Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.     

GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase

Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.     

Purification of GVBD-Inducing Protein from the Ciliate Tetrahymenathermophila

Germinal-vesicle-breakdown (GVBD) was induced if a 132,000-g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes. Using this induction of GVBD as a bioassay system, a GVBD-inducing substance was purified from the Tetrahymena by ultra-filtration, liquid chromatography, and electroelution from a band on native-PAGE gel. Proteins eluted from the single band on the native-PAGE gel induced GVBD in the absence of oocyte protein synthesis. This band resolved into two bands on SDS-PAGE: 60 and 112 kDa. The 60 kDa protein was the active fraction inducing GVBD. Immunoprecipitation of the 60 kDa protein prevented the GVBD-inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD-inducing substance. An immunoblot with anti-60 kDa monoclonal antibody and PSTAIR antibody showed that p13suc1-beads could remove cdc2 homologues from T. thermophila supernatant but could not remove the GVBD-inducing activity. The 60-kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division. The cyclic appearance of the 60-kDa protein in the T. thermophila cell cycle suggests that this protein has a cell cycle function.     

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.     

Protein phosphorylation and the regulation of basal body microtubule organizing centres in Tetrahymena.

Previous work suggests that changes in the phosphorylation state of some centrosomal proteins regulate centrosomal activity. The hypothesis that changes in the phosphorylation state of one or more basal body microtubule organizing centre (MTOC) components regulate its ability to nucleate cilia assembly in Tetrahymena thermophila was tested. The MPM-2 antibody, which recognizes phosphorylated epitopes in MTOCs in a variety of organisms, was used to probe immunoblots of cytoskeletal frameworks prepared from starved Tetrahymena, from starved deciliated Tetrahymena, and from a starved deciliated mutant Tetrahymena which failed to initiate ciliogenesis following deciliation. The MPM-2 antibody recognized an identical array of proteins in all blots. These results suggest that, unlike centrosomes, basal body MTOC activity is not regulated by changes in the phosphorylation state of component proteins.     

Conjugation involving dividing cells of Tetrahymena thermophila

Feulgen-stained preparations of mixtures of starved Tetrahymena thermophila cells of complementary mating types have revealed an atypical form of conjugation involving cells which have completed the nuclear events of cell division, but have not undergone cytokinesis. Both micronuclei in the dividing cells are induced to undergo meiosis, but in 21 of 23 cases, the anterior micronucleus was activated 1st, suggesting that the meiotic inducer is synthesized near the mating junction and diffuses posteriad. Despite the induction of two micronuclei, "triad" conjugants appear to regulate nuclear events so as to produce a normal outcome.     

The effects of supraoptimal temperatures on population growth and cortical patterning in Tetrahymena pyriformis and Tetrahymena thermophila: a comparison

In this investigation, we compare the multiplication rates and morphogenetic responses of the two most studied Tetrahymena species, T. pyriformis and T. thermophila, at supraoptimal temperatures. Although the upper temperature limits differ greatly in the two species, the pattern of growth responses to high temperature is for the most part similar, with some differences in detail. The transient recovery of cell division at the highest temperature that allows cell division, characteristic of T. pyriformis, is observed in a less distinct form in T. thermophila. Moreover, there is a remarkable difference in developmental response, with drastic abnormalities in patterning of oral structures during the transient recovery of cell division in T. pyriformis, and far more limited abnormalities under similar conditions in T. thermophila. The abnormalities result from spatial disorder in the alignment and orientation of basal body pairs within the early oral primordium, followed by failures in the realignment that normally occurs as oral structures (membranelles and undulating membrane) mature. Both the initial spatial disorder and the failures in realignment are far more severe in T. pyriformis than in T. thermophila.     

嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能分析

真核细胞中染色体浓缩调节因子（regulator of chromosome condensation 1, RCC1）是 RanGTPase 唯一的鸟嘌呤核苷酸交换因子. 染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成. 本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1（TTHERM_00530380）基因. 该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸. 实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高. 免疫荧光定位分析表明, HA RCC1在营养生长和饥饿时期,定位于大核和小核中|在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中. 过表达RCC1导致大核的无丝分裂异常, 细胞增殖变慢,最终产生无大核的后代细胞. 敲减RCC1导致了多小核的产生. 结果表明,RCC1参与调控了四膜虫细胞核的分裂, RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.     

