Fig 1.
Characteristic features of gastropod rhogocytes.
Note the plasma membrane (pm), nucleus (n), mitochondria (mi), Golgi bodies (gb), rough endoplasmic reticulum (rer), electron-dense granula (g), vesicles (v), uncoated vesicles (uv), coated vesicles (cv), coat (c), hemocyanin molecules (hc), extracellular lacunae (e), cytoplasmic bars (b), diaphragmatic slits (s), enveloping lamina of extracellular matrix (m), electron-dense material (a) at the edges of the bars. The slit apparatus resembles a gully grate (left scheme), but most often transversal cuts through the cytoplasmic bars are seen (right scheme).
Fig 2.
Light microscopy of paraffin-embedded tissue sections of L. stagnalis.
(A) Total scan of a diagonal cut through the animal (h, head; m, mantle; f, foot; v, visceral sac). (B-D) Head tissue sections; prominent structures such as eye, odontophore (o), radula (r) and esophagus (e) could be identified. (E, F) Mantle tissue sections; note that besides muscle cells (mc) and secretory cells (sc), many rhogocytes (rc) are visible. The rhogocytes are identified by their large nucleus and lamellar substructure. Hematoxylin & eosin stain (B-D, F), and Movat´s pentachrome stain (E) was applied.
Fig 3.
Detection of hemocyanin in L. stagnalis paraffin-embedded tissues and whole mounts.
(A, B) Immunohistochemistry on paraffin-embedded tissue sections. (A) Negative control. (B) Rabbit anti-Aplysia californica hemocyanin primary antibodies; note strong staining of rhogocytes (arrows). (C-E) Whole mount in situ hybridization of the extracted mantle. (Total length of the animals was ca. 2 cm.) (C) Negative control, with the DIG-labeled probe omitted. (D) 100 ng of the DIG-labeled probe was added; note the blue-purple staining. (E) 200 ng of the DIG-labeled probe was added; note deeper blue-purple staining. (F) Paraffin-embedded tissue section of the whole mount shown in (E); note specific labeling of a cell morphologically identified as rhogocyte. (G-I) In situ hybridization on paraffin-embedded tissue sections from other L. stagnalis individuals. The strongly stained cells (arrows) are morphologically identified as rhogocytes.
Fig 4.
Electron microscopy of L. stagnalis mantle tissue sections.
(A) Low magnification image with rhogocytes (rc), muscle cells (mc) and granular cells (gc). (B) Medium magnification showing a single rhogocyte; note the big nucleus (n), endoplasmatic reticulum (er) and electron-dense granula (g). The white frame indicates the area magnified in (C). (C) Close-up of an area with extracellular lacunae (e), cytoplasmic bars (b), and diaphragmatic slits (s) shown in longitudinal cuts. (D) Another single rhogocyte; the frame indicates the area magnified in (E). (E) Close-up of the area marked in (D), with a further magnification (frame) presented in the insert. It shows the cytoplasmic bars (b) and diaphragmatic slits (s) in a transversal cut. The extracellular lacunae (e) are filled with granular material. Note at both ends of the cytoplasmatic bars the electron-dense material (a). This material is likely to contain actin (see Fig 7). (F) Another rhogocyte. Note the magnified slit apparatus in the (white box), and the coated vesicle (cv) fused with the extracellular lacuna (black box).
Fig 5.
Electron microscopy of hemocyanin and rhogocytes.
