Gene network landscape of the ciliate Tetrahymena thermophila

Background

Genome-wide expression data of gene microarrays can be used to infer gene networks. At a cellular level, a gene network provides a picture of the modules in which genes are densely connected, and of the hub genes, which are highly connected with other genes. A gene network is useful to identify the genes involved in the same pathway, in a protein complex or that are co-regulated. In this study, we used different methods to find gene networks in the ciliate Tetrahymena thermophila, and describe some important properties of this network, such as modules and hubs.

Methodology/Principal Findings

Using 67 single channel microarrays, we constructed the Tetrahymena gene network (TGN) using three methods: the Pearson correlation coefficient (PCC), the Spearman correlation coefficient (SCC) and the context likelihood of relatedness (CLR) algorithm. The accuracy and coverage of the three networks were evaluated using four conserved protein complexes in yeast. The CLR network with a Z-score threshold 3.49 was determined to be the most robust. The TGN was partitioned, and 55 modules were found. In addition, analysis of the arbitrarily determined 1200 hubs showed that these hubs could be sorted into six groups according to their expression profiles. We also investigated human disease orthologs in Tetrahymena that are missing in yeast and provide evidence indicating that some of these are involved in the same process in Tetrahymena as in human.

Conclusions/Significance

This study constructed a Tetrahymena gene network, provided new insights to the properties of this biological network, and presents an important resource to study Tetrahymena genes at the pathway level.     

Phenotypic responses to temperature in the ciliate Tetrahymena thermophila

Understanding the effects of temperature on ecological and evolutionary processes is crucial for generating future climate adaptation scenarios. Using experimental evolution, we evolved the model ciliate Tetrahymena thermophila in an initially novel high temperature environment for more than 35 generations, closely monitoring population dynamics and morphological changes. We observed initially long lag phases in the high temperature environment that over about 26 generations reduced to no lag phase, a strong reduction in cell size and modifications in cell shape at high temperature. When exposing the adapted populations to their original temperature, most phenotypic traits returned to the observed levels in the ancestral populations, indicating phenotypic plasticity is an important component of this species thermal stress response. However, persistent changes in cell size were detected, indicating possible costs related to the adaptation process. Exploring the molecular basis of thermal adaptation will help clarify the mechanisms driving these phenotypic responses.     

New Tetrahymena basal body protein components identify basal body domain structure

Basal bodies organize the nine doublet microtubules found in cilia. Cilia are required for a variety of cellular functions, including motility and sensing stimuli. Understanding this biochemically complex organelle requires an inventory of the molecular components and the contribution each makes to the overall structure. We define a basal body proteome and determine the specific localization of basal body components in the ciliated protozoan Tetrahymena thermophila. Using a biochemical, bioinformatic, and genetic approach, we identify 97 known and candidate basal body proteins. 24 novel T. thermophila basal body proteins were identified, 19 of which were localized to the ultrastructural level, as seen by immunoelectron microscopy. Importantly, we find proteins from several structural domains within the basal body, allowing us to reveal how each component contributes to the overall organization. Thus, we present a high resolution localization map of basal body structure highlighting important new components for future functional studies.     

The formation of basal body domains in the membrane skeleton of Tetrahymena

Differentiated regions within the membrane skeleton are described around basal bodies in the ciliary rows of Tetrahymena. These domains, approximately 1 micron in diameter, are characterized by a relatively dense ultrastructure, the presence of a family of proteins called K antigens (Mr 39-44 x 10(3)) that are recognized by mAb 424A8, and the apparent exclusion of major membrane skeleton proteins that are present in most other regions of the cell (Mr 135, 125 x 10(3]. Mature basal body domains are asymmetric, reflecting the polarity of the cell as a whole. A similar differentiation of the membrane skeleton occurs in the oral apparatus, except here the K antigens surround four clusters of basal bodies (from which this cell takes its name) rather than the individual basal bodies. The development of new basal body domains in the cell cycle is described, with similarities and differences noted between somatic and oral regions of the cell. It is concluded that the capacity of this cell for precise topographic regulation of molecular events in the membrane skeleton makes it a useful model for the study of cortical differentiation in cells generally.     