Inactivation of a macronuclear intra-S-phase checkpoint in Tetrahymena thermophila with caffeine affects the integrity of the micronuclear genome

Aphidicolin (APH), an inhibitor of DNA polymerase α, arrested cell divisions in Tetrahymena thermophila. Surprisingly, low concentrations of APH induced an increase of macronuclear DNA content and cell size in non-dividing cells. In spite of the cell size increase, most proliferation of basal bodies, ciliogenesis and development of new oral primordia were prevented by the APH treatment. The division arrest induced by APH was partly overridden by caffeine (CAF) treatment, which caused the fragmentation ("pulverization") of the chromosomes in G2 micronuclei. Somatic progeny of dividers with pulverized micronuclei (APH+CAF strains) contained aneuploid and amicronucleate cells. The amicronucleate cells, after losing their oral structures and most of their cilia, and undergoing progressive disorganization of cortical structures, assumed an irregular shape ("crinkled") and were nonviable. "Crinkled" cells were not formed after APH + CAF treatment of the amicronuclear BI3840 strain, which contains some mic-specific sequences in its macronucleus. Most of the APH +CAF strains had a typical "*"- like conjugation phenotype: they did not produce pronuclei, but received them unilaterally from their mates and retained old macronuclei. However, 4 among 100 APH+CAF clones induced arrest at meiotic metaphase I in their wt mates. It is likely that the origin of such clones was enhanced by chromosome pulverization.     

Acquisition of cell polarity during cell cycle and oral replacement in Tetrahymena

The aim of this study was to search for a mechanism responsible for the acquisition of cell polarity in a ciliate Tetrahymena. Homologs of the mammalian genes coding for CDC42-GSK3beta- MARK/PAR1-MAPs proteins were found in the Tetrahymena genome (Eisen et al., 2006, and this study). These proteins belong to a pathway which controls assembly and disassembly of microtubule bundles and cell polarity in neural cells. In Tetrahymena, there are two types of morphogenesis: divisional and oral replacement (OR). In divisional morphogenesis, an elongation of longitudinal microtubule bundles (LMs) takes place during cell division. In contrast, in OR type morphogenesis, which occurs in starved non-dividing cells, a polar retraction of LMs occurs. In T. pyriformis, the frequency of developmental switch to OR morphogenesis increases in the presence of wortmannin, an inhibitor of the CDC42-GSK3beta-MARK pathway. In contrast, wortmannin when applied to dividing cells does not affect divisional morphogenesis. Using immunostaining with the antibody against mammalian mitotic phosphoproteins (MPM-2) we show that these proteins co-localize with the LMs and are distributed along the anterior-posterior gradient. In addition, we show that during OR type morphogenesis, the fate of LMs correlates with the anterior-posterior gradient of instability of the cortical structures. We used the conditional mouth-less mutant of T. thermophila (Tiedtke et al., 1988) to test if the presence of the oral apparatus is required for the maintenance of cell polarity. We discuss our results in relation to the hypothesis of GSK3-beta-MARK pathway involvement in the acquisition of cell polarity in Tetrahymena.     

Kinesin-II Is Preferentially Targeted to Assembling Cilia and Is Required for Ciliogenesis and Normal Cytokinesis in Tetrahymena

We cloned two genes, KIN1 and KIN2, encoding kinesin-II homologues from the ciliate Tetrahymena thermophila and constructed strains lacking either KIN1 or KIN2 or both genes. Cells with a single disruption of either gene showed partly overlapping sets of defects in cell growth, motility, ciliary assembly, and thermoresistance. Deletion of both genes resulted in loss of cilia and arrests in cytokinesis. Mutant cells were unable to assemble new cilia or to maintain preexisting cilia. Double knockout cells were not viable on a standard medium but could be grown on a modified medium on which growth does not depend on phagocytosis. Double knockout cells could be rescued by transformation with a gene encoding an epitope-tagged Kin1p. In growing cells, epitope-tagged Kin1p preferentially accumulated in cilia undergoing active assembly. Kin1p was also detected in the cell body but did not show any association with the cleavage furrow. The cell division arrests observed in kinesin-II knockout cells appear to be induced by the loss of cilia and resulting cell paralysis.     

The actin gene ACT1 is required for phagocytosis, motility, and cell separation of Tetrahymena thermophila

A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83:5160-5164, 1986), here called ACT1, was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1Delta mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1Delta cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1Delta cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.     

Members of the NIMA-related kinase family promote disassembly of cilia by multiple mechanisms

The genome of Tetrahymena thermophila contains 39 loci encoding NIMA-related kinases (NRKs), an extraordinarily large number for a unicellular organism. Evolutionary analyses grouped these sequences into several subfamilies, some of which have orthologues in animals, whereas others are protist specific. When overproduced, NRKs of three subfamilies caused rapid shortening of cilia. Ultrastructural studies revealed that each NRK triggered ciliary resorption by a distinct mechanism that involved preferential depolymerization of a subset of axonemal microtubules, at either the distal or proximal end. Overexpression of a kinase-inactive variant caused lengthening of cilia, indicating that constitutive NRK-mediated resorption regulates the length of cilia. Each NRK preferentially resorbed a distinct subset of cilia, depending on the location along the anteroposterior axis. We also show that normal Tetrahymena cells maintain unequal length cilia. We propose that ciliates used a large number of NRK paralogues to differentially regulate the length of specific subsets of cilia in the same cell.     

