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.

Entry and exit of proteins into flagella is gauged by CEP290 in the transition zone.

Terbufos (S-t-butylthiomethyl-O,O-diethyl phosphorodithioate) is a highly toxic organophosphate which is extensively used as an insecticide and nematicide. Chronic exposure to terbufos causes neuronal injury and predisposes to neurodegenerative diseases. Accumulating evidence has shown that the exposure to terbufos, as an occupational risk factor, may also cause reproductive disorders. However, the exact mechanisms of reproductive toxicity remain unclear. The present study aimed to investigate the toxic effect of terbufos on testicular cells and to explore the mechanism of toxicity on a cellular level. The cytotoxic effects of terbufos on mouse immortalized spermatogonia (GC-1), spermatocytes (GC-2), Leydig (TM3), and Sertoli (TM4) cell lines were assessed by MTT assays, caspase activation, flow cytometry, TUNEL assay, Western blot, and cell cycle analysis. The exposure to different concentrations of terbufos ranging from 50 to 800 μM for 6 h caused significant death in all the used testicular cell lines. Terbufos increased reactive oxygen species (ROS) production, reduced mitochondrial membrane potential, and initiated apoptosis, which was confirmed by a dose-dependent increase in the number of TUNEL-positive apoptotic cells. Blocking ROS production by N-acetyl cysteine (NAC) protected GC-1 cells from terbufos-induced cell death. The results demonstrated that terbufos induces ROS, apoptosis, and DNA damage in testicular cell lines and it should be considered potentially hazardous to testis. Together, this study provided potential molecular mechanisms of terbufos-induced toxicity in testicular cells and suggests a possible protective measure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1888-1898, 2016.

Intraflagellar transport (IFT) particles are multiprotein complexes that move bidirectionally along the cilium/flagellum. The Tetrahymena IFT172 gene encodes a protein with an N-terminal WD domain (WDD) and a C-terminal repeat domain (RPD). Epitope-tagged Ift172p localized to the basal body and in cilia along the axoneme, and IFT172 knockout cells lost cilia and motility. Using serial deletion constructs to rescue the knockout cells, we found that neither the WDD nor the RPD alone is sufficient to assemble cilia. Ift172p containing only the WDD or the RPD failed to enter cilia. Constructs with a partial truncation of the RPD still rescued although cilia were assembled less efficiently, indicating that the WDD and a part of the RPD are sufficient for anterograde transport. Partial truncation of the RPD caused the accumulation of truncated Ift172p itself and of Ift88p at ciliary tips, suggesting that IFT turnaround or retrograde transport was affected. These results implicate different regions of Ift172p in different steps of the IFT process. INTRODUCTIONCilia and flagella are microtubule (MT)-containing organelles protruding from the cell surface that generate repetitive beating motility and/or function as sensors (Sleigh, 1974;Rosenbaum and Witman, 2002). The scaffold of these organelles, the axoneme, contains nine sets of MT doublets arranged as a hollow cylinder. At the base of the axoneme is the basal body, a MT-organizing center that contains nine sets of MT triplets. The plus ends of the axonemal MTs are in the distal tip of the cilium and the minus ends are at the proximal region near the basal body. Because ribosomes are not detectable within cilia, and assembly occurs largely, if not entirely, at the ciliary tip, all of the proteins required to assemble the axoneme must be imported from the cell body and transported up the cilium (Johnson and Rosenbaum, 1992).Intraflagellar transport (IFT) is a bidirectional process that moves protein complexes (IFT particles) along the axoneme between the ciliary membrane and the outer doublet MTs (Kozminski et al., 1993). IFT is essential to build cilia (Pazour et al., 2000(Pazour et al., , 2002Brazelton et al., 2001;Deane et al., 2001;Haycraft et al., 2001Haycraft et al., , 2003Brown et al., 2003;Sun et al., 2004;Hou et al., 2007;Qin et al., 2007) and to regulate ciliary length (Marshall and Rosenbaum, 2001;Marshall et al., 2005). IFT machinery is also involved in cell signaling (Haycraft et al., 2005;Huangfu and Anderson, 2005;May et al., 2005;Wang et al., 2006) and cell cycle control Robert et al., 2007). Before entering cilia, IFT particles, MT motor proteins, and other cargos form complexes and dock at the basal body region (Deane et al., 2001;Iomini et al., 2001;Pedersen et al., 2006). Anterograde IFT, mediated by kinesin-2 (Lawrence et al., 2004), a heterotrimeric MT plus endmotor (Cole et al., 1993), moves these complexes from the cell body to the ciliary tip where axoneme assembly occurs (Kozminski et al., 1995;Pan et al., 2006). At the tip region, the IFT com...

