3D-Ultrastructure, Functions and Stress Responses of Gastropod (Biomphalaria glabrata) Rhogocytes

Abstract

Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastropods and other molluscs, with great variation in their number, shape and size. They are enveloped by a lamina of extracellular matrix. Their most characteristic feature is the “slit apparatus”, local invaginations of the plasma membrane bridged by cytoplasmic bars, forming slits of ca. 20 nm width. A slit diaphragm creates a molecular sieve with permeation holes of 20×20 nm. In blue-blooded gastropods, rhogocytes synthesize and secrete the respiratory protein hemocyanin, and it has been proposed–though not proven–that in the rare red-blooded snail species they might synthesize and secrete the hemoglobin. However, the cellular secretion pathway for respiratory proteins, and the functional role(s) of the enigmatic rhogocyte slit apparatus are still unclear. Additional functions for rhogocytes have been proposed, notably a role in protein uptake and degradation, and in heavy metal detoxification. Here we provide new structural and functional information on the rhogocytes of the red-blooded freshwater snail Biomphalaria glabrata. By in situ hybridization of mantle tissues, we prove that rhogocytes indeed synthesize hemoglobin. By electron tomography, the first three dimensional (3D) reconstructions of the slit apparatus are provided, showing detail of highly dense material in the cytoplasmic bars close to the slits. By immunogold labelling, we collected evidence that a major component of this material is actin. By genome databank mining, the complete sequence of a B. glabrata nephrin was obtained, and localized to the rhogocytes by immunofluorescence microscopy. The presence of both proteins fit the ultrastructure-based hypothesis that rhogocytes are related to mammalian podocytes and insect nephrocytes. Reactions of the rhogocytes to deprivation of food and cadmium toxification are also documented, and a possible secretion pathway of newly synthesized respiratory proteins through the slit apparatus is discussed.

Small-sized granules of biphasic bone substitutes support fast implant bed vascularization

“…38 In brief, the specific tissue regions within the bone substitute granules were cut into small pieces (3-6 mm 3 ). Subsequently, the tissue specimens were fixed in a freshly prepared medium of 1% glutaraldehyde in 0.1M cacodylate buffer pH 7.2 for a minimum of 2 h at room temperature.…”

Amorphous Ca2+ polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells

“…The methods and techniques applied for transmission electron microscopy analyses has been recently published (Kokkinopoulou et al, 2014). The cells were fixed in paraformaldehyde and glutaraldehyde, embedded in agarose and then in LR-white resin (no.…”

The Planorbid Snail Biomphalaria glabrata Expresses a Hemocyanin-Like Sequence in the Albumen Gland

“…Nonetheless, hemocyanin-like (cDNA and peptide) sequences as well as a large blood protein that resembles the characteristic ultrastructural shape of multimeric molluscan hemocyanin have been described from B . glabrata [16, 20, 21]. We conclude that these observations do not stem from a respiratory molluscan hemocyanin, but rather, find a basis in two novel hemocyanin-like genes, termed hcl -1 and hcl -2.…”

“…glabrata with a hemocyanin-like shape [16, 20] originates from the hcl-2 sequence. An antiserum raised against the heterologous keyhole limpet hemocyanin (KLH) recognizes a B .…”

“…The advent of hemoglobin has not expunged all traces of hemocyanin; partial hemocyanin-like EST sequences have been recorded from B . glabrata and a hemocyanin-like protein ultrastructure was visualized from the hemolymph [16], and in mantle rhogocytes [20]. Hathaway et al [21], using mass spectrometry, detected the presence of hemocyanin-like peptides in the albumen gland (AG) and egg mass fluid (EMF) of B .…”