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.

Trypanosoma, Paramecium and Tetrahymena: From genomics to flagellar and ciliary structures and cytoskeleton dynamics.

Cilia and flagella play an important role in motility, sensory perception, and the life cycles of eukaryotes, from protists to humans. However, much critical information concerning cilia structure and function remains elusive. The vast majority of ciliary and flagellar proteins analyzed so far are evolutionarily conserved and play a similar role in protozoa and vertebrates. This makes protozoa attractive biological models for studying cilia biology. Research conducted on ciliated or flagellated protists may improve our general understanding of cilia protein composition, of cilia beating, and can shed light on the molecular basis of the human disorders caused by motile cilia dysfunction. The Symposium "From genomics to flagellar and ciliary structures and cytoskeleton dynamics" at ECOP2019 in Rome presented the latest discoveries about cilia biogenesis and the molecular mechanisms of ciliary and flagellum motility based on studies in Paramecium, Tetrahymena, and Trypanosoma. Here, we review the most relevant aspects presented and discussed during the symposium and add our perspectives for future research.

The LisH Domain-Containing N-Terminal Fragment is Important for the Localization, Dimerization, and Stability of Katnal2 in Tetrahymena.

Katanin-like 2 protein (Katnal2) orthologs have a tripartite domain organization. Two highly conserved regions, an N-terminal LisH (Lis-homology) domain and a C-terminal AAA catalytic domain, are separated by a less conserved linker. The AAA domain of Katnal2 shares the highest amino acid sequence homology with the AAA domain of the canonical katanin p60. Katnal2 orthologs are present in a wide range of eukaryotes, from protists to humans. In the ciliate Tetrahymena thermophila, a Katnal2 ortholog, Kat2, co-localizes with the microtubular structures, including basal bodies and ciliary outer doublets, and this co-localization is sensitive to levels of microtubule glutamylation. The functional analysis of Kat2 domains suggests that an N-terminal fragment containing a LisH domain plays a role in the subcellular localization, dimerization, and stability of Kat2.

Silver nanoparticles are lethal to the ciliate model Tetrahymena and safe to the pike silverside Chirostoma estor.

Ciliate ectoparasites are one of the most important groups of pathogens in fish culture, and the traditional treatments are sometimes harmful to the fish and the environment. Thus, the search for novel compounds that are effective at low concentrations and safe for fish are necessary to optimise treatments in aquaculture. The antiprotozoal capacity of silver nanoparticles (AgNPs) against the ciliate Tetrahymena has been documented; however, their toxicity may vary with the synthesis methodology and nanoparticle size. The objectives of this study were a) to evaluate the acute toxicity in vitro of two AgNPs (Argovit™ and UTSA) on Tetrahymena sp., a biological model for ciliated ectoparasites of fish and b) to test the safety of lethal and higher doses of UTSA AgNPs for ciliates on the fish C. estor. Light microscopy and scanning electron microscopy (SEM) were used to determine whether AgNPs affected the structure of the cell surface of Tetrahymena. The mortality, histopathological alterations and metagenomics of the fish were used to determine the major effects of UTSA AgNPs. In Tetrahymena, the median lethal concentration (LC50) for Argovit™ was 2501 ± 1717 ng/L at 15 min and 796 ± 510 ng/L at 60 min, while the LC50 for UTSA AgNPs was 4 ± 2 and 1 ± 0.6 ng/L at 15 min and 60 min, respectively. A concentration of 3300 ng/L Argovit™ and 10.6 ng/L UTSA AgNPs for 15 and 60 min, respectively, was 100% effective against Tetrahymena. After 60 min of exposure to 0.25 and 0.50 ng/L UTSA AgNPs, the number of cilia significantly reduced, there were small holes on the cell surface, and the cellular membrane was ruptured. In fish exposed to lethal (10.6 ng/L) and higher (31.8 and 95.4 ng/L) doses of UTSA, the AgNPs did not affect fish survival after 96 h, and there were no signs of histopathological damage or gut microbial changes. This study is the first report on microscopic and ultrastructural changes in Tetrahymena after exposure to significantly low concentrations of UTSA AgNPs with antiprotozoal efficacy without evidence of harmful effects on fish. These results provide the basis for further studies of both pet aquarium and commercial fish that may validate these findings at a larger experimental scale, taking into account AgNPs bioaccumulation, safety for human consumption and environmental impact.

Tetrahymena dynamin-related protein 6 self-assembles independent of membrane association.