(A) Biochemically isolated, negatively stained L. stagnalis hemocyanin didecamers. Note the circular top views and the rectangular side views of the protein molecule. The panel shows side and top view of a gastropod hemocyanin didecamer simulated, as 7 Å resolution, using PDB-entry 4BED (deposited by our research group in 2013). Note wall (dark) and collar (light) of the molecule; the ten-fold repeating unit which is a subunit dimer (2x400 kDa) is indicated in blue. (B) A rhogocyte, with the area displayed magnified in (C) indicated by a frame. (C) Close-up of the framed area in (B), showing an ER compartment with side views of two pseudo-crystalline hemocyanin arrays (arrowheads). Note the surrounding solitary hemocyanin molecules (asterisk). The insert shows an array at higher magnification. (D) Another rhogocyte; the frame marks the area magnified in (E). (E) Close-up of the framed area in (D), showing two endomembrane compartments with top views of pseudo-crystalline hemocyanin arrays. The insert shows a further enlargement; note details of outer wall and inner collar of the protein cylinder. (F) Section of a rhogocyte (rc), a muscle cell (mc) and a hemolymph space (h) in between, labeled with immunogold particles recognizing gastropod hemocyanin. Note that the hemolymph space and compartments of the rhogocyte are strongly marked.
Fig 6.
Electron microscopy of mantle rhogocyte regions containing hemocyanin.
(A) Cluster of rhogocytes in the connective tissue. The framed area is further enlarged in (B). (B) Close-up view of the area framed in (A), showing the slit apparatus in transversal cut (black box) and longitudinal cut (white box). Magnifications of both areas are also shown. Note masses of hemocyanin-like particles at both sides of the slit apparatus. (C) Rhogocyte showing large peripheral ER cisternae filled with particle. The framed area is further magnified in (D). (D) Close-up view of the framed area seen in (C). The insert shows a further enlargement of the region indicated by a black box. Different views of hemocyanin molecules are visible. (E) Another rhogocyte containing hemocyanin-rich ER cisternae. The framed area is further magnified in (F). (F) Close-up view of the lacuna framed in (E) and filled with hemocyanin. In the magnified box, different views of hemocyanin molecules can be distinguished.
Fig 7.
Localization of actin and nephrin in the slit apparatus.
(A-C) Electron micrographs of LR-white embedded mantle tissue sections of L. stagnalis. (A) A group of muscle cells (mc) labeled with a gold-coupled monoclonal anti-actin antibody (black dots in enlarged area). (B) A rhogocyte within mantle connective tissue. Note the enlarged area (insert), showing anti-actin immunogold labeling at the edges of the cytoplasmic bars (arrows) covering the extracellular lacuna (e). The black frame indicates the area magnified in (C). (C) Close-up of the region framed in (B), showing anti-actin immunogold labeling (arrows) at the cytoplasmic bars (b) close to the diaphragmatic slits (s). Also note boxed enlargement. e, extracellular lacuna; pm, plasma membrane. (D-F) Immunofluorescence microscopy of a frozen mantle tissue section. (D) Phase contrast optics. (E) Epifluorescence optics, showing DAPI (diamidino-2-phenylindole) staining to visualize the cell nuclei. (F) Epifluorescence optics, showing a positive reaction (red signal) of the periphery of a rhogocyte with polyclonal anti-nephrin antibodies. Also note enlargement.
Fig 8.
Electron microscopy of mantle tissue sections fixed by high pressure freezing and freeze substitution.
(A) A single rhogocyte composed from a series of electron micrographs (n, nucleus). The black arrows indicate two pseudo-crystalline hemocyanin arrays that are magnified in the inserts with black frames. White frames indicate areas that are enlarged in (B-E). (B) Close-up of area B framed in (A), showing an elaborate extracellular lacuna (e) with slit apparatus (sa). m, enveloping lamina of extracellular matrix. Note the adjacent compartment with a pseudo-crystalline hemocyanin array (arrow) that seems to be accompanied by solitary hemocyanin didecamers. Coated vesicles (cv) are also present; they apparently lack hemocyanin. (C) Close-up of area C framed in (A), with rich rough ER (rer) and two pseudo-crystalline hemocyanin arrays in a compartment that seems to contain also free didecamers (arrow; see also inserts in (A)). (D) Close-up of area D framed in (C), featuring a small vesicle (v) within the extracellular lacuna (e). sa, slit apparatus; cv, coated vesicle. (E) Close-up of area E framed in (A), showing Golgi bodies (gb), mitochondria (mi) and many vesicles.
