and Gusev, G. P. (1998). Chloride transport in red blood cells of lamprey Lampreta fluviatilis: evidence for a novel anion-exchange system. J. Exp. Biol. 201,693 -700.
Brion, L. P., Cammer, W., Satlin, L. M., Suarez, C., Zavilowitz, B. J. and Schuster, V. L. (1997). Expression of carbonic anhydrase IV in carbonic anhydrase II-deficient mice. Am. J. Physiol. Renal Physiol. 42,F234 -F245.
Cameron, B. A., Gilmour, K., Forster, R., Ko, K. and Tufts, B. L. (2000). Unique distribution of the anion exchange protein in the sea lamprey, Petromyzon marinus. J. Comp. Physiol. B 170,497 -504.[CrossRef][Medline]
Cavanaugh, C. M., Gardiner, S. L., Jones, M. L., Jannasch, H. W. and Waterbury, J. B. (1981). Prokaryotic cells in the hydrothermal vent tubeworm Riftia pachyptila Jones: possible chemoautotrophic symbionts. Science 213,340 -342.
Chen, R. F. and Kernohan, J. C. (1967). Combination of bovine carbonic anhydrase with a fluorescent sulfonamide. J. Biol. Chem. 24,5813 -5823.
Childress, J. J., Lee, R. W., Sanders, N. K., Felbeck, H., Oros, D. R., Toulmond, A., Desbruyères, D., Kennicut II, M. C. and Brooks, J. (1993). Inorganic carbon uptake in hydrothermal vent tubeworms facilitated by high environmental PCO2. Nature 362,147 -149.[CrossRef]
De Cian, M. C., Andersen, A. C., Toullec, J. Y., Biegala, I., Caprais, J. C., Shillito, B. and Lallier, F. H. (in press). Isolated bacteriocyte cell suspensions from the hydrothermal vent tubeworm Riftia pachyptila, a potent tool for cellular physiology in a chemoautotrophic symbiosis. Mar. Biol.
Dermietzel, R., Leibstein, A., Siffert, W., Zamboglou, A. and Gros, G. (1985). A fast screening method for histochemical localization of carbonic anhydrase. J. Histochem. Cytochem. 33,93 -98.[Abstract]
Felbeck, H. (1981). Chemoautotrophic potential of the hydrothermal vent tubeworm, Riftia pachyptila Jones (Vestimentifera). Science 213,336 -338.
Fisher, C. R., Childress, J. J. and Sanders, N. K. (1988). The role of vestimentiferan hemoglobin in providing an environment suitable for chemoautotrophic sulfide-oxidizing endosymbionts. Symbiosis 5,229 -246.
Gardiner, S. L. and Jones, M. L. (1993). Vestimentifera. In Microscopic Anatomy of Invertebrates: Onychophora, Chilopoda and lesser Protostomata., vol.12 (ed. S. L. Gardiner), pp.371 -460. New York: Wiley-Liss.
Goffredi, S. K. and Childress, J. J. (2001). Activity and inhibitor sensitivity of ATPases in the hydrothermal vent tubeworm Riftia pachyptila: a comparative approach. Mar. Biol. 138,259 -265.[CrossRef]
Goffredi, S. K., Childress, J. J., Desaulniers, N. T., Lee, R.
W., Lallier, F. H. and Hammond, D. (1997). Inorganic carbon
acquisition by the hydrothermal vent tubeworm Riftia pachytila
depends upon high external PCO2 and upon
proton-equivalent ion transport by the worm. J. Exp.
Biol. 200,883
-896.
Goffredi, S. K., Childress, J. J., Lallier, F. H. and Desaulniers, N. T. (1999a). The ionic composition of the hydrothermal vent tube worm Riftia pachyptila: Evidence for the elimination of SO42- and H+ and for a Cl-/HCO3- shift. Physiol. Biochem. Zool. 72,296 -306.[CrossRef][Medline]
Goffredi, S. K., Girguis, P. R., Childress, J. J. and
Desaulniers, N. T. (1999b). Physiological functioning of
carbonic anhydrase in the hydrothermal vent tubeworm Riftia pachyptila.Biol. Bull. 196,257
-264.
Hagerstrand, H., Danieluk, M., BobrowskaHagerstrand, M., Holmstrom, T., KraljIglic, V., Lindqvist, C. and Nikinmaa, M. (1999). The lamprey (Lampetra fluviatilis) erythrocyte; morphology, ultrastructure, major plasma membrane proteins and phospholipids, and cytoskeletal organization. Mol. Membr. Biol. 16,195 -204.[CrossRef][Medline]
Henry, R. P. (1996). Multiple roles of carbonic anhydrase in cellular transport and metabolism. Annu. Rev. Physiol. 58,523 -538.[CrossRef][Medline]
Henry, R. P. and Cameron, J. N. (1982). The distribution and partial characterization of carbonic anhydrase in selected aquatic and terrestrial decapod crustaceans. J. Exp. Zool. 221,309 -321.
