Identification of a Tetrahymena species infecting guppies, pathology, and expression of beta-tubulin during infection.

Tetrahymenosis is caused by the ciliated protozoan Tetrahymena and is responsible for serious economic losses to the aquaculture industry worldwide. However, information regarding the molecular mechanism leading to tetrahymenosis is limited. In previous transcriptome sequencing work, it was found that one of the two ß-tubulin genes in T. pyriformis was significantly expressed in infected fish, we speculated that ß-tubulin is involved in T. pyriformis infecting fish. Herein, the potential biological function of the ß-tubulin gene in Tetrahymena species when establishing infection in guppies was investigated by cloning the full-length cDNA of this T. pyriformis ß-tubulin (BTU1) gene. The full-length cDNA of T. pyriformis BTU1 gene was 1873 bp, and the ORF occupied 1134 bp, whereas 5' UTR 434 bp, and 3' UTR 305 bp whose poly (A) tail contained 12 bases. The predicted protein encoded by T. pyriformis BTU1 gene had a calculated molecular weight of 42.26 kDa and pI of 4.48. Moreover, secondary structure analysis and tertiary structure prediction of BTU1 protein were also conducted. In addition, morphology, infraciliature, phylogeny, and histopathology of T. pyriformis isolated from guppies from a fish market in Harbin were also investigated. Furthermore, qRT-PCR analysis and experimental infection assays indicated that the expression of BTU1 gene resulted in efficient cell proliferation during infection. Collectively, our data revealed that BTU1 is a key gene involved in T. pyriformis infection in guppies, and the findings discussed herein provide valuable insights for future studies on tetrahymenosis.

Global and local functions of the Fused kinase ortholog CdaH in intracellular patterning in Tetrahymena.

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning.

Tracking Native Tetrahymena Ribozyme Folding with Fluorescence.

Folding of the Tetrahymena group I intron ribozyme and other structured RNAs has been measured using a catalytic activity assay to monitor the native state formation by cleavage of a radiolabeled oligonucleotide substrate. While highly effective, the assay has inherent limitations present in any radioactivity- and gel-based assay. Administrative and safety considerations arise from the radioisotope, and data collection is laborious due to the use of polyacrylamide gels. Here we describe a fluorescence-based, solution assay that allows for more efficient data acquisition. The substrate is labeled with 6-carboxyfluorescein (6FAM) fluorophore and black hole quencher (BHQ1) at the 5' and 3' ends, respectively. Substrate cleavage results in release of the quencher, increasing the fluorescence signal by an average of 30-fold. A side-by-side comparison with the radioactivity-based assay shows good agreement in monitoring Tetrahymena ribozyme folding from a misfolded conformation to the native state, albeit with increased uncertainty. The lower precision of the fluorescence assay is compensated for by the relative ease and efficiency of the workflow. In addition, this assay will allow institutions that do not use radioactive materials to monitor native folding of the Tetrahymena ribozyme, and the same strategy should be amenable to native folding of other ribozymes.

Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications.

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins.

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 42 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.

Snapshots of the second-step self-splicing of Tetrahymena ribozyme revealed by cryo-EM.

Group I introns are catalytic RNAs that coordinate two consecutive transesterification reactions for self-splicing. To understand how the group I intron promotes catalysis and coordinates self-splicing reactions, we determine the structures of L-16 Tetrahymena ribozyme in complex with a 5'-splice site analog product and a 3'-splice site analog substrate using cryo-EM. We solve six conformations from a single specimen, corresponding to different splicing intermediates after the first ester-transfer reaction. The structures reveal dynamics during self-splicing, including large conformational changes of the internal guide sequence and the J5/4 junction as well as subtle rearrangements of active-site metals and the hydrogen bond formed between the 2'-OH group of A261 and the N2 group of guanosine substrate. These results help complete a detailed structural and mechanistic view of this paradigmatic group I intron undergoing the second step of self-splicing.

Two Predators, One Prey - the Interaction Between Bacteriophage, Bacterivorous Ciliates, and Escherichia coli.

