Tetrahymena Research Articles

Lysosome-related organelles (LROs) are a heterogeneous family of organelles found in many cell types, whose similarities to lysosomes include acidification by vacuolar-type ATPases (V-ATPases). However, some organelles with hallmarks of LROs are nonetheless non-acidic. Here we investigate this phenomenon using the ciliate Tetrahymena thermophila, which have secretory LROs called mucocysts. Using three approaches, we show that mature mucocysts, poised for exocytosis, are non-acidic. However, mucocysts forming in the cytoplasm are acidic, and a specific V-ATPase a-subunit is present and indispensable for mucocyst biogenesis. In the absence of this subunit, cells show defects in at least two features of mucocyst formation, namely heterotypic vesicle fusion of mucocyst precursors, and proprotein processing. The stage specificity of acidification can be explained by our finding that several other canonical V-ATPase subunits are present in the forming, but not mature, mucocysts. Our data argue that a specific V-ATPase complex is targeted to newly-forming, immature mucocysts and subsequently disassembles at a later stage in the maturation pathway.

Lipid droplets are increasingly recognized as necessary organelles. However, the cellular pathways that regulate lipid droplets have only been defined in select fungi, algae, plants, and animals. Our experiments expand the study of lipid droplets to an evolutionarily distinct model organism, the ciliate Tetrahymena thermophila. We identify conserved pathways that promote lipid droplet homeostasis while also uncovering features that suggest adaptation. We show that Tetrahymena accumulate lipid droplets in response to nutrient deprivation, including starvation and the stationary phase. Pulse-chase experiments with a fluorescent fatty acid analogue demonstrate lipid trafficking to lipid droplets in starved cultures. Unlike other cell types, starved Tetrahymena appear to use both peroxisomes and mitochondria (not vacuoles) for further fatty acid catabolism. We observe cooccurence of the fluorescent fatty acid analogue with markers of peroxisomes and a subpopulation of mitochondria, suggesting specialized catabolic roles for both organelles. We demonstrate a decrease in survival following starvation in the presence of inhibitors of mitochondrial fatty acid import or peroxisomal fatty acid metabolism. Together, our experiments add Tetrahymena to the expanding list of eukaryotes that increase lipid droplets in response to nutrient depletion while also uncovering important and distinct roles for mitochondrial and peroxisomal catabolism in survival pathways.

The human pathogen Campylobacter jejuni can be packaged within multilamellar bodies (MLB), also called fecal pellets, produced by ciliates such as Tetrahymena pyriformis when these microorganisms are co-cultivated. This packaging increases the survival of C. jejuni in oxygenic conditions and potentially protects it against other stressors. Traditional methods for detecting and quantifying these pellets, such as transmission electron microscopy and fluorescence microscopy, are time-consuming and labor intensive. In this study, we devised an approach for utilizing flow cytometry to distinguish and quantify C. jejuni-containing pellets produced by both T. pyriformis and T. thermophila. Co-cultures of each Tetrahymena species with four different C. jejuni strains, along with monoculture controls, were incubated for 24 hours, stained with SYTO9 and analyzed using flow cytometry. The results revealed ciliate species-specific and bacterial strain-specific differences in the number of pellets and their fluorescence intensity. Transmission electron microscopy (TEM) confirmed that this variability in fluorescence corresponds to differences in the number of bacteria per pellet. Our method provides a rapid and efficient means of quantifying bacteria-containing MLBs, which would facilitate the screening and comparison of a large quantity of C. jejuni strains and different conditions for studying the packaging of C. jejuni by ciliates.

