Phagocytosis In Tetrahymena Research Articles

Experimental Design using India Ink to Study the Effects of Phagocytosis in Tetrahymena

Experimental Design using India Ink to Study the Effects of Phagocytosis in Tetrahymena

Investigating Phagocytosis in Tetrahymena

Investigating Phagocytosis in Tetrahymena

Investigating Phagocytosis in Tetrahymena: An Experimental System Suitable for Introductory & Advanced Instruction

Tetrahymena, a freshwater ciliated protozoan, is in many ways an ideal candidate for the study of cells by students. Readily obtained and cultured, Tetrahymena can be used to study ciliary motion, organelle structure and function, cell morphology, cell proliferation, and cell behaviors (Leick & Hellung-Larsen 1992; Wheatley et al. 1994). Because of the ease with which Tetrahymena can be grown and manipulated, it is especially suitable for use in inquiry-based labs and student projects. Phagocytosis in Tetrahymena is an especially dramatic cell behavior that students can observe directly with a compound microscope. When hungry Tetrahymena encounter food, they use their cilia to sweep it into each cell's oral groove. This process can be visualized by feeding stained yeast cells India ink (Keenan 1984) or Chlorella to Tetrahymena. Phagocytosis can be quantitated by counting the number of vacuoles that form in a defined time period. In this paper, I describe how our students use the India ink method, described below, to investigate phagocytosis and vacuole formation in Tetrahymena first to learn the basis of microscope use, and then to pursue various experimental projects.

On the evolution of opioid mechanisms and immune defenses.

Results from our laboratory suggest that opioid-modulated phagocytosis originated early in evolution and fundamental aspects of this mechanism have been conserved. β-endorphin-like immunoreactivity detected in the ciliate Tetrahymena appears to modulate phagocytosis. We have compared the effects of opioids on phagocytosis in Tetrahymena and in Fc-mediated phagocytosis by peritoneal murine macrophages. These effects are remarkably similar: a) opioid agonists inhibit phagocytosis by a naloxone-reversible mechanism with effective concentrations in the nanomolar range, b) the effect of acute exposure is dose-dependent and biphasic, c) chronic exposure to morphine results in a state akin to desensitization and tolerance, d) the effect of morphine on tolerant cells may be stimulatory under certain circumstances, e) withdrawal of morphine from tolerant cells may inhibit phagocytosis suggesting that tolerant cells may require morphine to perform this function at a basal level. Pharmacological data suggest that the opioid receptor in Tetrahymena is mu-like and may be associated with a Gi-like protein since pertussis toxin pre-treatment will counteract the inhibitory effect of opioids. Our work suggests that the influence of opioids on the immune system originated early in evolution since phagocytosis may be considered the primeval cell defense mechanism.

Effect of adrenocorticotropic hormone (ACTH) and insulin on the phagocytic capacity of Tetrahymena.

Adrenocorticotropic hormone (ACTH) and insulin negatively influenced the phagocytic activity of Tetrahymena. The two hormones had diverse effects after 4 hr of treatments on no-test-particle containing, "0-cells". At this time the number of "0 cells" was significantly lower in the ACTH-treated groups, while in the insulin-treated groups there was an increase of "0-cells" compared to the control and to the results of the starting experiment. Considering previous results, when small molecular weight hormones, if did at all, positively influenced phagocytosis in Tetrahymena, the experiments call the attention to the differences caused by the size of the signal molecules. In the light of the literary data on hormone effects to phagocytosis in mammals and men, the similarity of the effects in species being very far from each other in evolution, could be concluded.

A Novel Opioid Mechanism Seems to Modulate Phagocytosis in Tetrahymena

We have previously reported that a beta-endorphin-like substance inhibits phagocytosis in Tetrahymena perhaps by a mu-like opioid receptor. We now report a further characterization of the elements involved in the signal transduction mechanism of this opioid. Affinity chromatography followed by immunoblots of both intracellular extracts and extracellular medium reveal the presence of two main proteins of 64 and 75 kDa. These molecular weights are much higher than that of any known opioid peptide or precursor protein and suggest that we may be dealing with either a novel opioid or with proteins that by chance cross-react with anti-beta-endorphin antibody. Nevertheless, when the biological activity of these proteins was tested it was found that they had an effect similar to that of mammalian beta-endorphin, namely inhibition of phagocytosis by a naloxone-reversible mechanism. We have probed a size-selected Tetrahymena library with a pro-opiomelanocortin probe and have obtained several positive clones; the sequencing of their inserts should establish whether we are dealing with a bona fide member of the opioid family. Another aspect we have been studying is the G-proteins which appear to be involved in the modulation of phagocytosis. We have found, by means of Western blotting (using an antibody against the conserved GTP-binding region of the alpha-subunit), two bands of 51 and 59 kDa; no alpha-subunit of 59 kDa had been reported previously and may represent a novel G-protein. In spite of these differences, the opioid signal transduction mechanism appears to remarkably resemble that present in more complex organisms.

