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Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water

W. S. Marshall^*, E. M. Lynch and R. R. F. Cozzi

Department of Biology, St Francis Xavier University, Antigonish, NS, Canada B2G 2W5

View larger version (98K): [in a new window]   Fig. 1. The distribution of kfCFTR in mitochondria-rich (MR) chloride cells in killifish opercular epithelium. Images are confocal laser scans of kfCFTR revealed by immunofluorescence of mouse anti-human CFTR and goat anti-mouse IgG Oregon Green 488 and counterstained for mitochondria with Mitotracker Red (green fluorescence + red counterstain yields yellow). (A) Membrane from a SW-adapted animal has kfCFTR immunofluorescence at 4.5 µm from the surface of the chloride cells, localized to the apical crypt (white arrows). The asterisk indicates a gap in the pavement cell layer. (B) The same frame as for A but at a depth of 7.5 µm at the plane of the nucleus of the MR cell. Blue arrows indicate the mitochondria-rich chloride cells below the apical crypt. (C) Diagram of approximate depths of the optical sections, AP, apical crypt level; CY, cytosol level. (D,E) Similar kfCFTR immunostaining, but in MR cells from fish acclimated to FW. (D) An optical section at 4.5 µm from the surface has positive kfCFTR immunofluorescence evenly distributed in the chloride cells (white arrows) as well as in the pavement cells at the plane of the pavement cell nuclei (orange arrow). (E) The same frame as for D but 10.5 µm into the tissue at the level of the MR cell nuclei. Blue arrows indicate the MR cells with diffusely distributed positive immunofluorescence for kfCFTR. (F,G) Similar immunostaining but for a fish transferred from FW to SW for 48 h. (F) An optical section 9.0 µm from the surface, with ring-shaped kfCFTR-positive immunofluorescence (white arrows); (G) 13.5 µm from the surface, with kfCFTR immunofluorescence diffusely distributed in MR cells (blue arrows). Scale bars, 20 µm.

View larger version (122K): [in a new window]   Fig. 2. The distribution of Na+,K+,2Cl- cotransporter in SW-adapted and FW-acclimated killifish opercular epithelium. Images are confocal laser scans of NKCC revealed by immunofluorescence of mouse anti-human NKCC and goat anti-mouse IgG Oregon Green 488 in MR cells counterstained for mitochondria with Mitotracker Red (green fluorescence with red counterstain yields yellow). (A) In SW-adapted killifish, NKCC immunofluorescence at 7.5 µm from the surface of the chloride cells is evenly distributed throughout the cytoplasm of MR cells (white arrows). (B) In FW-acclimated killifish, NKCC immunofluorescence at 9.0 µm from the surface of the chloride cells is eccentrically distributed in the chloride cells and localized between pairs of MR cells (white arrows). Scale bar, 20 µm.

View larger version (18K): [in a new window]   Fig. 3. Evaluation of kfCFTR immunofluorescent MR cells into those containing diffuse only, punctate only, or both diffuse and punctate distribution. Bars indicate the percentage of cells in each category; the numbers of cells measured are shown. The number of animals in each group were FW, 9; FW to SW for 24 h, 4; FW to SW for 48 h, 6; SW, 5. In FW animals, most cells had only diffuse kfCFTR immunofluorescence; 8.1 % (18 cells) had both. The percentage of cells with punctate distribution increased with acclimation to SW, and in fully adapted SW animals all cells had only apical punctate kfCFTR localization.

View larger version (15K): [in a new window]   Fig. 4. The cellular location of punctate kfCFTR immunofluorescence in FW controls (a small minority of cells) and SW animals (all cells), as determined by optical sections in the confocal microscope, was in apical crypts approximately 7 µm deep. The transitional phases included many cells with punctate kfCFTR immunofluorescence well below the surface. It would appear kfCFTR distribution in FW is diffuse, then condenses in the first 24 h after salinity transfer, prior to moving kfCFTR to the apical membrane at approximately 48 h. Columns with different letters are significantly different (P<0.05 or better; single-classification ANOVA followed by a Bonferroni post-test).

View larger version (40K): [in a new window]   Fig. 5. Western blot analysis of CFTR, NKCC and Na+,K+-ATPase in opercular epithelium (20 µg) and gill tissue (20 µg for CFTR and NKCC, 10 µg for Na+,K+-ATPase) from SW-adapted killifish. Proteins were separated on a 7.0 % polyacrylamide gel, transferred onto an Immobilion-P membrane and probed with anti-human CFTR monoclonal antibody (1.0 µg ml—1). Proteins were visualized following incubation in BCIP/NBT Blue substrate development solution. Lanes 1, 2 and 3 show heart (H) (negative control), opercular epithelium (Oe) and gill tissue (G), respectively. The anti-hCFTR antibody is specific for a protein of approx. 150 kDa and two lower molecular mass bands in the opercular epithelium and in the gill. Lanes 4, 5 and 6 show the same tissues probed with the NKCC T4 antibody with the main band at approx. 150 kDa and two lower molecular mass bands. Lanes 7, 8 and 9 show the same tissues probed with the Na+,K+-ATPase 5 antibody. Visible proteins appear at approx. 120 kDa (major band) and 101, 72 and 68 kDa. Molecular mass markers (kDa) were: 205, 116, 97.4, 84, 66, 55, 45 and 33 (from top to bottom). For means and S.E.M. (N=3), see text.