Wild aquatic birds are a major reservoir of the influenza A virus in natural ecosystems, facilitating its entry into the aquatic microbial food web through their feces. Free-living protozoa and particularly bacterivorous ciliates are essential players of the microbial food web. This study investigates the interactions between the Influenza A(H1N1)pdm09 virus and the ciliated protozoan Tetrahymena pyriformis at the population and ultrastructural levels. Co-cultivation of Influenza A(H1N1)pdm09 and T. pyriformis resulted in a decline and eventual complete elimination of the viral population. The inactivation of the virus was not mediated by products excreted by T. pyriformis but required A(H1N1)pdm09 endocytosis. Viruses ingested by protozoa lost their virulence within 48 hours post infection (hpi) and, as determined by hemagglutination assays, were entirely inactivated within 72 hpi. When lysates infected with A(H1N1)pdm09 T. pyriformis were applied to MDCK cells 1.5 and 24 hpi the undamaged part of ingested virions caused a cytopathic effect. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) of infected T. pyriformis cells revealed large food vacuoles, including multiple undamaged and partly processed virus particles, at 1.5 and 24 hpi. Furthermore, TEM identified coated and half-coated small one-virus endosomes that predominated at 48 hpi. These results demonstrated that A(H1N1)pdm09 inactivation by T. pyriformis includes two types of endosomes that dominated at different periods of interpopulation interactions. The process of A(H1N1)pdm09 inactivation in protozoan cells occurs rapidly, but not instantaneously, that suggesting a dual role of protozoa in the fate of influenza A viruses in natural ecosystems, both as predators and as potential vectors.

The nexin-dynein regulatory complex (N-DRC) is an essential axonemal structure for ciliary and flagellar motility. Coiled-coil domain containing 153 (CCDC153) has recently been identified as a new N-DRC component in Tetrahymena thermophila. However, the physiological function of its mammalian homolog remains unknown. Here, we generated a Ccdc153 knockout mouse model and explored its functional association with motile cilia. We found that CCDC153 was highly expressed in the motile cilia-abundant tissues and localized to the axonemal lumen in motile cilia. However, Ccdc153 knockout mice were viable and exhibited normal brain ventricles and fertility. Overall, our results suggest that CCDC153 is dispensable for ciliary motility in brain ventricles and sperm movement, indicating that CCDC153 is not a potential causative gene in human ciliopathies.

Abstract Phenotypic plasticity is a widespread strategy used by organisms to cope with environmental fluctuations. Empirical studies have mostly focused on describing the amplitude of phenotypic change through reaction norms, which ignore the temporal dynamics of plasticity. Although the speed of plastic responses has recurrently been predicted to modulate their adaptiveness, it remains largely understudied. Here, we retraced the time course of plasticity across four traits in 12 isogenic strains of the ciliate Tetrahymena thermophila to test how the temporal dynamics of plasticity mediate its adaptiveness under fluctuations. We decomposed plastic responses into 3 parameters: a lag and a rate describing their temporal dimension and the canonical plastic capacity. All showed high intraspecific variability. We found the plastic capacity to be positively correlated to the rate of plasticity and not to the time required for plastic changes to be implemented. We then linked the dynamics of plasticity to how strains performed across a gradient of fluctuation periods. The temporal parameters of plasticity significantly explained performance in fluctuating conditions, more so than the plastic capacity alone. Interestingly, strains mounting morphological plasticity at a slower rate tended to be less sensitive to fluctuations. This study demonstrates that a better understanding of how organisms cope with environmental change requires us to consider and incorporate the temporal dynamics of plasticity in theories and experiments.

Multiciliogenesis requires large-scale biosynthesis of motility-powering axonemal inner and outer dynein arm motors (IDAs and ODAs) before their intraflagellar transport (IFT) into cilia. ODAs are inhibited by the packaging chaperone Shulin during ciliogenesis in Tetrahymena thermophila. How Shulin is released for ODAs to become active inside cilia remains unclear. Here we uncover a molecular mechanism for ODA activation. We establish interactions between DNAAF9 (human Shulin) and mammalian ODA subunits, IFT proteins and the ciliary small guanosine triphosphatase (GTPase) ARL3 using proteomics and in vitro reconstitutions. Mutagenesis combined with biochemical and structural studies reveal that DNAAF9 and Shulin preferentially bind active Arl3-GTP highlighting a cross-species conservation of this interaction. GTP-loaded Arl3 can access, bind and displace Shulin from the packaged ODA-Shulin complex. We propose that, once the inhibited ODA complex enters growing cilia, Arl3-GTP displaces Shulin (DNAAF9) and sequesters it away from ODAs, promoting activation of their motility specifically inside cilia.