Characterization and properties of cholesterol desaturases from the ciliate Tetrahymena thermophila

Live Tetrahymena thermophila transforms exogenous cholesterol into 7,22-bis, dehydrocholesterol (DHC) by desaturation at positions C7(8) and C22(23) of the cholesterol moiety. In this first report on expression, isolation, characterization, and reconstitution of Tetrahymena's cholesterol desaturases in cell-free extracts, we describe conditions for increasing the expression of both desaturases based on the addition of specific sterols to the culture medium. Reactions performed in vitro, with isolated microsomes, yield only the mono-unsaturated derivatives, 7-DHC and/or 22-DHC. However, selectivity towards one product can be improved with the addition of specific compounds: beta-mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent-solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroyl-phosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b(5). NADH or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b(5) in these reactions.     

Methylation of replicating and nonreplicating DNA in the ciliate Tetrahymena thermophila.

Methylation of adenine in replicating and nonreplicating DNA of the ciliate Tetrahymena thermophila was examined. In growing cells, 87% of the methylation occurred on the newly replicated daughter strand, but methylation was also detectable on the parental strand. Methylation of nonreplicating DNA from starved cells was demonstrated.     

Restricted distribution and limited gene flow in the model ciliate Tetrahymena thermophila

The biogeography of microbial eukaryotes has long been debated, but few phylogeographic data have been available to assess whether protists tend to have ubiquitous or endemic distributions. We addressed this issue in the ciliate Tetrahymena thermophila, a highly successful model system in cell and molecular biology. We found that this species has a distribution that is restricted to the Eastern United States, with high diversity in the northeast and low diversity across the rest of its distribution. We find high levels of population subdivision, low rates of migration and significant isolation by distance, supporting the moderate endemicity model of protist biogeography. This restricted gene flow may be a result of small population size, which would reduce the probability of migration events, or the inability to establish after migration. This work lays the foundation for T. thermophila to become a valuable model system for studying population biology.     

Surface display of a parasite antigen in the ciliate Tetrahymena thermophila.

The ciliated protozoan, Tetrahymena thermophila, offers an attractive medium for the expression of heterologous proteins and could prove particularly useful for the display of foreign proteins on the cell surface. Although progress has been made in transformation of Tetrahymena with heterologous DNA, methods that permit reliable expression of foreign genes have been lacking. Using a mutant strain of T. thermophila carrying a negatively selectable allele of a beta-tubulin gene, we have been able to direct foreign genes to this locus by homologous recombination. Transformed cell lines producing foreign proteins were readily identified and, in at least one case, targeting of proteins to the plasma membrane was accomplished.     

Cdk3, a conjugation-specific cyclin-dependent kinase,is essential for the initiation of meiosis in Tetrahymena thermophila

Meiosis is an important process in sexual reproduction. Meiosis initiation has been found to be highly diverse among species. In yeast, it has been established that cyclin-dependent kinases (Cdks) and cyclins are essential components in the meiosis initiation pathway. In this study, we identified 4 Cdks in the model ciliate, Tetrahymena thermophila, and we found one of them, Cdk3, which is specifically expressed during early conjugation, to be essential for meiosis initiation. Cdk3 deletion led to arrest at the pair formation stage of conjugation. We then confirmed that Cdk3 acts upstream of double-strand break (DSB) formation. Moreover, we detected that Cdk3 is necessary for the expression of many genes involved in early meiotic events. Through proteomic quantification of phosphorylation, co-expression analysis and RNA-Seq analyses, we identified a conjugation-specific cyclin, Cyc2, which most likely partners with Cdk3 to initiate meiosis.     

The two domains of centrin have distinct basal body functions in Tetrahymena

The basal body is a microtubule-organizing center responsible for organizing the cilium, a structure important for cell locomotion and sensing of the surrounding environment. A widely conserved basal body component is the Ca(2+)-binding protein centrin. Analyses of centrin function suggest a role in basal body assembly and stability; however, its molecular mechanisms remain unclear. Here we describe a mutagenic strategy to study the function and essential nature of the various structural features of Cen1 in the ciliate Tetrahymena. We find that the two domains of Cen1 are both essential, and examination of strains containing mutant CEN1 alleles indicates that there are two predominant basal body phenotypes: misorientation of newly assembled basal bodies and stability defects. The results also show that the two domains of Cen1 are able to bind Ca(2+) and that perturbation of Ca(2+) binding affects Cen1 function. In all, the data suggest that the two domains of Cen1 have distinct functions.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Rotokinesis, a novel phenomenon of cell locomotion-assisted cytokinesis in the ciliate Tetrahymena thermophila

