, 2002). In this context, it had been proposed that glucose synthesis from glycogen by a glycogen phosphorylase (Lorenzo-Morales et al., 2008) has an important role in encystment. The exocyst, on the other hand, contains a combination

of proteins and polysaccharides (Neff & Neff, 1969). The cyst morphology has been used as a taxonomical tool, based on its size and number of endocyst arms (Pussard & Pons, 1977). Molecular mechanisms of encystment have been partially described. It had been described that autophagosomes are structures that mediate encystment, in both small and large vacuolar structures, with the involvement of actin dynamics (Bouyer buy RG7422 et al., 2009), a ubiquitin-like protein (ATG8) (Moon et al., 2009) and a specific serine protease (Moon et al., 2008). Because of the taxonomical and biological relevance of Acanthamoeba cysts, the structural organization of the cyst has been characterized in previous studies using both TEM and

SEM (Bowers & Korn, 1968, 1969; Chavez-Munguia et al., 2005, 2007). The use of chemical fixation for processing of the samples used in these ultrastructural studies, despite being designed Protein Tyrosine Kinase inhibitor to preserve and stabilize the structural features of the sample, can cause changes in the material due to the slow rate of fixative diffusion through the samples (Lupetti, 2005). In order to overcome such artifacts, a rapid freezing rate must be achieved in which there is no significant damage or distortion caused by the

formation of ice crystals (Pinto da Silva & Kachar, 1980). Such a condition can be obtained using the quick-freeze/freeze-fracture/deep-etching technique (QF-DE), which allows a tridimensional visualization of very well-preserved Adenosine triphosphate cellular structures (Heuser, 1981; Kubo et al., 1998). Here, the use of the QF-DE technique to characterize the fine structural components of the cyst of Acanthamoeba polyphaga is presented. Acanthamoeba polyphaga (ATCC 30461) was cultivated in peptone–yeast extract–glucose medium, pH 6.5 (Alfieri et al., 2000), at 28 °C in 25 cm2 tissue culture flasks without shaking. Trophozoites (105 amoebae mL−1) were incubated in six-well plates for 54 h in Neff’s encystment solution (Neff et al., 1964) as described previously by Rocha-Azevedo & Silva-Filho (2007). For the following assays, cysts were sedimented by centrifugation, and fixed with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2. Fixed cysts were washed in 0.1 M cacodylate buffer and postfixed in 1% osmium tetroxide/0.8% potassium ferrocyanide/5 mM CaCl2 in the same buffer, at room temperature. After 2 h, the material was washed, dehydrated in ascending acetone series (15%, 30%, 50%, 70%, 90% and 100%) and embedded in Polybed 812 resin (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were stained with uranyl acetate, followed by lead citrate, before observation in a Jeol 1200 EX electron microscope operating at 80 kV.