The significant upregulation of TtCBS1 in Tetrahymena thermophila under cadmium stress indicates that this enzyme is a key player in the organism's defense mechanism against cadmium toxicity. Cadmium sulfide (CdS) nanoparticles were synthesized using the TtCbs1 single-enzyme system in vitro. Cysteine and glutathione play a critical role in controlling the growth of biosynthetic CdS particle size. Understanding the precise mechanisms by which cysteine and glutathione control particle size could lead to the development of more precise and efficient biomineralization processes. The synthesized CdS quantum dots exhibited significant photocatalytic activity. This work highlights the potential of cystathionine β-synthase from protists in metal detoxification and environmental remediation.

We describe the identification and three-dimensional (3D) structure determination of an approx. 1 MDa, hollow, elliptical protein cage discovered while surveying proteins isolated from the ciliary matrix of the ciliate Tetrahymena thermophila. By using mass spectrometry, AlphaFold, and cryo-electron microscopy, we identified the cage-like protein and determined its stoichiometry, mid-resolution 3D structure, and protein interactions. A sequence survey revealed several thousand homologs, with conservation across eukaryotic microbes spanning green algae, fungi, amoebozoans, choanoflagellates, and SAR organisms, as well as deep homology to genes in gram-negative predominantly marine prokaryotes and microbial mats, implying an ancient origin and arguing against a eukaryote-specific function. We subsequently isolated and solved the structure to high resolution from the slime mold Dictyostelium discoideum. Based on these observations, we named this assembly the CAGE complex (for Conserved Assembly in Gram-negative bacteria and Eukaryotes). We speculate on potential roles as a chaperone, container, or protease trap, but the biological function of the CAGE complex has yet to be determined.

Tissue- or cell type- specific expression of transgenes is often essential for interrogation of biological phenomenon or predictable engineering of multicellular organisms but can be stymied by cryptic enhancers that make identification of promoters that generate desired expression profiles challenging. In plants the months-to-years long timeline associated with prototyping putative tissue-specific promoters in transgenic lines deepens this challenge. We have developed a novel strategy calledRibozyme Enabled Tissue Specificity(RETS) that leverages the knowledge of where and when genes are expressed derived from transcriptomic studies to enable tissue-specific expression without needing characterized promoters. It uses a split self-splicing ribozyme based on a group I intron fromTetrahymena thermophilato enable conditional reconstitution of a transgene mRNA in the presence of a secondary tissue-specific mRNA of choice. We elucidate the design features that enable flexible swapping of transgenes and targets, enhancing transgene expression, and circumventing host RNA interference responses. We then show that these innovations enable tissue-specific and dose-dependent expression of transgenes inArabidopsis thaliana. Finally, we demonstrate the utility of RETS both for creating genetically encoded biosensors to study the spatiotemporal patterns of gene expressionin plantaas well as for engineering tissue-specific changes in organ size. RETS provides a novel avenue to study expression patterns of native loci with non-destructive imaging, complementing the weakness of existing approaches. Additionally, the spatiotemporal control of transgene expression afforded by RETS enables precision engineering of plant phenotypes which will facilitate enhancing crops without the trade-offs associated with constitutive expression.

Inhibitory effects of isobavachalcone against Tetrahymena thermophila: Mechanistic insights.

N6-adenine (6mA) DNA methylation plays an important role in gene regulation and genome stability. The 6mA methylation in Tetrahymena thermophila is mainly mediated by the AMT complex, comprised of the AMT1, AMT7, AMTP1, and AMTP2 subunits. To date, how this complex assembles on the DNA substrate remains elusive. Here we report the structure of the AMT complex bound to the OCR protein from bacteriophage T7, mimicking the AMT-DNA encounter complex. The AMT1-AMT7 heterodimer approaches OCR from one side, while the AMTP1 N-terminal domain, assuming a homeodomain fold, binds to OCR from the other side, resulting in a saddle-shaped architecture reminiscent of what was observed for prokaryotic 6mA writers. Mutation of the AMT1, AMT7, and AMTP1 residues on the OCR-contact points led to impaired DNA methylation activity to various extents, supporting a role for these residues in DNA binding. Furthermore, structural comparison of the AMT1-AMT7 subunits with the evolutionarily related METTL3-METTL14 and AMT1-AMT6 complexes reveals sequence conservation and divergence in the region corresponding to the OCR-binding site, shedding light on the substrate binding of the latter two complexes. Together, this study supports a model in which the AMT complex undergoes a substrate binding-induced open-to-closed conformational transition, with implications in its substrate binding and processive 6mA methylation.