Bacterivorous ciliates and lytic bacteriophages are two major predators in aquatic environments, competing for the same type of prey. This study investigated the possible interaction of these different microorganisms and their influence on the activity of each other. Therefore, two bacterivorous ciliates, Paramecium sp. RB1 and Tetrahymena sp. RB2, were used as representative ciliates; a T4-like Escherichia coli targeting lytic bacteriophage as a model virus; and E. coli ATCC 25922 as a susceptible bacterial host and prey. The growth of the two ciliates with E. coli ATCC 25922 as prey was affected by the presence of phage particles. The grazing activity of the two ciliates resulted in more than a 99% reduction of the phage titer and bacterial cell numbers. However, viable phage particles were recovered from individual washed cells of the two ciliates after membrane filtration. Therefore, ciliates such as Paramecium sp. RB1 and Tetrahymena sp. RB2 can remove bacteriophages present in natural and artificial waters by ingesting the viral particles and eliminating bacterial host cells required for viral replication. The ingestion of phage particles may marginally contribute to the nutrient supply of the ciliates. However, the interaction of phage particles with ciliate cells may contribute to the transmission of bacteriophages in aquatic environments.

Basal body organization and cell geometry during the cell cycle in Tetrahymena thermophila.

Tetrahymena thermophila possesses arrays of motile cilia that promote fluid flow for cell motility. These consist of intricately organized basal bodies (BBs) that nucleate and position cilia at the cell cortex. Tetrahymena cell geometry and spatial organization of BBs play important roles in cell size, swimming, feeding, and division. How cell geometry and BB organization are established and maintained remains poorly understood, and prior studies have been limited due to difficulties in accurate BB identification and small sample size. We therefore developed an automated image processing pipeline that segments single cells, distinguishes unique BB populations, assigns BBs into distinct ciliary rows, and distinguishes new from mature BBs. We identified unique features to describe the variation of cell shape and BB spatial organization in unsynchronized single-cell images. The results reveal asymmetries in BB distribution and ingression of the cytokinetic furrow within the cell. Moreover, we establish novel spatial and temporal waves in new BB assembly through the cell cycle. Finally, we used measurements from single cells across the cell cycle to construct a generative model that allows synthesis of movies depicting single cells progressing through the cell cycle. Our approach is expected to be of particular value for characterizing Tetrahymena mutants.

Snapshots of the first-step self-splicing of Tetrahymena ribozyme revealed by cryo-EM.

Tetrahymena ribozyme is a group I intron, whose self-splicing is the result of two sequential ester-transfer reactions. To understand how it facilitates catalysis in the first self-splicing reaction, we used cryogenic electron microscopy (cryo-EM) to resolve the structures of L-16 Tetrahymena ribozyme complexed with a 11-nucleotide 5'-splice site analog substrate. Four conformations were achieved to 4.14, 3.18, 3.09 and 2.98 Å resolutions, respectively, corresponding to different splicing intermediates during the first enzymatic reaction. Comparison of these structures reveals structural alterations, including large conformational changes in IGS/IGSext (P1-P1ext duplex) and J5/4, as well as subtle local rearrangements in the G-binding site. These structural changes are required for the enzymatic activity of the Tetrahymena ribozyme. Our study demonstrates the ability of cryo-EM to capture dynamic RNA structural changes, ushering in a new era in the analysis of RNA structure-function by cryo-EM.

Molecular identification of Tetrahymena species.

Mitochondrial cox1 689 bp barcodes are routinely used for identification of Tetrahymena species. Here, we examine whether two shorter nuclear sequences, the 5.8S rRNA gene region and the intergenic region between H3 and H4 histone genes, might also be useful either singly or in combination with each other or cox1. We obtained sequences from ~300 wild isolates deposited at the Tetrahymena Stock Center and analyzed additional sequences obtained from GenBank. The 5.8S rRNA gene and portions of its transcribed flanks identify isolates as to their major clade and uniquely identify some, but not all, species. The ~330 bp H3/H4 intergenic region possesses low intraspecific variability and is unique for most species. However, it fails to distinguish between two pairs of common species and their rarer counterparts, and its use is complicated by the presence of duplicate genes in some species. The results show that while the cox1 sequence is the best single marker for Tetrahymena species identification, 5.8S rRNA, and the H3/H4 intergenic regions sequences are useful, singly or in combination, to confirm cox1 species assignments or as part of a preliminary survey of newly collected Tetrahymena. From our newly collected isolates, the results extend the biogeographical range of T. shanghaiensis and T. malaccensis and identify a new species, Tetrahymena arleneae n. sp. herein described.