Henry, R. P., Gilmour, K. M., Wood, C. M. and Perry, S. F. (1997). Extracellular carbonic anhydrase activity and carbonic anhydrase inhibitors in the circulatory system of fish. Physiol. Zool. 70,650 -659.[Medline]
Jones, M. L. (1981). Riftia pachyptila Jones: observations on the Vestimentiferan worm from the Galapagos rift. Science 213,333 -336.
Jones, M. L. (1988). The Vestimentifera, their biology, systematic and evolutionary patterns. Oceanol. Acta 8,69 -82.
Just, F. and Walz, B. (1994). Localization of carbonic anhydrase in the salivary glands of the cockroach, Periplaneta americana. Histochem. 102,271 -277.
Kochevar, R. E., Govind, N. S. and Childress, J. J. (1993). Identification and characterization of two carbonic anhydrases from the hydrothermal vent tubeworm Riftia pachyptila Jones. Mol. Mar. Biol. Biotechnol. 2, 10-19.
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Lebovitz, R. M., Takeyasu, K. and Fambrough, D. M.
(1989). Molecular characterization and expression of the
(Na-K)-ATPase -subunit in Drosophila melanogaster. EMBO
J. 8,193
-202.[Abstract]
Lucas, J. M. and Knapp, L. W. (1996). Biochemical characterization of purified carbonic anhydrase from the octocoral Leptogorgia virgulata. Mar. Biol. 126,471 -477.
Lutz, R. A., Shank, T. M., Fornari, D. J., Haymon, R. M., Lilley, M. D., Vondamm, K. L. and Desbruyeres, D. (1994). Rapid growth at deep-sea vents. Nature 371,663 -664.[CrossRef]
Maren, T. H. (1967). Carbonic anhydrase: chemistry, physiology and inhibition. Phyiol. Rev. 47,595 -781.
Nelson, N. (1992). The vacuolar
H+-ATPase one of the most fundamental ion pumps in nature.
J. Exp. Biol. 204,25
-37.
Peters, T., Forster, R. E. and Gros, G. (2000).
Hagfish (Myxine glutinosa) red cell membrane exhibits no bicarbonate
permeability as detected by 18O exchange. J. Exp.
Biol. 203,1551
-1560.
Reynolds, W. S., Schwarz, J. A. and Weis, V. M. (2000). Symbiosis-enhanced gene expression in cnidarian-algal associations: cloning and characterization of a cDNA, sym 32, encoding a possible cell adhesion protein. Comp. Biochem. Physiol. 126A,33 -44.
Romero, M. F., Henry, D., Nelson, S., Harte, P. J., Dillon, A.
K. and Sciortino, C. M. (2000). Cloning and characterization
of a Na+-driven anion exchanger (NDAE1) A new bicarbonate
transporter. J. Biol. Chem.
275,24552
-24559.
Scott, K. M., Bright, M., Macko, S. A. and Fisher, C. R. (1999). Carbon dioxide use by chemoautotrophic endosymbionts of hydrothermal vent vestimentiferans: affinities for carbon dioxide, absence of carboxysomes, and delta C-13 values. Mar. Biol. 135, 25-34.[CrossRef]
Shillito, B., Lubbering, B., Lechaire, J. P., Childress, J. J. and Gaill, F. (1995). Chitin localization in the tube secretion system of a repressurized deep-sea tube worm. J. Struct. Biol. 114,67 -75.[CrossRef]
Toulmond, A., Lallier, F. H., De Frescheville, J., Desbruyères, D., Childress, J. J., Lee, R. and Sanders, N. K. (1994). Unusual carbon dioxide-combining properties of body fluids in the hydrothermal vent tubeworm Riftia pachyptila.Deep-Sea Res. 41,1447 -1456.
Tufts, B. L., Gervais, M. R., Moss, A. G. and Henry, R. P. (1999). Carbonic anhydrase and red blood cell anion exchange in the neotenic aquatic salamander, Necturus maculosus. Physiol. Biochem. Zool. 72,317 -327.[CrossRef][Medline]
Weis, V. M. (1991). The induction of carbonic
anhydrase in the symbiotic sea anemone Aiptasia pulchella. Biol.
Bull. 180,496
-504.
Weis, V. M., Smith, G. J. and Muscatine, L. (1989). A `CO2 supply' mechanism in zooxanthellate cnidarians: role of carbonic anhydrase. Mar. Biol. 100,195 -202.