Proteogenomic Reassessment Provides Novel Insight into the Life Cycle of Tetrahymenathermophila.

Radial spokes, RS1, RS2, and RS3, are T-shaped, multiprotein complexes that transmit regulatory signals from the central apparatus to outer doublet complexes, including dynein arms. Radial spokes, especially RS3, differ in their morphology, protein composition, and RS base-docked IDAs. Spokes’ defects alter cilia beating frequency, waveform, and amplitude, leading, in humans, to primary ciliary dyskinesia and male infertility. The role of the particular spokes remains unclear. Ciliate Tetrahymena thermophila has three RSP3 paralogs and two or three paralogs of some other RSPs. Using multiple complementary approaches, we demonstrated that Tetrahymena forms RS1 and RS2 subtypes, each with a core composed of various RSP3 paralogs and one type of RSP3-less RS3. We elucidated proteomes of RS1 and RS2 subtypes, RS3, and identified novel RS proteins, including enzymatic proteins involved in local regulation of ADP/ATP levels, the guanylate nucleotide pathway, and protein phosphorylation, whose presence further diversifies RSs’ properties and likely functions.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05871-x.

Therapeutic strategies for Neurofibromatosis Type I (NF1) that correct the underlying pathogenic NF1 variant hold promise for restoring neurofibromin function, reducing tumor burden, and improving patient outcomes by addressing the root cause of the disease rather than its symptoms. Beyond gene editing, transcript reprogramming via RNA trans-splicing has gained attention, particularly with the recent FDA approval of two trans-splicing-based drugs for IND phase 1/2a trials. This study tests whether trans-splicing group I intron ribozymes from Tetrahymena thermophila can be used to repair pathogenic variants of NF1 (pre-)mRNA by 3'-tail replacement. Splice sites on the NF1 mRNA were identified computationally and validated biochemically, and an efficiency-enhancing Extended Guide Sequence (EGS) of the corresponding ribozyme was identified in a combinatorial experiment. The correct trans-splicing product of this ribozyme was validated in HEK293 NF1-/- cells expressing mNf1. This study established a splice site and activity-enhancing extended guide sequences for the repair of NF1 mRNA. Further optimization of the ribozyme, as well as improved delivery methods, may establish ribozyme-based RNA repair as a viable strategy for NF1 treatment.

Ciliates utilise motile cilia, which are highly dynamic organelles protruding from the cell surface, to swim helically in a three-dimensional (3D) space. The 3D nature of their swimming behaviour and rapid ciliary beatings make its quantitative analysis difficult. Here, we quantified the 3D motion of a microbead bound to a ciliary tip in a live immobilised Tetrahymena thermophila cell using 3D tracking optical microscopy. We found that the tip of individual ciliate cilia, consisting of the 9+2 structure of the axoneme, shows semicircular counterclockwise rotation in a single plane when looking down on the cilium. The rotational trajectories of the tip consist of fast and slow strokes, with the tip path during the fast and slow strokes being an arc and linear, respectively. The direction of the fast stroke of the ciliary tip, with respect to the cell body, was from the right-anterior to the left-posterior region, which is consistent with the direction that would induce right-handed helical swimming of the Tetrahymena.

