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Expression and localization of carbonic anhydrase and ATPases in the symbiotic tubeworm Riftia pachyptila

Marie-Cécile De Cian*, Ann C. Andersen, Xavier Bailly and François H. Lallier

Equipe Ecophysiologie, CNRS-UPMC UMR 7127 CEOBM, Station Biologique, BP 74, F-29682 Roscoff Cedex, France



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  		Fig. 1. (A) Sagittal section showing the body parts of the whole vestimentiferan tubeworm Riftia pachyptila and the labeling of total RNA by Gallocyanin. The four main body compartments of the worm are indicated, with their respective RNA synthesis activity. (B) Corresponding densitometry analysis of the section processed with NIH Image, with a binary transform at 50% threshold. Maximal transcription activity was detected in the trophosome lobules, at the basis of the joined branchial lamellae and in the epithelium of the wings. The obturaculum, vestimentum and body wall showed basal expression levels. Scale bar, 2 mm.

 


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  		Fig. 2. (A) SDS-polyacrylamide gel of total protein extracts of Riftia pachyptila tissues (load approx. 7 µg) stained with silver nitrate, and (B) corresponding immunoblot probed with rabbit anti-chick CA II. CA cross-reacted with a protein of a 27 kDa in the plume and one of 28 kDa in the trophosome. The vestimentum and the trunk body wall were weakly immunoreactive to CA antibody. No cross-reaction was observed with pre-immune serum (data not shown). The positions of marker proteins (kDa) are shown. BW, body wall; Pl, plume; S2, soluble protein fraction; S3, membrane-associated protein fraction; Tr, Trophosome; V, vestimentum.

 


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  		Fig. 3. (A) Western blot analysis of Na+K+-ATPase. Proteins of molecular mass approx. 115 kDa were probed with anti-chicken Na+K+-ATPase antibody in the plume and in the trophosome membrane fractions (S3) but not in the soluble fractions (S2). (B) Western blot analysis of V-H+ATPase. Two bands of approximately 59 and 57 kDa were visualized with anti-yeast V-H+ATPase antibody for the plume fraction, but only one band of 57 kDa in the trophosome. The positions of marker proteins (kDa) are shown.

 


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  		Fig. 4. Localization of carbonic anhydrase in trophosome lobules of Riftia pachyptila. (A—D) In situ hybridization of carbonic anhydrase mRNA with DIG-labeled riboprobe. (A) Negative control, showing the organization of the trophosome with numerous lobules (L) bathing in coelomic fluid (CF). (B—D) Section of a lobule composed of a central efferent vessel (EV), peripheral afferent vessels (AV), bacteriocytes (BC) and peritoneal cells (PC) in the extreme periphery of the lobule. Arrowheads indicate positive hybridization with the riboprobe in the bacteriocytes (C) and in the peritoneal cells (D). (E) Corresponding protein immunolocalization using rabbit anti-chick CA II antiserum with (F) a detailed portion (boxed area in E) focusing on CA labeling in the bacteriocytes filling the lobule. (G,H) Histochemical localization of CA with DNSA (G) and corresponding result of competition with Ethoxyzolamide (H). Scale bar, 150 µm (A); 50 µm (B,E,G,H); 15 µm (C,D,F).

 


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  		Fig. 5. Localization of carbonic anhydrase in branchial tissue of Riftia pachyptila. (A) Negative control with sense riboprobe on a transverse section of the plume showing its anatomical organisation. Lamellae are attached to the central obturaculum (Ob), and are composed of filaments (Fl) joined proximally but free distally. (B—D) In situ hybridization of CA mRNA with DIG-labeled riboprobe. (B) Transverse section of a single filament; the arrowhead indicates maximum staining intensity observed in a pinnule. (C,D) Epithelial cells are clearly stained by CA riboprobe, contrasting with the weak staining of extracellular matrix (C) and vascular vessels (D). (E) Negative control for CA immunolocalization using rabbit anti-chick CA II antiserum. (F) Corresponding protein immunolocalization restricted to the apical region of the epithelial cells. (G) Protein localization with DNSA, and (H) control section with DNSA on slides pre-incubated with the competitive CA inhibitor ethoxyzolamide. AV, afferent blood vessel; CF, coelomic fluid; EV, efferent blood vessel. Scale bar, 200 µm (A); 40 µm (E,G,H); 20 µm (B—D,F).

 


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  		Fig. 6. Immunocytochemical analyses of V-H+ATPase and Na+K+-ATPase in cryosections of trophosome of Riftia pachyptila. (A) Lobule section stained with DAPI. Bacteriocytes are characterized by a more diffuse staining than peritoneal cells and cells surrounding the efferent vessel (EV), due to the staining of bacterial DNA; white arrowheads indicate bacteriocyte cell membranes. (B) Corresponding section showing the immunolocalization of V-H+ATPase in the lobule. Staining was abundant all around the bacterial vacuoles, membrane and vacuolar sublocalization being hardly distinguishable using light microscopy, even at higher magnification (C). (D,E) Immunolocalization of Na+K+-ATPase, where staining was restricted to the periphery of the lobule (D) and appeared to line host cell membranes (E). AV, afferent blood vessel; EV, efferent blood vessel. Scale bar, 20 µm (A,B,D); 6 µm (C,E).

 


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  		Fig. 7. Immunocytochemical analyses of V-H+ATPase and Na+K+-ATPase in cryosections of the branchial filaments of Riftia pachyptila. (A) Transverse section of branchial filament stained for V-H+ATPase. (B) Magnification of cell membrane area from A showing the apical localization of the V-H+ATPase (arrowheads). (C) Localization of Na+K+-ATPase in transversal section of branchial filament showing its basal localization in the epidermis (arrowhead). CF, coelomic fluid; C, cuticle. Scale bar, 40 µm (A,C); 10 µm (B).

 

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© The Company of Biologists Ltd 2003