Magnetic Separation of Phagosomes of Defined Age from Tetrahymena thermophila

ABSTRACT. We describe a new mass isolation procedure for both pure and stage-specific phagosomes from Tetrahymena thermophila . We prepared magnetic iron dextran particles about 1 μm in diameter to label the phagosomes. The oral apparatus of the cells concentrated these particles so readily that after 1 min the majority of the cells had formed a single phagosome. A short wash removed non-ingested particles, enabling us to follow the age-dependent changes of a single labeled phagosome through the cell. Phagosomes of different ages, including very young and nascent phagosomes, were removed easily from the non-magnetic cell debris of mechanically homogenized cells by means of a permanent magnet. The isolated phagosomes are pure as tested by enzymatic assays and light and electron microscopy. Since the yield of pure phagosomes of all ages is high (∼ 90%), this method could be generally applied for phagosome isolation from ciliates.     

Tetrahymena Thermophila: Growth In Synthetic Nutrient Medium In the Presence and Absence of Glucose

ABSTRACT This report contains the newest directions for preparation of the synthetic nutrient medium for some Tetrahymena species that do not require lipids. In the standard medium T. thermophila. strains SB 210 and 281, multiply at 37°C with doubling times of around 2 h and at 26°C around 5 h. We have established multiplication rates as functions of variations in the composition of the medium. In media in which all components are present at one-third of the normal concentrations and only the essential amino acids are included, growth and multiplication become sharply dependent on glucose in strain SB 281. Such media may be used for selection and enrichment of certain specified cell lines.     

The Golgi Apparatus of Tetrahymena Thermophila

ABSTRACT. Electron microsocpic investigations reveal that the Golgi apparatus of Tetrahymena thermophila consists of numerous tiny dictyosomes, each consisting of one or two cisternae. the dictyosomes are localized predominantly in the cell cortex closely associated with the mitochondria, arranged in meridians alternating with the ciliary meridians. We estimated about 300-400 of these dictyosomes in the periphery of a cell, a value corresponding to the number of somatic cilia per cell. Cytochemical assays of thiamine pyrophosphatase and acid phosphatase, both marker enzymes of trans Golgi cisternae, resulted in deposits of lead or cerium phosphate in the outermost cisternae of the dictyosomes. In addition, cisternae located at the bases of the basal body/parasomal sac arrangements are stained. This indicates that these cisternae may belong to the Golgi apparatus of the cell.     

Processing and Secretion of Lysosomal Acid α-Glucosidase In Tetrahymena Wild Type and Secretion-Deficient Mutant Cells

ABSTRACT. The proteolytic processing and secretion of a lysosomal enzyme, acid α-glucosidase, was studied by pulse-chase labeling with [³⁵S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid α-glucosidase into the cultured medium during starvation. the secretion was found to be repressed by addition of ammonium chloride (NH₄Cl). Acid α-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH₄Cl-treated cells. NH₄Cl did not affect the processing of the precursor acid α-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid a-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid α-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.     

Constitutive Secretion of Acid Hydrolases in Tetrahymena thermophila

THE role of lysosomal enzymes in intracellular digestion is now well established [11]. Most often we think of lysosomal hydrolases in catabolism of endogenous or foreign material taken up by endocytosis. There is however, a number of reports dealing with the release of acid hydrolases into the extracellular fluid in a variety of eukaryote cells. These cells range from Saccharomyces cerevisiae [15], Dictyostelium discoideum [10], Leishmania donovani [20], Acanthamoeba castellani [22], Entamoeba histolytica [12, 31], and species of Tetrahymena [1–3, 6] to mammalian cells in culture [49]. Concerning the latter, fibroblasts and hepatocytes in culture release acid hydrolases to the extracellular medium, but only if the synthesis of a specific recognition marker is impaired in the cells. This marker (man-nose-6-phosphate) is used for receptor mediated segregation of lysosomal enzymes into the lysosomal compartments. If the receptor or the marker are lacking, the hydrolases fail to enter the lysosomal compartment, and are secreted in immature form together with molecules belonging to the constitutive secretory pathway of the cells [8, 49]. Such a release of acid hydrolases seems to occur spontaneously from mammalian osteoclasts [4]. Macrophages, on the other hand, need a specific stimulation for their release process [40]. In lower eukaryotes the release may