Figure 1.
Light microscopy of paraffin-embedded tissue sections of B. glabrata.
(A) Total scan of a diagonal cut through the animal. (B) Total scan as in (A), but from a section 0.4 mm closer towards the body surface. d, dart sac; f, foot; g, ganglion; h, head; i, intestine with food mass; lf, lime fold; lr, lime ridge; m, mantle; o, ovo-testis; p, propodium; v, visceral hump. (C) Mantle tissue section, showing many scattered cells (arrow, double arrow) embedded in the connective tissue between muscle cells (asterisk). (D, E) At higher magnification, weakly stained cells filled with a homogeneous material (arrows) are discernable from somewhat smaller, strongly stained cells that are filled with a dense lamellar material (double arrows). Corresponding electron microscopical images (see Fig. 3) revealed that the strongly stained cells are rhogocytes and that the lamellar material is mostly endoplasmic reticulum and dense granula. The weakly stained cells are mucus glands. Movat’s pentachrome staining (A, B, D) and hematoxylin & eosin staining (C, E) were applied.
Figure 2.
Detection of B. glabrata hemoglobin (BgHb) in tissue sections and whole mounts.
(A, B) Indirect immunohistochemistry with rabbit anti-BgHb antibodies on mantle tissue sections. The background staining might result from hemoglobin freely dissolved in the hemolymph spaces. The strongly stained cells (arrows) are morphologically identified as rhogocytes. The insert shows an enlargement of the region in the red box. (C) In situ hybridization with antisense BgHb1-h cDNA (see Table 1) performed on a mantle tissue section. Note strong reaction of cells that are morphologically identified as rhogocytes (arrows). (D) A section next to that seen in (C), stained with Movat’s pentachrome to visualize rhogocytes (arrows). (E) Whole mount in situ hybridization using antisense BgHb2-i cDNA (see Table 1). Note blue staining of head (→h), foot (→f) and mantle (→m), and negative reaction of the visceral hump (→v). Total length of the animal was 2 cm. (F) Three different paraffin-embedded tissue sections of a whole mount as shown in (E), but treated with antisense BgHb1-h cDNA. Note specific labelling of cells morphologically identified as rhogocytes (arrows).
Table 1.
Antisense cDNA probes applied to in situ hybridization.
Figure 3.
Electron microscopy of a foot tissue section showing a typical rhogocyte.
(A) Overview of the rhogocyte. Note the large nucleus (→n), electron-dense granula (→g) and the abundant endoplasmic reticulum (→ER). (B) Enlargement of the region indicated by a black box in (A), showing that the endoplasmic reticulum (→ER) is lined with ribosomes (i.e. rough ER). Note in the adjacent ER lacuna the flocculate material that might be protein (→p). The insert in (B) shows a further enlargement of the region indicated by the black box in (B) to visualize individual ribosomes. (C) Enlargement of the region indicated by a white box in (A), showing several extracellular lacunae (→e) with cytoplasmatic bars (→b) and 20 nm slits (→s). In the bars, adjacent to the slits, electron-dense material is visible that was later shown to contain actin (→a). Also note the lamina of extracellular matrix (→m) and the coat (→c) lining the plasma membrane at the extracellular lacunae. In the adjacent cytoplasm, the protein-like material is seen (→p). The insert in (C) shows a further enlargement of the region indicated by a black box in (C) to visualize the slit diaphragm (→d) as two parallel small rods. Also note, in this insert, the actin-containing electron-dense material (→a) and the lamina of extracellular matrix (→m). The gully grate-like cytoplasmic bars are cut here in transversal section; for a longitudinal section, see Fig. 4B.
Figure 4.
Electron microscopy of mantle tissue sections.
(A) Rhogocyte, with the nucleus not visible in this section. (B) Enlargement of the area marked in (A), showing a longitudinal cut through several cytoplasmic bars (for a transversal cut, see Fig. 3C). It is obvious that the cytoplasmic bars (long arrow) border slits (short arrows) and not holes. Note that these slits can be very long (double arrow). (C) Rhogocyte, with the large nucleus visible. (D) Enlargement of the area marked in (C), showing several endoplasmic reticulum lacunae filled with circular structures ca. 50 nm in diameter (→F). These structures might be cylindrical hemocyanin molecules viewed from the top. However, in some areas these structures show open connections (→G) which is not explained by the typical hemocyanin structure (see, however, Fig. 10). Also note the more amorphous protein-like material in other lacunae (→E) that is interpreted as hemoglobin. (E–G) Enlargements of the regions marked in (D).
Figure 5.
3D ultrastructure of a rhogocyte region with slit apparatus.
(A) Electron tomogram slice of chemically fixed mantle tissue, superimposed by a 3D reconstruction performed in IMOD. (B) The corresponding 3D model visualized and segmented in IMOD. Note the enveloping lamina of extracellular matrix (→m, salmon), the extracellular lacuna (→e) filled with protein-like particles (→p, blue), the coated plasma membrane (→c, cyan), the bridging cytoplasmic bars (→b, green), the slits (→s) with the diaphragm (→d, magenta). Vesicles (→v, yellow) inside the extracellular lacuna and the neighboring cytoplasm are also seen. Highly dense material (→a, purple) is observed at the cytoplasmic bars adjacent to the slits; it might contain actin bundles (see Fig. 7).