Fig 9.
3D reconstruction of a rhogocyte region with slit apparatus.
(A,B) Electron tomogram slices of two mantle rhogocytes (rc) and adjacent hemolymph spaces (h) of L. stagnalis, superimposed with their corresponding 3D reconstruction (calculated in IMOD). Note the enveloping lamina of extracellular matrix (m, red), plasma membrane (pm, cyan), extracellular lacuna (e), cytoplasmic bars (b, green), diaphragmatic slits (s, pink), coated vesicles (cv, yellow). Also note the highly dense, possibly actin-rich material in peripheral parts of the cytoplasmic bars (a, purple). (A) Note the large vesicle with clearly visible coat (c) and granular content (blue) in open contact with the extracellular lacuna. In the neighboring cytoplasm, confined coated vesicles are present. (B) A peculiar situation is seen here, with a seemingly doubled slit apparatus and two concatenated extracellular lacunae. The insert shows details of a diaphragmatic slit, with a double-layer slit diaphragm (black arrows); this resembles images from B. glabrata rhogocytes [3]. Several putative hemocyanin molecules (hc) are visible in the hemolymph space. Note that such structures are absent from the coated vesicles. Also note that they appear to be too large to pass through the present diaphragmatic slits.
Fig 10.
Response of rhogocytes to deprivation of food and cadmium stress.
Electron micrographs of mantle tissue sections from L. stagnalis after starvation (A,B) and experimental cadmium contamination (C-F). (A) Total view of a rhogocyte from a snail deprived of food for 96 h. The insert (white box) shows that the cell surface is rather plain in that areas with invaginations and slit apparatus are rarely seen. The area within the black frame is magnified in (B). (B) Close-up of the indicated area in (A). Note the high number of small endomembrane compartments filled with a pseudo-crystalline hemocyanin array (arrows). The insert in (B) shows a further enlargement with top views of the hemocyanin stacks. (C) Total view of a rhogocyte from a cadmium-contaminated snail (48 h, 0.05mg/ml of CdCl2). The framed area is magnified in (D). (D) Close-up of the area framed in (C). Note many electron-dense granula (g) and mitochondria (mi). (E) A cluster rhogocytes from a cadmium-contaminated snail (96 h, 0.1mg/ml of CdCl2). The three small ovoid rhogocytes might stem from recent cell divisions. The larger rhogocyte with an irregular shape seems to be active in decontamination, as deduced from the framed area magnified in (F). (F) Close-up of the area framed in (E), showing a deeply folded cell surface with slit apparatus, suggesting exceptionally high ultrafiltration rates exhibited by this cell.
Table 1.
Number of electron-dense granula in 15 individual rhogocytes from untreated control snails and CdCl2 exposed snails.
Fig 11.
Hypothesis for the passage of material through the slit apparatus.
We assume that there are three different cellular stages: (1) Uncoated vesicles transport newly synthesized hemocyanin stacks to the plasma membrane; the stacks dissociate into single didecamers; exocytosis and accumulation of hemocyanin within the extracellular lacunae (their further release into the hemolymph is blocked by the slit apparatus); colloidosmotic pressure in the lacunae increases; water loss of the cytoplasm through exocytosis and enlargement of the extracellular lacunae yields the wrinkled rhogocyte population (observed by Sairi and coworkers [52]). (2) Pressure-induced contraction of the actin-rich cytoplasmic bars yields enlargement of the diaphragmatic slits. Free hemocyanin and occasional vesicles are released through the slits and the enveloping lamina of extracellular matrix (orange) into the hemolymph. The extracellular lacunae shrink. (3) Endocytosis of solvent by coated vesicles, with uptake of heavy metal ions; passage of hemocyanin through the slits is blocked again by the relaxed bars which prohibits its uptake from the hemolymph; the cell regains its original water content; the extracellular lacunae shrink further (ovoid rhogocyte population); hemocyanin biosynthesis starts again.