monolayer Selleck GDC-941 is comprised of midgut epithelial cells and is surrounded on its basal side by a well-established extracellular space. Muscle cells and tracheoles were found adjacent to the extracellular space ( Fig. 1C). Columnar and goblet cells are the most abundant cell types and no specific distribution pattern was observed ( Fig. 1C–E). Both cells define the monolayer height and present luminal-oriented microvilli. They differ by the presence of the goblet cell cavity (GV), a specific luminal space (besides EcS and EnS), rich in microvilli. Vesicles could be detected in the columnar cell, suggesting a trafficking route, perhaps involving multivesicular bodies ( Fig. 1D). Regenerative cells were a less often observed cell type limited to the basal side of the cellular monolayer. As vesicles could be observed inside the epithelial cells cytoplasm, we proceeded towards detecting PolyP stores
using both the modified exopolyphosphatase GSK458 research buy PolyP-binding domain (PPBD) (Saito et al., 2005) and DAPI staining on OCT embedded sections. DAPI has been used as PolyP reporter as its interaction with PolyP yields fluorescence in a different wavelength than the blue emission from DAPI–DNA (Allan and Miller, 1980 and Aschar-Sobbi et al., 2008). Although PolyP stores were present along both columnar and goblet cells, goblet cell cavities and its surroundings were the major regions of accumulation of HAS1 PolyP stores (Fig. 2A and B). To confirm storage of PolyP inside epithelial cells, tissue homogenates were analyzed using a recombinant yeast exopolyphosphatase-based assay (Ruiz et al., 2001b). PolyP strongly concentrated in the posterior midgut of A. gemmatalis but was also detected in the anterior midgut ( Fig. 3A). In that regard, both regions were used in the following experiments. After mechanical lysis and decantation, we could obtain
a fraction rich in PolyP granules as detected by DAPI staining ( Fig. 3B). Under the transmission electron microscope, midgut PolyP granules presented an electron dense morphology ( Fig. 3C, inset) similar to PolyP granules from other models. X-ray microanalysis showed an elemental composition identical to previously found spherite profiles ( Fig. 3C). In that regard, detectable levels of metallic atoms like calcium, magnesium, potassium, sodium, and zinc were present. Phosphorous and chloride were also detected. Manganese, iron and sulfur were less often detected ( Fig. 3D). In our samples, calcium peaks were only observed inside spherites and allowed us to use calcium as a spherite reporter in subsequent experiments. A specific group of polyP-containing organelles from protozoans have been shown to contain bafilomycin A1-sensitive V-ATPases (Docampo et al., 2005 and Scott et al., 1995a) and vanadate-sensitive Ca+2-ATPases (Docampo et al., 1995b) important for metal uptake.