The mechanism responsible for final cell separation at the end of cytokinesis is currently unknown. Knockout strains of the ciliate, Tetrahymena thermophila lacking the kinesin-II homologous molecular motors, Kin1p and Kin2p are paralyzed due to their complete loss of cilia and undergo frequent cytokinesis failures. Observations of live dividing cells revealed that cleavage furrow ingression is normal in kinesin-II double knockout cells until the final stage of cell separation (Brown et al., 1999). During closer inspection of dividing cells using video differential interference contrast microscopy, we found that wild-type cells undergo an extremely complex motile behavior near the end of cytokinesis. This process, which we have named rotokinesis, appears to facilitate the physical separation of daughter cells. Here we present recent work onTetrahymena rotokinesis, and review studies in other organisms which suggest that the use of cell locomotion in the completion of cytokinesis is a general phenomenon of motile cell types.     

Experimental identification and analysis of macronuclear non-coding RNAs from the ciliate Tetrahymena thermophila

The ciliate Tetrahymena thermophila is an important eukaryotic model organism that has been used in pioneering studies of general phenomena, such as ribozymes, telomeres, chromatin structure and genome reorganization. Recent work has shown that Tetrahymena has many classes of small RNA molecules expressed during vegetative growth or sexual reorganization. In order to get an overview of medium-sized (40-500 nt) RNAs expressed from the Tetrahymena genome, we created a size-fractionated cDNA library from macronuclear RNA and analyzed 80 RNAs, most of which were previously unknown. The most abundant class was small nucleolar RNAs (snoRNAs), many of which are formed by an unusual maturation pathway. The modifications guided by the snoRNAs were analyzed bioinformatically and experimentally and many Tetrahymena-specific modifications were found, including several in an essential, but not conserved domain of ribosomal RNA. Of particular interest, we detected two methylations in the 5'-end of U6 small nuclear RNA (snRNA) that has an unusual structure in Tetrahymena. Further, we found a candidate for the first U8 outside metazoans, and an unusual U14 candidate. In addition, a number of candidates for new non-coding RNAs were characterized by expression analysis at different growth conditions.     

Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila.

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.     

Purification and partial characterization of glyceraldehyde-3-phosphate dehydrogenase from the ciliate Tetrahymena thermophila

In the present study, we purified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is involved in cellular energy production and has important housekeeping functions, from the ciliate Tetrahymena thermophila using a three-step procedure. The enzyme was purified ~68 folds by ammonium sulfate precipitation, followed by two steps of column chromatography (DEAE-cellulose and Mono-S). The purified enzyme is a homotetramer with a molecular weight of ~120 kDa. Isoelectric focusing analysis showed the presence of only one basic GAPDH isoform with an isoelectric point of 8.8. Western blot analysis showed a single 32-kDa band corresponding to the enzyme subunit using a monospecific polyclonal antibody against the T. thermophila GAPDH. The maximum of enzyme activity occurred at pH 8.0 and at 30-35°C. The apparent K(m) values for both NAD(+) and D-glyceraldehyde-3-phosphate were 0.102 ± 0.012 and 0.360 ± 0.018 mM, respectively. The maximal velocity (V(max)) was 39.40 ± 2.95 U/mg. The T. thermophila GAPDH is inhibited by oxidative and nitrosative stress reagents.     

Cellular responses of the ciliate, Tetrahymena thermophila, to far infrared irradiation.

Infrared rays from sunlight permeate the earth's atmosphere, yet little is known about their interactions with living organisms. To learn whether they affect cell structure and function, we tested the ciliated protozoan, Tetrahymena thermophila. These unicellular eukaryotes aggregate in swarms near the surface of freshwater habitats, where direct and diffuse solar radiation impinge upon the water-air interface. We report that populations irradiated in laboratory cultures grew and mated normally, but major changes occurred in cell physiology during the stationary phase. Early on, there were significant reductions in chromatin body size and the antibody reactivity of methyl groups on lysine residues 4 and 9 in histone H3. Later, when cells began to starve, messenger RNAs for key proteins related to chromatin structure, intermediary metabolism and cellular motility increased from two- to nearly nine-fold. Metabolic activity, swimming speed and linearity of motion also increased, and spindle shaped cells with a caudal cilium appeared. Our findings suggest that infrared radiation enhances differentiation towards a dispersal cell-like phenotype in saturated populations of Tetrahymena thermophila.     

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila

Summary A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.