Poc1 bridges basal body inner junctions to promote triplet microtubule integrity and connections.

Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT linkages which stabilize the structure. Poc1 is a conserved protein important for BB structural integrity in the face of ciliary forces transmitted to BBs. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. The full localization of the inner scaffold protein Fam161A requires Poc1. As ciliary forces are increased, Fam161A is reduced, indicative of a force-dependent molecular remodeling of the inner scaffold. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces.

Construction of Tetrahymena strains with highly active arsenic methyltransferase genes for arsenic detoxification in aquatic environments.

Biomethylation is an effective means of arsenic detoxification by organisms living in aquatic environments. Ciliated protozoa (including Tetrahymena species) play an important role in the biochemical cycles of aquatic ecosystems and have a potential application in arsenic biotransformation. This study compared arsenic tolerance, accumulation, methylation, and efflux in 11 Tetrahymena species. Nineteen arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferase (arsM) genes, of which 12 are new discoveries, were identified, and protein sequences were studied. We then constructed recombinant cell lines based on the Tetrahymena thermophila (T. thermophila) wild-type SB210 strain and expressed each of the 19 arsM genes under the control of the metal-responsive the MTT1 promoter. In the presence of Cd2+ and As(V), expression of the arsM genes in the recombinant cell lines was much higher than in the donor species. Evaluation of the recombinant cell line identified one with ultra-high arsenic methylation enzyme activity, significantly higher arsenic methylation capacity and much faster methylation rate than other reported arsenic methylated organisms, which methylated 89% of arsenic within 6.5 h. It also had an excellent capacity for the arsenic detoxification of lake water containing As(V), 56% of arsenic was methylated at 250 µg/L As(V) in 48 h. This study has made a significant contribution to our knowledge on arsenic metabolism in protozoa and demonstrates the great potential to use Tetrahymena species in the arsenic biotransformation of aquatic environments.

The three-dimensional structure of the Vint domain from Tetrahymena thermophila suggests a ligand-regulated cleavage mechanism by the HINT fold.

Vint proteins have been identified in unicellular metazoans as a novel hedgehog-related gene family, merging the von Willebrand factor type A domain and the Hedgehog/INTein (HINT) domains. We present the first three-dimensional structure of the Vint domain from Tetrahymena thermophila corresponding to the auto-processing domain of hedgehog proteins, shedding light on the unique features, including an adduct recognition region (ARR). Our results suggest a potential binding between the ARR and sulfated glycosaminoglycans like heparin sulfate. Moreover, we uncover a possible regulatory role of the ARR in the auto-processing by Vint domains, expanding our understanding of the HINT domain evolution and their use in biotechnological applications. Vint domains might have played a crucial role in the transition from unicellular to multicellular organisms.

Independent and Complementary Functions of Caf1b and Hir1 for Chromatin Assembly in Tetrahymena thermophila.

Histones and DNA associate to form the nucleosomes of eukaryotic chromatin. Chromatin assembly factor 1 (CAF-1) complex and histone regulatory protein A (HIRA) complex mediate replication-couple (RC) and replication-independent (RI) nucleosome assembly, respectively. CHAF1B and HIRA share a similar domain but play different roles in nucleosome assembly by binding to the different interactors. At present, there is limited understanding for the similarities and differences in their respective functions. Tetrahymena thermophila contains transcriptionally active polyploid macronuclei (MAC) and transcriptionally silent diploid micronuclei (MIC). Here, the distribution patterns of Caf1b and Hir1 exhibited both similarities and distinctions. Both proteins localized to the MAC and MIC during growth, and to the MIC during conjugation. However, Hir1 exhibited additional signaling on parental MAC and new MAC during sexual reproduction and displayed a punctate signal on developing anlagen. Caf1b and Hir1 only co-localized in the MIC with Pcna1 during conjugation. Knockdown of CAF1B impeded cellular growth and arrested sexual reproductive development. Loss of HIR1 led to MIC chromosome defects and aborted sexual development. Co-interference of CAF1B and HIR1 led to a more severe phenotype. Moreover, CAF1B knockdown led to the up-regulation of HIR1 expression, while knockdown of HIR1 also led to an increase in CAF1B expression. Furthermore, Caf1b and Hir1 interacted with different interactors. These results showed that CAF-1 and Hir1 have independent and complementary functions for chromatin assembly in T. thermophila.

Mismatch Repair Protein Msh6Tt Is Necessary for Nuclear Division and Gametogenesis in Tetrahymena thermophila.

DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in E. coli or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. Tetrahymena undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6Tt localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6Tt only localized in MICs and newly developing MACs. MSH6Tt knockout led to aberrant nuclear division during vegetative growth. The MSH6TtKO mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. MSH6Tt knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6Tt interacted with Msh2Tt and MMR-independent factors. Downregulation of MSH2Tt expression affected the stability of Msh6Tt. In addition, MSH6Tt knockout led to the upregulated expression of several MSH6Tt homologs at different developmental stages. Msh6Tt is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in Tetrahymena.

CEP104/FAP256 and associated cap complex maintain stability of the ciliary tip.

Cilia are essential organelles that protrude from the cell body. Cilia are made of a microtubule-based structure called the axoneme. In most types of cilia, the ciliary tip is distinct from the rest of the cilium. Here, we used cryo-electron tomography and subtomogram averaging to obtain the structure of the ciliary tip of the ciliate Tetrahymena thermophila. We show that the microtubules at the tip are highly crosslinked with each other and stabilized by luminal proteins, plugs, and cap proteins at the plus ends. In the tip region, the central pair lacks typical projections and twists significantly. By analyzing cells lacking a ciliary tip-enriched protein CEP104/FAP256 by cryo-electron tomography and proteomics, we discovered candidates for the central pair cap complex and explained the potential functions of CEP104/FAP256. These data provide new insights into the function of the ciliary tip and the mechanisms of ciliary assembly and length regulation.

Recombinant production of hormonally active human insulin from pre-proinsulin by Tetrahymena thermophila.

Alternative cell factories, such as the unicellular ciliate eukaryotic Tetrahymena thermophila, may be required for the production of protein therapeutics that are challenging to produce in conventional expression systems. T. thermophila (Tt) can secrete proteins with the post-translational modifications necessary for their function in humans. In this study, we tested if T. thermophila could process the human pre-proinsulin to produce hormonally active human insulin (hINS) with correct modifications. Flask and bioreactor culture of T. thermophila were used to produce the recombinant Tt-hINS either with or without an affinity tag from a codon-adapted pre-proinsulin sequence. Our results indicate that T. thermophila can produce a 6 kDa Tt-hINS monomer with the appropriate disulfide bonds after removal of the human insulin signal sequence or endogenous phospholipase A signal sequence, and the C-peptide of the human insulin. Additionally, Tt-hINS can form 12 kDa dimeric, 24 kDa tetrameric, and 36 kDa hexameric complexes. Tt-hINS-sfGFP fusion protein was localized to the vesicles within the cytoplasm and was secreted extracellularly. Assessing the affinity-purified Tt-hINS activity using the in vivo T. thermophila extracellular glucose drop assay, we observed that Tt-hINS induced a significant reduction (approximately 21 %) in extracellular glucose levels, indicative of its functional insulin activity. Our results demonstrate that T. thermophila is a promising candidate for the pharmaceutical and biotechnology industries as a host organism for the production of human protein drugs.

Mismatch Repair Protein Msh2 Is Necessary for Macronuclear Stability and Micronuclear Division in Tetrahymena thermophila.

Mismatch repair (MMR) is a conserved mechanism that is primarily responsible for the repair of DNA mismatches during DNA replication. Msh2 forms MutS heterodimer complexes that initiate the MMR in eukaryotes. The function of Msh2 is less clear under different chromatin structures. Tetrahymena thermophila contains a transcriptionally active macronucleus (MAC) and a transcriptionally silent micronucleus (MIC) in the same cytoplasm. Msh2 is localized in the MAC and MIC during vegetative growth. Msh2 is localized in the perinuclear region around the MIC and forms a spindle-like structure as the MIC divides. During the early conjugation stage, Msh2 is localized in the MIC and disappears from the parental MAC. Msh2 is localized in the new MAC and new MIC during the late conjugation stage. Msh2 also forms a spindle-like structure with a meiotic MIC and mitotic gametic nucleus. MSH2 knockdown inhibits the division of MAC and MIC during vegetative growth and affects cellular proliferation. MSH2 knockdown mutants are sensitive to cisplatin treatment. MSH2 knockdown also affects micronuclear meiosis and gametogenesis during sexual development. Furthermore, Msh2 interacts with MMR-dependent and MMR-independent factors. Therefore, Msh2 is necessary for macronuclear stability, as well as micronuclear mitosis and meiosis in Tetrahymena.

Tubulysins are Essential for the Preying of Ciliates by Myxobacteria.

