Although encystation (or cyst formation) can be an essential step of the life span cycle of by density gradient centrifugation and found to become delicate to nystatin and oseltamivir. most lipids are acquired URB754 by this parasite from a growth medium or from the small intestinal milieu (9). Some of the acquired lipids undergo remodeling by the head group and fatty acid exchange reactions. Fatty acids can undergo chain shortening or elongation before incorporation into the plasma membranes (10,C12). Most recently, we have demonstrated that glucosylceramide transferase (GlcT1), an enzyme of the sphingolipid pathways, serves as a key regulator of encystation and viable cyst production by (13). However, it is not known how the process of encystation is initiated and if the plasma membranes of trophozoites participate in this process. Because membrane rafts are present in the majority of eukaryotic cells and involved in cellular differentiation, we postulate that assembles raft-like microdomains and the molecules that URB754 are associated with giardial rafts take part in the URB754 encystation process. In this paper, we show for the first time that has the ability to assemble cholesterol- and GM1 ganglioside-enriched membrane microdomains. Disassembly of these microdomains affects encystation and cyst production. Depletion of cholesterol from the culture medium also interferes with raft assembly and cyst formation and produces atypical (non-type I) cysts that express both trophozoite and cyst proteins instead of mostly cyst proteins. The addition of cholesterol rescues this process by assembling raft-like microdomains and generating cysts with classical oval morphologies. MATERIALS AND METHODS Materials. Lipid raft (LR) inhibitors (i.e., nystatin and filipin III) were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO). Oseltamivir (Tamiflu; a viral neuraminidase inhibitor) and myriocin (an inhibitor of sphingolipid synthesis) were purchased from Selleckchem (Houston, TX) and Sigma-Aldrich, respectively. Stock solutions of nystatin (25 mM), filipin III (25 mM), and oseltamivir (12.18 mM) were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich). Myriocin (12.45 mM) was dissolved in methanol (Sigma-Aldrich). All other reagents were of analytical grade and obtained in the highest-purity grades from Sigma-Aldrich. Adult bovine serum (ABS; catalogue no. SH30075.03) and dialyzed fetal bovine serum (DFBS; catalogue no. 26400-044) were purchased from HyClone (UT, USA) and Gibco Invitrogen Inc. (Carlsbad, CA), respectively. A fluorescent LR labeling kit (Vybrant Alexa Fluor 488) and 1,1-dilinoleyl-3,3,3,3-tetramethylindocarbocyanine perchlorate [Dil9,12-C18(3), ClO4; FAST Dil oil] were purchased from Gibco Invitrogen (Carlsbad, CA). Fluorescein isothiocyanate (FITC)-conjugated trophozoite antibody (antirat polyclonal antibody; catalogue no. A900; Troph-O-Glo; Waterborne Inc., New Orleans, LA), Alexa Fluor 568-conjugated donkey antimouse antibody, and anti-ganglioside GM1 rabbit polyclonal antibody were purchased from Waterborne Inc. (New Orleans, LA), Gibco Invitrogen (Carlsbad, CA), and Abcam (Cambridge, MA), respectively. Mouse monoclonal cyst antibody and FITC-conjugated goat antirabbit secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cell culture. trophozoites (ATCC 30957, strain WB), clone C6, were cultivated in TYI-S-33 medium supplemented with 5% ABS or DFBS URB754 and 0.5 mg/ml adult bovine bile (14, 15). The antibiotic piperacillin (100 g/ml) was added during routine culture Rabbit polyclonal to PPA1. of (16). Parasites were detached by chilling on ice, harvested by centrifugation at 1,500 for 10 min at 4C, repeatedly washed in phosphate-buffered saline (PBS), and counted with the help of a hemocytometer under a light microscope (phase-contrast). encystation was carried out by culturing trophozoites in TYI-S-33 medium supplemented with 5% ABS (which is cholesterol enriched) or DFBS (which has a low level of cholesterol) and bovine bile (i.e., 5 mg/ml; high-bile medium) at pH 7.8 as described previously by Kane et al. (17). Treatment with inhibitors. To examine the effects of inhibitors on growth and encystation, trophozoites were inoculated (1 106 cells/ml) in 4-ml glass vials containing TYI-S-33 medium (1 ml, no serum, pH 7.1) and treated with various concentrations (0, 5, 10, 20, and 50 M) of inhibitors (nystatin, filipin III, and oseltamivir) for.