Biogenic amines stimulate regeneration of cilia in Tetrahymena thermophila

Serotonin and catecholamines affect the regeneration of cilia in Tetrahymena thermophila in a dose-dependent manner: micromolar concentrations are stimulatory, whereas millimolar concentrations have little or no effect. This conclusion is based on motility measurements in regenerating cells and on ciliary counts in scanning electron micrographs. In addition, the recognition mechanism for each hormone appears to be specific and independent. Our results suggest an evolutionary link with hormonal mechanisms in multicellular eukaryotes.     

Calcium Regulates the Regeneration of Cilia in Tetrahymena thermophila

A study of calcium metabolism in Tetrahymena during the regeneration of cilia evidenced that the process is inhibited by nifedipine and trifluoperazine. This suggests that calcium ions play an important regulatory role in this process. This was confirmed by studies on calcium uptake and efflux which showed that there was a net increase in calcium uptake prior to the reinitiation of motility. The increase coincided with a period of sensitivity to the calcium antagonist TMB-8 and with an increase in the intracellular level of cGMP. The process was also inhibited by neomycin and stimulated by phorbol esters, which suggests that hydrolysis of phosphatidylinositol phosphates may take place as part of the calcium regulatory network during the regeneration of cilia.     

Induction of tubulin synthesis during cilia regeneration in growing Tetrahymena

Log growth Tetrahymena pyriformis GL were deciliated by means of a calcium pulse and allowed to regenerate their cilia in a non-nutrient recovery medium. Polyribosome profiles show only small amounts of polysomes up to 30 min after suspension in recovery medium. After this time the number of polysomes increases continuously as protein synthetic activity and motility are recovered. Labeling of whole cells with l-[³⁵S]methionine and comparison of the resulting electrophoretic patterns reveals a marked induction of tubulin synthesis as cilia regeneration proceeds. At its peak, tubulin accounts for 7–10% of the incorporated label but this peak occurs 35 min after the cells become greater than 90% motile and about 25 min after the cilia reach full length. These results are discussed with respect to the regulatory mechanism of tubulin induction and induction of tubulin synthesis in starved Tetrahymena.     

Glutathione in Tetrahymena thermophila

In Tetrahymena , glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.     

A temperature-sensitive mutation affecting cilia regeneration, nuclear development, and the cell cycle of Tetrahymena thermophila is rescued by cytoplasmic exchange.

A temperature-sensitive mutation was isolated that blocks cilia regeneration and arrests growth in Tetrahymena thermophila. Protein and RNA synthesis and ATP production appeared to be largely unaffected at the restrictive temperature, suggesting that the mutation is specific for cilia regeneration and growth. At the restrictive temperature, mutant cells arrested at a specific point in the cell cycle, after macronuclear S phase and shortly before micronuclear mitosis. Arrested cells did not undergo nuclear divisions, DNA replication, or cytokinesis, so the mutation appears to cause true cell cycle arrest. Surprisingly, the mutation does not appear to affect micronuclear mitosis directly but rather some event(s) prior to micronuclear mitosis that must be completed before cells can complete the cell cycle. The cell cycle arrest was transiently complemented by wild-type cytoplasm exchanged during conjugation with a wild-type cell. Each starved, wild-type cell apparently contained enough rescuing factor to support an average of six cell divisions. Thus, this mutation affects assembly and/or function of at least one but not all of the microtubule-based structures in T. thermophila.     

Glutathione in Tetrahymena thermophila

In Tetrahymena, glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.     

Control of initiation and elongation of cilia during ciliary regeneration in Tetrahymena.

Tetrahymena thermophila strain B could regenerate approximately 10% of its somatic ciliary mass in concentrations of cycloheximide believed to block all cytoplasmic protein synthesis. A quantitative study of the relative numbers and lengths of cilia regenerated in the presence and absence of cycloheximide under a variety of conditions suggested that specific initiation and elongation protein factors are involved in the control of ciliary morphogenesis in Tetrahymena.     