Activation of prostacyclin synthesis by mechanical stimulation in the ciliated protozoan Tetrahymena thermophila

We detected a HPLC peak corresponding to 6-keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI(2)), in [1-(14)C]arachidonate metabolites from the ciliated protozoan Tetrahymena thermophila. Quantitative analysis of 6-keto-PGF(1alpha) by enzyme immunoassay revealed that the synthesis and release were rapidly activated by the mechanical stimulation of a short centrifugation. The activation was suppressed significantly by a cyclooxygenase inhibitor, ibuprofen, and was independent of the extracellular Ca(2+). External addition of PGI(2) and its stable analogue, beraprost, caused a transient increase in the tumbling frequency of swimming. Other prostanoids, PGE(2) and PGF(2alpha), have no effect on the swimming. These results indicate that a free-living ciliate, T. thermophila, synthesizes and has a specific sensitivity to PGI(2).     

Effect of cell population density on G2 arrest in Tetrahymena

The ciliated protozoan, Tetrahymena pyriformis strain GL-C, has been used to study the effect of cell population density during starvation on the synchrony obtained after refeeding and on the number of cells arrested in G2 phase of the cell cycle. At high cell densities two peaks of division indices were observed after refeeding while only one was observed at low cell densities. Cell division began earlier in cultures starved at high cell densities. Most importantly, the proportion of cells in G2 was considerably higher in populations starved at high cell densities. When tritiated thymidine was present during the refeeding period, radioautographs of cell samples at different times showed that the first cells to exhibit division furrows contained unlabeled nuclei. The first peak in the division index after refeeding was observed only at higher cell densities and is attributed to the cells arrested in G2. These results suggest that Tetrahymena is an excellent organism to study the concept of resting stages in the cell cycle and their control.     

Deciliation interferes with cell-cycle progression in Tetrahymena

The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied. It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation. Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later. This was shown by pulse-labelling the cells with [3H]thymidine at various times after deciliation. Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose. At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment. This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division. The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure. The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells. This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation. Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells.     

Developmental regulation of gene expression in Tetrahymena

The onset of gene expression in Tetrahymena thermophila during macronuclear differentiation was investigated by assay of galactokinase in conjugating deoxygalactose-resistant heterokaryons. Our results distinguish three successive states of galactokinase gene expression for cells developing a new macronucleus: stage 0, refractory to induction; stage 1, inducible by refeeding; and stage 2, induced. The refractory period ends at 12 to 13 hr after the onset of conjugation; this corresponds to the time of pair separation, and occurs several hours after the new macronuclei have become morphologically distinguishable. Stage 1 cells behave indistinguishably from mature starved cells. Inhibitor studies suggest that galactokinase synthesis is induced coincidentally with the induction of bulk protein synthesis during conjugation: thus it behaves developmentally like a typical protein; and that galactokinase mRNA is probably transcribed within 1 hr prior to its translation. Thus, when conjugating cells are refed during the refractory period, some developmental condition prevents the swift induction of protein (and galactokinase) synthesis observed upon refeeding starved (nonmating) cells. The possible nature of this developmental phenomenon is discussed.     

Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis.

Deciliated starved Tetrahymena recover motility with kinetics similar to those of growing cells and, like growing cells, require RNA and protein synthesis for regeneration. Comparisons of polysome profiles and electrophoretic analyses of newly synthesized proteins indicate, however, that the basal level of protein synthesis in starved cells is markedly lower than that in growing cells. This difference allows demonstration of changes in protein synthesis following deciliation of starved cells which cannot be detected (if they occur at all) in growing cells. Deciliation of starved cells induces a specific and orderly program of protein synthesis. The synthesis of an 80,000 dalton protein (deciliation-induced protein, DIP) begins shortly after deciliation, comprises 15% of the protein synthesized from 20-60 min, and declines around 60 min after deciliation, shortly after most cells have begun to regenerate cilia. The synthesis of a 55,000 dalton protein is also induced during regeneration and has been identified as tubulin using a well characterized antibody made to ciliary tubulin. Tubulin synthesis is undetectable during the first hour after deciliation even though 60-80% of the cells regain mobility and regenerate short but clearly visible cilia. Tubulin synthesis begins 60 min after deciliation and continues for 2 hr. At its peak, tubulin comprises 7-8% of the protein synthesized. The results of actinomycin D addition at different times after deciliation suggest that RNA required for DIP synthesis is synthesized early (0-30 min), while RNA required for tubulin is synthesized later and over a longer period (30-90 min). Thus deciliation of starved cells, an event occurring at the cell periphery, initiates a well defined and reproducible series of events culminating in cilia formation. This system should be useful in elucidating the molecular mechanisms regulating gene expression and organelle biogenesis in Tetrahymena.