We have used in vitro mutagenesis and gene replacement to study the function of the nucleotide-binding domain (NBD) of γ-tubulin in Tetrahymena thermophila. In this study, we show that the NBD has an essential function and that point mutations in two conserved residues lead to over-production and mislocalization of basal body (BB) assembly. These results, coupled with previous studies (Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and K. Oegema. 2004. Dev. Cell. 7:815–829; La Terra, S., C.N. English, P. Hergert, B.F. McEwen, G. Sluder, and A. Khodjakov. 2005. J. Cell Biol. 168:713–722), suggest that to achieve the precise temporal and spatial regulation of BB/centriole assembly, the initiation activity of γ-tubulin is normally suppressed by a negative regulatory mechanism that acts through its NBD.

Intraflagellar transport (IFT) moves multiple protein particles composed of two biochemically distinct complexes, IFT-A and IFT-B, bi-directionally within cilia and is essential for cilia assembly and maintenance. We identified an ORF from the Tetrahymena macronuclear genome sequence, encoding IFT122A, an ortholog of an IFT-A complex protein. Tetrahymena IFT122A is induced during cilia regeneration, and epitope-tagged Ift122Ap could be detected in isolated cilia. IFT122A knockout cells still assembled cilia, albeit with lower efficiency, and could regenerate amputated cilia. Ift172p and Ift88p, two IFT-B complex proteins that localized mainly to basal bodies and along the cilia in wild-type cells, became preferentially enriched at the ciliary tips in IFT122A knockout cells. Our results indicate that Tetrahymena IFT122A is not required for anterograde transport-dependent ciliary assembly but plays a role in returning IFT proteins from the ciliary tip to the cell body.

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 ACT1⌬ 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 ACT1⌬ 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 ACT1⌬ 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.Actin, although not abundant (26), has been detected at numerous intracellular sites within ciliates. These sites include the oral apparatus (8,21,29,41,45), phagosomes (food vacuoles) (8, 31), the cytoproct (8, 28), basal bodies (29,40,41,64), and cilia (41, 46, 64) of both Tetrahymena (reviewed in reference 15) and Paramecium, and the division furrow (22, 28) of Tetrahymena. However, despite the numerous places where actin has been found, its role in the life of these cells is incompletely understood.Perhaps the best-established function of ciliate actin is associated with phagocytosis, as cytochalasins and other drugs that affect actin polymerization (9) inhibit formation of food vacuoles in Tetrahymena pyriformis (49, 50), T. vorax (23), T. thermophila (72), and Paramecium tetraurelia (8,14). Cytochalasin B also binds to isolated oral apparatuses of T. thermophila (21), and inhibits the formation of this organelle, but does so at a much higher concentration than is sufficient to inhibit feeding (20). A recent study also has shown that latrunculin, an inhibitor of actin polymerization, interferes with the posteriorly directed movement of phagosomes within this cell (31).From past work it is unclear whether perturbing actin function interferes with cell division. Cytochalasin B did not prevent cell division of...

Previous methods to measure protozoan numbers mostly rely on manual counting, which suffers from high variation and poor efficiency. Although advanced counting devices are available, the specialized and usually expensive machinery precludes their prevalent utilization in the regular laboratory routine. In this study, we established the ImageJ-based workflow to quantify ciliate numbers in a high-throughput manner. We conducted Tetrahymena number measurement using five different methods: particle analyzer method (PAM), find maxima method (FMM), trainable WEKA segmentation method (TWS), watershed segmentation method (WSM) and StarDist method (SDM), and compared their results with the data obtained from the manual counting. Among the five methods tested, all of them could yield decent results, but the deep-learning-based SDM displayed the best performance for Tetrahymena cell counting. The optimized methods reported in this paper provide scientists with a convenient tool to perform cell counting for Tetrahymena ecotoxicity assessment.