Transcriptome analysis of the binucleate ciliate Tetrahymena thermophila with asynchronous nuclear cell cycles.

Tetrahymena thermophila harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the "cell cycles" of the diploid micronucleus and polyploid macronucleus are offset. Micronuclear S phase initiates just before cytokinesis and is completed in daughter cells before onset of macronuclear DNA replication. Mitotic micronuclear division occurs mid-cell cycle, while macronuclear amitosis is coupled to cell division. Here we report the first RNA-seq cell cycle analysis of a binucleated ciliated protozoan. RNA was isolated across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the 26,000+ predicted genes were shown to be cell cycle regulated. Proteins present in both nuclei exhibit a single mRNA peak that always precedes their macronuclear function. Nucleus-limited genes, including nucleoporins and importins, are expressed before their respective nucleus-specific role. Cyclin D and A/B gene family members exhibit different expression patterns that suggest nucleus-restricted roles. Periodically expressed genes cluster into seven cyclic patterns. Four clusters have known PANTHER gene ontology terms associated with G1/S and G2/M phase. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication, and cell division.

Tethering CST to the telomerase catalytic core-the p50 linchpin.

Emerging models of telomere and associated proteins in Tetrahymena have broadened our understanding of telomeric processes. In this issue of Structure, Ma et al. pinpoint a region in p50 that associates with the CST complex. The interaction mediated by p50 is crucial for the optimal positioning of TERT to maintain homeostasis at the chromosome ends.

Cfap91-Dependent Stability of the RS2 and RS3 Base Proteins and Adjacent Inner Dynein Arms in Tetrahymena Cilia.

Motile cilia and eukaryotic flagella are specific cell protrusions that are conserved from protists to humans. They are supported by a skeleton composed of uniquely organized microtubules-nine peripheral doublets and two central singlets (9 × 2 + 2). Microtubules also serve as docking sites for periodically distributed multiprotein ciliary complexes. Radial spokes, the T-shaped ciliary complexes, repeat along the outer doublets as triplets and transduce the regulatory signals from the cilium center to the outer doublet-docked dynein arms. Using the genetic, proteomic, and microscopic approaches, we have shown that lack of Tetrahymena Cfap91 protein affects stable docking/positioning of the radial spoke RS3 and the base of RS2, and adjacent inner dynein arms, possibly due to the ability of Cfap91 to interact with a molecular ruler protein, Ccdc39. The localization studies confirmed that the level of RS3-specific proteins, Cfap61 and Cfap251, as well as RS2-associated Cfap206, are significantly diminished in Tetrahymena CFAP91-KO cells. Cilia of Tetrahymena cells with knocked-out CFAP91 beat in an uncoordinated manner and their beating frequency is dramatically reduced. Consequently, CFAP91-KO cells swam about a hundred times slower than wild-type cells. We concluded that Tetrahymena Cfap91 localizes at the base of radial spokes RS2 and RS3 and likely plays a role in the radial spoke(s) positioning and stability.

Moderate activity of RNA chaperone maximizes the yield of self-spliced pre-RNA in vivo.

CYT-19 is a DEAD-box protein whose adenosine-triphosphate (ATP)-dependent helicase activity facilitates the folding of group I introns in precursor RNA (pre-RNA) of Neurospora crassa (N. crassa). In the process, they consume a substantial amount of ATP. While much of the mechanistic insight into CYT-19 activity has been gained through the studies on the folding of Tetrahymena group I intron ribozyme, the more biologically relevant issue, namely the effect of CYT-19 on the self-splicing of pre-RNA, remains largely unexplored. Here, we employ a kinetic network model, based on the generalized iterative annealing mechanism (IAM), to investigate the relation between CYT-19 activity, rate of ribozyme folding, and the kinetics of the self-splicing reaction. The network rate parameters are extracted by analyzing the recent biochemical data for CYT-19-facilitated folding of Tetrahymena ribozyme. We then build extended models to explore the metabolism of pre-RNA. We show that the timescales of chaperone-mediated folding of group I ribozyme and self-splicing reaction compete with each other. As a consequence, in order to maximize the self-splicing yield of group I introns in pre-RNA, the chaperone activity must be sufficiently large to unfold the misfolded structures, but not too large to unfold the native structures prior to the self-splicing event. We discover that despite the promiscuous action on structured RNAs, the helicase activity of CYT-19 on group I ribozyme gives rise to self-splicing yields that are close to the maximum.