Small RNAs of Tetrahymena thermophila

Highly purified nuclear and cytoplasmic RNAs were obtained from Tetrahymena thermophila BVII containing only a minimal amount of cross-contamination. In the nuclear RNA fraction we have detected at least 6 distinct snRNAs. Some of the RNA species showed microheterogeneity. SnRNAs of Tetrahymena thermophila are very similar to rat snRNAs, as far as length is concerned. Our cytoplasmic small RNA fraction contained two RNAs, 7S and T7, reported recently (18) as nuclear, particularly nucleolar RNAs. Moreover, we could detect only one cytoplasmic small RNA species Tc1, Tc2 was not observed.Neither the nuclear nor the cytoplasmic small RNA species are degradation products of ribosomal RNA as was shown by Northern blotting and following hybridization with pGY17 containing the entire transcribed region of the ribosomal DNA of Tetrahymena thermophila.     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

Kinetics of the DFPase activity in Tetrahymena thermophila

Crude homogenates of the ciliate protozoon, Tetrahymena thermophila, can hydrolyze the potent acetylcholinesterase inhibitors O,O-diisopropylphosphorofluoridate (DFP) and O-1,2,2-trimethylpropylmethylphosphonofluoride (soman). Characterization of the enzymatic activity of the homogenate has been performed. The DFPase operates over a pH range of 4 to 10 and an ionic range of 0-500 mM NaCl. Rate of reaction increases three- to four-fold from 25 degrees C to 40 degrees C and is still present at 55 degrees C. These results indicate that the enzymatic activity operates over a broad range of environmental conditions, making it an attractive material for use in the detoxification and detection of organofluorophosphates. DFPases may be important in the metabolism of naturally occurring organophosphates.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

Oral Assembly in Left-Handed Tetrahymena thermophila

We have investigated oral development in a non-genically derived left-handed (LH) form of Tetrahymena thermophila , in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells'anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a "sculptured" appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180° rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama [36, 37]. We present a model (Fig. 37) for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for "secondary" OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

嗜热四膜虫减数分裂研究进展

减数分裂是真核生物有性生殖过程的关键步骤,染色体的行为变化贯穿整个减数分裂的过程。近些年来,借助先进的分子生物学技术和细胞学实验手段,通过对突变细胞株的筛选和评价,单细胞真核模式生物原生动物嗜热四膜虫(Tetrahymena thermophila)减数分裂方面的研究取得了长足的进展。本文主要介绍嗜热四膜虫减数分裂的过程,以及在此过程中伴随染色体行为变化的相关基因的功能,从而为进一步探讨嗜热四膜虫减数分裂的分子机制提供有效信息。     

Oral assembly in left-handed Tetrahymena thermophila

We have investigated oral development in a non-genetically derived left-handed (LH) form of Tetrahymena thermophila, in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells' anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a "sculptured" appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180 degrees rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama. We present a model for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for "secondary" OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

Septins stabilize mitochondria in Tetrahymena thermophila

We describe phylogenetic and functional studies of three septins in the free-living ciliate Tetrahymena thermophila. Both deletion and overproduction of septins led to vacuolization of mitochondria, destabilization of the nuclear envelope, and increased autophagy. All three green fluorescent protein-tagged septins localized to mitochondria. Specific septins localized to the outer mitochondrial membrane, to septa formed during mitochondrial scission, or to the mitochondrion-associated endoplasmic reticulum. The only other septins known to localize to mitochondria are human ARTS and murine M-septin, both alternatively spliced forms of Sep4 (S. Larisch, Cell Cycle 3:1021-1023, 2004; S. Takahashi, R. Inatome, H. Yamamura, and S. Yanagi, Genes Cells 8:81-93, 2003). It therefore appears that septins have been recruited to mitochondrial functions independently in at least two eukaryotic lineages and in both cases are involved in apoptotic events.     

The Tetrahymena thermophila phagosome proteome

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.     

Development in Electrofused Conjugants of Tetrahymena thermophila

Electric shock can create parabiotic fusions of living Tetrahymena cells. In this study, cells were mated and successful pairs were electrofused with either vegetatively growing cells or other mating pairs. In particular, we electrofused pairs from normal [diploid x diploid] matings with vegetatively dividing cells in G- or M-phase of the cell cycle. We also fused [diploid x diploid] conjugants with mating pairs involving an aneuploid partner [diploid x "star"], which typically undergo an abortive conjugal pathway termed genomic exclusion. Using such parabiotic fusions we identified and characterized two developmentally critical landmarks: 1) the "abort" signal, which is initiated in pairs with nuclear defects (this first becomes evident soon after the completion of Meiosis I or the beginning of Meiosis II); and 2) the "terminal commitment point", a developmental stage in normal [diploid x diploid] pairs after which conjugation no longer responds to a parabiotically transmitted abort signal (this correlates with the onset of the second postzygotic nuclear division). Finally we demonstrate that a conjugal-arrest-activity varies with the vegetative cell cycle, reaching its highest level of activity during M-phase and dropping just after cytokinesis.