Tubulysins are bioactive secondary metabolites produced by myxobacteria that promote microtubule disassembly. Microtubules are required for protozoa such as Tetrahymena to form cilia and flagella. To study the role of tubulysins in myxobacteria, we co-cultured myxobacteria and Tetrahymena. When 4000 Tetrahymena thermophila and 5.0 × 108 myxobacteria were added to 1 ml of CYSE medium and co-cultured for 48 h, the population of T. thermophila increased to more than 75,000. However, co-culturing tubulysin-producing myxobacteria, including Archangium gephyra KYC5002, with T. thermophila caused the population of T. thermophila to decrease from 4000 to less than 83 within 48 h. Almost no dead bodies of T. thermophila were observed in the culture medium. Co-culturing of T. thermophila and the A. gephyra KYC5002 strain with inactivation of the tubulysin biosynthesis gene led to the population of T. thermophila increasing to 46,667. These results show that in nature, most myxobacteria are preyed upon by T. thermophila, but some myxobacteria prey on and kill T. thermophila using tubulysins. Adding purified tubulysin A to T. thermophila changed the cell shape from ovoid to spherical and caused cell surface cilia to disappear.

A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila.

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.

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.

A split ribozyme that links detection of a native RNA to orthogonal protein outputs.

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we use laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We further resolve a thermodynamic model to guide RENDR design, show how input signals can be transduced into diverse outputs, demonstrate portability across different bacteria, and use RENDR to detect antibiotic-resistant bacteria. This work shows how transcriptional signals can be monitored in situ and converted into different types of biochemical information using RNA synthetic biology.

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.

Glyphosate metabolism in Tetrahymena thermophila: A shotgun proteomic analysis approach.

Glyphosate is one of the most widely used herbicides in the world. However, because of its overuse and resistance to degradation, high levels of glyphosate residues in the environment are reported. Therefore, this study aimed to investigate the effects of glyphosate on proteomic aspects of Tetrahymena thermophila and their uses as bioindicators of freshwater ecosystem. First, an acute toxicity test was performed to determine the median inhibition concentration (IC50 ). The toxicity test results showed that glyphosate inhibited the growth (proliferation) of T. thermophila. The 96 h-IC50 value of glyphosate was 171 mg L-1 . No visible changes in aggregation behavior and cell morphology were observed under glyphosate exposure. In addition, the effects of low and high dose glyphosate concentrations (77.5 mg L-1 , 171 mg L-1 ) on the proteomic changes of T. thermophila was investigated using a label-free shotgun proteomic approach. A total of 3191 proteins were identified, 2791 proteins were expressed in the control, 2651 proteins were expressed in 77.5 mg L-1 glyphosates, and 3012 proteins were expressed in 171 mg L-1 glyphosates. Under glyphosate exposure at both low and high dose glyphosate, 400 unique proteins were upregulated. The majority of these proteins was classified as proteins associated with oxidative stress response and intracellular transport indicating the shifts in the internal metabolism. Proteomics revealed that the glyphosate metabolism by T. thermophila is a multi-step process involving several enzymes, which can be divided into four phases, including modification (phase I), conjugation (phase II), transport (phase III), and degradation (phase IV). The accumulation of various biochemical reactions contributes to overall glyphosate resistance. With the proteomics approach, we have found that T. thermophila was equipped with glyphosate detoxification and degradation mechanisms.

Metabolic alteration of Tetrahymena thermophila exposed to CdSe/ZnS quantum dots to respond to oxidative stress and lipid damage.

CdSe/ZnS Quantum dots (QDs) are possibly released to surface water due to their extensive application. Based on their high reactivity, even small amounts of toxicant QDs will disturb water microbes and pose a risk to aquatic ecology. Here, we evaluated CdSe/ZnS QDs toxicity to Tetrahymena thermophila (T. thermophila), a model organism of the aquatic environment, and performed metabolomics experiments. Before the omics experiment was conducted, QDs were found to induce inhibition of cell proliferation, and reactive oxygen species (ROS) production along with Propidium iodide labeled cell membrane damage indicated oxidative stress stimulation. In addition, mitochondrial ultrastructure alteration of T. thermophila was also confirmed by Transmission Electron Microscope results after 48 h of exposure to QDs. Further results of metabolomics detection showed that 0.1 µg/mL QDs could disturb cell physiological and metabolic metabolism characterized by 18 significant metabolite changes, of which twelve metabolites improved and three decreased significantly compared to the control. Kyoto Encyclopedia of Genes and Genomes analysis showed that these metabolites were involved in the ATP-binding cassette transporter and purine metabolism pathways, both of which respond to ROS-induced cell membrane damage. In addition, purine metabolism weakness might also reflect mitochondrial dysfunction associated with energy metabolism and transport abnormalities. This research provides deep insight into the potential risks of quantum dots in aquatic ecosystems.

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.

Establishing a High-Throughput Locomotion Tracking Method for Multiple Biological Assessments in Tetrahymena.