BackgroundLegionella pneumophila, an intracellular pathogen responsible for the pneumonia-like Legionnaires’ disease in humans, inhabits aquatic environments, including man-made water systems such as water fountains, foot spas, and tap water, and exists as part of biofilms or as a protozoan parasite. As a bacterivore, Tetrahymena thermophila provides a favorable environment for Legionella to establish a replicative niche (Legionella-containing vacuole; LCV) under environmental stress. Conversely, the L. pneumophila Ofk308 strain, isolated from an Ashiyu foot spa, has been found to be cytotoxic to the ciliate T. thermophila CU427. This study aimed to identify the cytotoxicity-related genes of Legionella and elucidate their mechanisms specific to the Tetrahymena host.MethodsA comparative analysis using RNA-sequencing was conducted with two Legionella strains, Philadelphia-1 and Ofk308, to select several candidate genes. Deletion mutants of Ofk308 were constructed by homologous recombination. Eight out of ten candidate gene deletion mutants were successfully generated. These mutants were analyzed for cytotoxicity against T. thermophila and intracellular bacterial growth at 2 h, 24 h, and 48 h postinfection.Results and DiscussionsAmong the deletion mutants, Δ vicinal oxygen chelate (VOC) and msrB/A exhibited reduced cytotoxicity. Furthermore, LCVs formed in T. thermophila infected with DVOC and msrB/A were smaller in size compared to those formed by the parental strain Ofk308, suggesting a role in both cytotoxicity and intracellular growth. Multiple factors contribute to the cytotoxicity exhibited by the Ofk308 strain in protozoan host cells, and gene expression analysis may reveal additional relevant factors.

Abstract Antibiotics exert a considerable ecotoxicological impact, even at very low concentrations. However, adequately removing these substances to minimize the underlying hazards is challenging due to their hydrophilicity and strong chemical stability. To cope with this hurdle the present investigation utilized 25 antibiotics and their known human CYP metabolites (35) to evaluate ecotoxicity by implementing T.E.S.T. endpoints including; Fathead minnow (EC50), Daphnia magna (LC50), and Tetrahymena pyriformis (ICG50). Subsequently, theoretical degradation was assessed using multivalent computational techniques coupled with Steccherinum murashkinskyi‐derived laccase. Ecotoxicological hazards were assessed with estimated concentrations of antibiotics/metabolites ranging from 1.14E‐03 (0.00114) mg/L to 370.13 mg/L for selected endpoints. Laccase‐assisted docked complexes exhibited binding affinity in a range of −4.80 ± 0.00 to −9.40 ± 0.10 Kcal/mol (p < 0.05). Determining the accessible surface area (ΔASA) implies adequate ligand bindings. A few crucial residues; ILE, SER, PRO, THR, GLY, LEU, PHE, LYS, ALA, ASN, ASP, VAL, GLN, TRP, ARG, GLU, and HIS were identified as the most notable active site residues in the binding process. These residues contribute to various chemical interactions, including hydrogen bonds, alkyl interactions, Pi‐alkyl, Pi‐Pi T‐shaped, Pi‐Pi stacked, Pi‐anion, halogen, and Pi‐sigma type. Computational findings necessitate validation through conventional assays for practical deployment coupled with a laccase‐based system.

PCNA's dual legacy in ciliates: Conserved replication scaffold and lineage-specific genome architect.

Post-translational modifications (PTMs) to tubulin subunits in microtubule filaments are thought to comprise a component of the tubulin code that specifies microtubule functions in cell physiology and animal development. Acetylation of Lysine-40 (K40) on α-tubulin (αTub-K40ac) and glutamylation of both α- and β-tubulin are two tubulin PTMs of interest to the field. Antibodies that recognize these PTMs have been indispensable tools to study the localization of these PTMs as well as their biological functions. Although widely used, these antibodies are procured from commercial sources and thus have drawbacks including availability, high cost, and lack of reproducibility. To mitigate these downsides, we report the protein sequences of GT335 (anti-glutamylation) and 6-11B-1 (anti-αTub-K40ac) monoclonal antibodies and describe the use of these sequences to generate recombinant monoclonal antibody (rMAb) versions of GT335 and 6-11B-1. We demonstrate through western blotting and immunofluorescence of cultured mammalian cells and Tetrahymena thermophila that rMAb-GT335 and rMAb-611B1 match the specific activity of the commercially available antibodies. Our work provides the field with a renewable source of antibodies with high specificity and affinity towards tubulin glutamylation and acetylation and opens the door to more reproducible and large-scale studies of the function and regulation these tubulin PTMs.