Figure 6.
3D ultrastructure of a rhogocyte periphery with different locations of vesicles.
Electron tomograms with superimposed 3D reconstructions are shown. (A) Region showing the coated plasma membrane (→c, cyan), several extracellular lacunae (→e), diaphragmatic slits (→s) and the lamina of extracellular matrix (→m, salmon). Note vesicles (yellow) present in the cytoplasm (→v1), the extracellular lacunae (→v2), between slit apparatus and lamina (→v3), and outside of the latter in the adjacent hemolymph (→v4). A vesicle probably fused with the slit apparatus is also seen (→v5). (B) Region showing, in an extracellular lacuna (→e), a vesicle (→v6, yellow) in open contact with a diaphragmatic slit (→s) formed by neighboring cytoplasmic bars (→b, green). The vesicle contains protein-like material (→p, blue) that is likely to be transported through the slit. Note similar vesicles (→v7, red) outside of the lamina of extracellular matrix (→m).
Figure 7.
Immunogold localization of actin in the slit apparatus.
(A) Electron microscopy of a rhogocyte in a mantle tissue section labeled with immunogold particles against actin. (B) The region marked by a box in (A) imaged at higher magnification, showing gold labeling. (C) Part of the slit apparatus of three different rhogocytes, showing anti-actin immunogold labeling in the electron-dense regions of the cytoplasmic bars adjacent to the slits.
Figure 8.
Primary structure of B. glabrata nephrin.
Its sequence relationships to human nephrin (AF190637.1) as deduced from a pair-wise alignment are shown by symbols for identical (*), isofunctional (:) and similar (.) amino acids. Note that gaps in the alignment are not considered here. The predicted structural domains as defined by Kestilä et al. [28] are indicated by thick lines (black, extracellular immunoglobulin-like domains; orange dotted, large linker; red, extracellular ferredoxin-III-like domain; blue, transmembrane helix; grey dotted, intracellular domain). Also indicated are the cysteines (red letters), most of which form a disulfide bridge within the immunoglobulin-like domains. Moreover, the potential attachment sites for N-linked glycans are highlighted (blue letters). The sequence is available under GenBank accession number KJ829367. Also shown is a predicted 3D structure as obtained by homology modelling; regions with no template available are sketched. (The same color code as above, except for the immunoglobulin-like domains.) Cysteines forming disulfide bridges are highlighted.
Figure 9.
Immunofluorescence microscopy of a mantle frozen tissue section with anti-nephrin antibodies.
(A) Cluster of rhogocytes at 250x primary magnification, showing positive immune reaction with guinea pig anti-nephrin antibodies. Note negative reaction of the mantle epithelium (arrow). (A’) The same section as in (A), labeled with the DNA stain DAPI (diamidino-2-phenylindole) to visualize the cell nuclei (arrow, mantle epithelium). (B–B”) The boxed area in (A, A’) at 1000x primary magnification, shown in phase contrast optics (B), epifluorescence optics with anti-nephrin antibodies (B’), and epifluorescence optics with DAPI stain (B”). Two rhogocytes are highlighted in boxes. Note that the positive reaction is mostly restricted to the cell periphery.
Figure 10.
Responses of rhogocytes to deprivation of food and cadmium stress.
(A) Electron microscopy of a rhogocyte from a snail deprived of food for 96 hours. Electron-dense granula and extracellular lacunae with slit apparatus are rarely seen, but vesicles containing hemocyanin-like particles are abundant (box and arrows). (B) Higher magnification of the region marked in (A) by a box. (C) Enlargement of the region marked in (B) by an asterisk. Note that the 50 nm rings show no connections, in contrast to those in Fig. 4G. (D) B. glabrata hemocyanin molecule extracted from an electron microscopical image of negatively stained hemolymph proteins (for details, see [20]). Note that its outer diameter (ca. 35 nm) corresponds to the diameter of the internal material of the 50 nm rings. In other words, the black annulus visible in (C) is heavy metal stain and not the protein cylinder wall, and corresponds to the dark uranyl acetate halo surrounding the molecule in (D). The thin white circle in (C, D) indicates the dimension of 50 nm. (E) 3D model derived from electron tomography of the area shown in (B). Note that the hemocyanin-like particles now appear as stacks of short cylinders. This is compatible with the quaternary structure of B. glabrata hemocyanin which is a hollow cylinder 35 nm in diameter and 18 nm in height (see [18]). (F) Rhogocyte of a cadmium-contaminated animal (0.05 mg/l CdCl2, 48 h). Note the large number of electron-dense granula (for comparison, see Fig. 3; for quantification, see Table 2). (G) Rhogocyte of a cadmium-contaminated animal (0.05 mg/l CdCl2, 96 h), suggesting significant increase of the filtrating cell surface. The insert shows an enlargement of the boxed area.
Table 2.
Number of electron-dense granula in individual rhogocytes from untreated and CdCl2-contaminated animals.