Protozoa are eukaryotic, unicellular microorganisms that have an important ecological role, are easy to handle, and grow rapidly, which makes them suitable for ecotoxicity assessment. Previous methods for locomotion tracking in protozoa are largely based on software with the drawback of high cost and/or low operation throughput. This study aimed to develop an automated pipeline to measure the locomotion activity of the ciliated protozoan Tetrahymena thermophila using a machine learning-based software, TRex, to conduct tracking. Behavioral endpoints, including the total distance, velocity, burst movement, angular velocity, meandering, and rotation movement, were derived from the coordinates of individual cells. To validate the utility, we measured the locomotor activity in either the knockout mutant of the dynein subunit DYH7 or under starvation. Significant reduction of locomotion and alteration of behavior was detected in either the dynein mutant or in the starvation condition. We also analyzed how Tetrahymena locomotion was affected by the exposure to copper sulfate and showed that our method indeed can be used to conduct a toxicity assessment in a high-throughput manner. Finally, we performed a principal component analysis and hierarchy clustering to demonstrate that our analysis could potentially differentiate altered behaviors affected by different factors. Taken together, this study offers a robust methodology for Tetrahymena locomotion tracking in a high-throughput manner for the first time.

Structural basis for MTA1c-mediated DNA N6-adenine methylation.

DNA N6-adenine methylation (6 mA) has recently been found to play a crucial role in epigenetic regulation in eukaryotes. MTA1c, a newly discovered 6 mA methyltransferase complex in ciliates, is composed of MTA1, MTA9, p1 and p2 subunits and specifically methylates ApT dinucleotides, yet its mechanism of action remains unknown. Here, we report the structures of Tetrahymena thermophila MTA1 (TthMTA1), Paramecium tetraurelia MTA9 (PteMTA9)-TthMTA1 binary complex, as well as the structures of TthMTA1-p1-p2 and TthMTA1-p2 complexes in apo, S-adenosyl methionine-bound and S-adenosyl homocysteine-bound states. We show that MTA1 is the catalytically active subunit, p1 and p2 are involved in the formation of substrate DNA-binding channel, and MTA9 plays a structural role in the stabilization of substrate binding. We identify that MTA1 is a cofactor-dependent catalytically active subunit, which exhibits stable SAM-binding activity only after assembly with p2. Our structures and corresponding functional studies provide a more detailed mechanistic understanding of 6 mA methylation.

Molecular evolutionary analysis of the SM and SNARE vesicle fusion machinery in ciliates shows concurrent expansions in late secretory machinery.

Protists in the phylum Ciliophora possess a complex membrane-trafficking system, including osmoregulatory Contractile Vacuoles and specialized secretory organelles. Molecular cell biological investigations in Tetrahymena thermophila have identified components of the protein machinery associated with the secretory organelles, mucocysts. The Qa-SNARE Syn7lp plays a role in mucocyst biogenesis as do subunits of the CORVET tethering complex (specifically Vps8). Indeed, Tetrahymena thermophila possesses expanded gene complements of several CORVET components, including Vps33 which is also a Sec1/Munc18 (SM) protein that binds Qa-SNAREs. Moreover, the Qa-SNAREs in Paramecium tetraurelia have been localized to various endomembrane organelles. Here, we use comparative genomics and phylogenetics to determine the evolutionary history of the SM and Qa-SNARE proteins across the Ciliophora. We identify that the last ciliate common ancestor possessed the four SM proteins and six Qa-SNAREs common to eukaryotes, including the uncommonly retained Syntaxin 17. We furthermore identify independent expansion of these protein families in several ciliate classes, including concurrent expansions of the SM protein-Qa SNARE partners Sec1:SynPM in the oligohymenophorean ciliates lineage, consistent with novel Contractile Vacuole specific innovations. Overall, these data are consistent with SM proteins and Qa-SNAREs being a common set of components for endomembrane modulation in the ciliates.

Zfp1, a Cys2His2 zinc finger protein is required for meiosis initiation in Tetrahymena thermophila.

Meiosis is an important and highly conserved process that occurs during eukaryotic sexual reproduction. Diverse mechanisms are responsible for meiosis initiation among eukaryotes, and transcription factors have been established to have an important role in many species. However, the specific function of transcription factors in initiating meiosis in ciliates is unknown. Here we show that a putative Cys2His2 zinc finger-containing transcription factor encoded by the ZFP1 gene is specifically expressed during sexual reproduction in Tetrahymena thermophila. Meiosis is not initiated in the cells lacking ZFP1. Transcriptome sequencing analyses reveal that Zfp1 is required for the expression of many meiosis-specific genes. Our results indicate that Zfp1 could be a transcriptional activator required for meiosis initiation in T. thermophila.