Pharmacological characterization of an opioid receptor in the ciliate Tetrahymena.

A pharmacological characterization has been performed of the opioid receptor involved in modulation of phagocytosis in the protozoan ciliate Tetrahymena. Studies on inhibition of phagocytosis by mammalian prototypic opioid agonists revealed that morphine and beta-endorphin have the highest intrinsic activity, whereas all the other opioids tested can only be considered partial agonists. However, morphine (a mu-receptor agonist) is twice as potent as beta-endorphin (a delta-receptor agonist). Furthermore, the sensitivity for the opioid antagonist naloxone, determined in the presence of morphine and beta-endorphin, is very similar to the sensitivity exhibited by mammalian tissues rich in mu-opioid receptors. We suggest that the opioid receptor coupled to phagocytosis in Tetrahymena is mu-like in some of its pharmacological characteristics and may serve as a model system for studies on opioid receptor function and evolution.

Effect of chronic opioid treatment on phagocytosis in Tetrahymena

Opioid inhibition of phagocytosis in the protozoan ciliate Tetrahymena is antagonized by naloxone and this antagonism can be surmounted by increasing agonist concentration, which suggests a receptor-mediated mechanism. Desensitization of the opioid effect is time dependent in addition to concentration dependent. Chronic exposure to opioids results in the development of tolerance to the inhibitory effect of the agonists, and withdrawal of the latter results in a decrease in phagocytic capacity, which suggests that a state akin to dependence has been developed in these cells. Naloxone appears to behave as a partial agonist in tolerant cells, and there seems to exist cross-tolerance to mu and delta agonists.

Effects of copper on phagocytosis inTetrahymena

Addition of copper, corresponding to 100 ppm, to the normal 2% proteose peptone medium is tolerated byTetrahymena. This concentration of copper stimulates phagocytosis to a maximum value which is reached gradually during the first 1 hour exposure, and which is maintained during continuous exposures. Cell proliferation is resumed after a lag period, although at a decreased rate. Cells exposed to copper contain small refractile granules, previously proposed to represent an ion-regulating system; the number of granules remains constant in proliferating cells. Higher concentrations of copper also resulted in an elevated rate of phagocytosis but at the same time cell mortality was observed; this lack of transition between inhibited phagocytosis and cell mortality may be ascribed to the physiological role of copper. The high amount of organic matter in the growth medium protects against the toxic effects of copper, thus in the absence of organic matterTetrahymena tolerated only a 100-fold lower concentration of copper than that tolerated in the growth medium. However, cells which had initiated granule formation (for example for regulation of calcium) prior to starvation and exposure to copper, were more resistant to copper than cells which had not yet activated this mechanism, perhaps because of the low capacity of starved cells for protein synthesis.

Mutant with heat-sensitive capacity for phagocytosis in tetrahymena: isolation and genetic characterization.

SUMMARYA mutant ofTetrahymenawith heat-sensitive phagocytosis was obtained using a tantalum-particle enrichment procedure. The mutant phenotype is most likely determined by a somatic (macronuclear) mutation(s). The inability of the mutant to sustain cell division and to phagocytize at 37 °C are most likely determined by the same mutation. The phenotype of the mutant is stably inherited under vegetative propagation at 30 °C. At 37 °C, the mutation affects the development of the oral apparatus, the phagocytotic organelle. This mutant has proven useful for the study of cellular functions related to phagocytosis.

Cytochalasin B: aspects of phagocytosis in nutrient uptake in Tetrahymena.

ABSTRACT Cytochalasin B (37 μg per ml) reduces the rate of food vacuole formation, i.e. the rate of phagocytosis, in Tetrahymena pyriformis. Cytochalasin B in this concentration suppresses multiplication rates in a nutrient medium consisting of 2 % proteose peptone, but multiplication is unaffected if this medium is supplemented with glucose and high concentrations of nucleosides. Thus nutrients in high concentrations circumvent the necessity for phagocytosis in Tetrahymena.