Torque generating properties of Tetrahymena ciliary three-headed outer-arm dynein.

Eukaryotic cilia/flagella are cellular bio-machines that drive the movement of microorganisms. Molecular motor axonemal dyneins in the axoneme, which consist of an 9 + 2 arrangement of microtubules, play an essential role in ciliary beating. Some axonemal dyneins have been shown to generate torque coupled with the longitudinal motility of microtubules across an array of dyneins fixed to the coverglass surface, resulting in a corkscrew-like translocation of microtubules. In this study, we performed three-dimensional tracking of a microbead coated with axonemal outer-arm dyneins on a freely suspended microtubule. We found that microbeads coated with multiple outer-arm dyneins exhibited continuous right-handed helical trajectories around the microtubule. This unidirectional helical motion differs from that of other types of cytoplasmic dyneins, which exhibit bidirectional helical motility. We also found that, in an in vitro microtubule gliding assay, gliding microtubules driven by outer-arm dyneins tend to turn to the left, causing a curved path, suggesting that the outer-arm dynein itself is able to rotate on its own axis. Two types of torque generated by the axonemal dyneins, corresponding to the forces used to rotate the microtubule unidirectionally with respect to the long and short axes, may regulate ciliary beating with complex waveforms.

Topological crossing in the misfolded Tetrahymena ribozyme resolved by cryo-EM.

The Tetrahymena group I intron has been a key system in the understanding of RNA folding and misfolding. The molecule folds into a long-lived misfolded intermediate (M) in vitro, which has been known to form extensive native-like secondary and tertiary structures but is separated by an unknown kinetic barrier from the native state (N). Here, we used cryogenic electron microscopy (cryo-EM) to resolve misfolded structures of the Tetrahymena L-21 ScaI ribozyme. Maps of three M substates (M1, M2, M3) and one N state were achieved from a single specimen with overall resolutions of 3.5 Å, 3.8 Å, 4.0 Å, and 3.0 Å, respectively. Comparisons of the structures reveal that all the M substates are highly similar to N, except for rotation of a core helix P7 that harbors the ribozyme's guanosine binding site and the crossing of the strands J7/3 and J8/7 that connect P7 to the other elements in the ribozyme core. This topological difference between the M substates and N state explains the failure of 5'-splice site substrate docking in M, supports a topological isomer model for the slow refolding of M to N due to a trapped strand crossing, and suggests pathways for M-to-N refolding.

PIWI-Directed DNA Elimination for Tetrahymena Genetics.

Piwi-bound small RNAs induce programmed DNA elimination in the ciliated protozoan Tetrahymena. Using the phenomenon called codeletion, this process can be reprogrammed to induce ectopic DNA elimination at basically any given genomic location. Here, we describe the usage of codeletion for genetic studies in Tetrahymena and for investigations of the molecular mechanism of Piwi-directed programmed DNA elimination.

Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN21,2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand3 and TPP1 (in complex with TIN24) recruiting telomerase via interaction with telomerase reverse transcriptase5 (TERT). The telomere DNA ends are replicated and maintained by telomerase6, for the G-strand, and subsequently DNA polymerase α-primase7,8 (PolαPrim), for the C-strand9. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN110-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.

Box-shaped ribozyme octamer formed by face-to-face dimerization of a pair of square-shaped ribozyme tetramers.

Naturally occurring ribozymes with defined three-dimensional (3D) structures serve as promising platforms for the design and construction of artificial RNA nanostructures. We constructed a hexameric ribozyme nanostructure by face-to-face dimerization of a pair of triangular ribozyme trimers, unit RNAs of which were derived from the Tetrahymena group I ribozyme. In this study, we have expanded the dimerization strategy to a square-shaped ribozyme tetramer by introducing four pillar units. The resulting box-shaped nanostructures, which contained eight ribozyme units, can be assembled from either four or two components of their unit RNAs.

Performance Comparison of Five Methods for Tetrahymena Number Counting on the ImageJ Platform: Assessing the Built-in Tool and Machine-Learning-Based Extension.

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.