Self-assembly on target membranes is one of the important properties of all dynamin family proteins. Drp6, a dynaminrelated protein in Tetrahymena, controls nuclear remodelling and undergoes cycles of assembly/disassembly on the nuclear envelope. To elucidate the mechanism of Drp6 function, we have characterized its biochemical and biophysical properties using size exclusion chromatography, chemical cross-linking and electron microscopy. The results demonstrate that Drp6 readily forms high-molecular-weight self-assembled structures as determined by size exclusion chromatography and chemical cross-linking. Negative stain electron microscopy revealed that Drp6 assembles into rings and spirals at physiological ionic strength. We have also shown that the recombinant Drp6 expressed in bacteria is catalytically active and its GTPase activity is not enhanced by low salt. These results suggest that, in contrast to dynamins but similar to MxA, Drp6 self-assembles in the absence of membrane templates, and its GTPase activity is not affected by ionic strength of the buffer. We discuss the self-assembly structure of Drp6 and explain the basis for lack of membrane-stimulated GTPase activity.

Encystment/excystment response and serotypic variation in the gastropod parasite Tetrahymena rostrata (Ciliophora, Tetrahymenidae).

Tetrahymena rostrata, which is characterized by a particular encystment-excystment cycle involving autogamy, has been recently found infecting the kidney of edible Helix aspersa snails under farming conditions. In this work, the effects of several factors on its encystment/excystment behaviour and the occurrence of different serotypes were investigated. The encystment/excystment response under starvation conditions was seriously affected by temperature. While a peak of encystment at 48 h followed by a progressive spontaneous excystment was observed at 18 and 25 °C, the encystment response was practically inhibited at 5 °C and clearly slowed down at 10 °C. At 30 °C, most of surviving ciliates remained encysted throughout the experiment, with spontaneous excystment being detected only after switching the temperature to 18 °C. Soil components also affected the encystment/excystment behaviour at 18 °C, with spontaneous excystment occurring in the presence of a sterile-filtered soil extract or mineral water but being strongly minimized with a non-filtered soil extract. Resting cysts formed in the latter extract exhibited a 3­4 times thicker and ultrastructurally more complex wall than that formed in mineral water and retained the excystment ability for about 4 weeks. Incomplete desiccation did not affect significantly the encystment response, while the mucus and kidney extracts from snails as well as a ciliate extract strongly stimulated a rapid excystment. Finally, two different serotypes infecting H. aspersa in heliciculture farms of Galicia (NW Spain) were identified, but no differences were observed between the encystment/excystment responses exhibited by two isolates belonging to each serotype.

Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena.

Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light-electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates.

The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia.

Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility.

FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.

3D structural analysis of flagella/cilia by cryo-electron tomography.

Three dimensional arrangement of proteins inside the axoneme is essential information to elucidate the flagellar/ciliary bending mechanism. Cryo-electron tomography provides structural information in situ at ~30 Å resolution and thus is a suitable method for flagella/cilia research. The biggest challenge in cryo-electron tomography is the low signal-to-noise ratio. Therefore, subtomogram averaging, an in silico processing step to detect identical structural units based on 3D image analysis, aligns them three dimensionally and average is required to improve the signal-to-noise ratio. When structural units are not exactly identical, they must be classified. In this chapter, we describe our strategy to extract, align, classify, and average molecular structures from cryo-electron tomograms of flagella/cilia, utilizing longitudinal periodicity and pseudo-ninefold symmetry in the axoneme.

Survival characteristics of diarrheagenic Escherichia coli pathotypes and Helicobacter pylori during passage through the free-living ciliate, Tetrahymena sp.

Free-living protozoa have been implicated in the survival and transport of pathogens in the environment, but the relationship between non-Shiga toxin-producing Escherichia coli or Helicobacter pylori and ciliates has not been characterized. Six diarrheagenic pathotypes of E. coli and an isolate of H. pylori were evaluated for their susceptibility to digestion by Tetrahymena, an aquatic ciliate. Tetrahymena strain MB125 was fed E. coli or H. pylori, and the ciliate's egested products examined for viable bacterial pathogens by the BacLight(™) LIVE/DEAD (™) assay, a cell elongation method, and by colony counts. All six diarrheagenic E. coli pathotypes survived digestion, whereas H. pylori was digested. Growth of E. coli on agar plates indicated that the bacteria were able to replicate after passage through the ciliate. Transmission electron micrographs of E. coli cells as intact rods vs. degraded H. pylori cells corroborated these results. Scanning electron microscopy revealed a net-like matrix around intact E. coli cells in fecal pellets. These results suggest a possible role for Tetrahymena and its egested fecal pellets in the dissemination of diarrheagenic E. coli in the environment. This bacterial-protozoan interaction may increase opportunities for transmission of diarrheagenic E. coli to mammalian hosts including humans.

Comparative structural analysis of eukaryotic flagella and cilia from Chlamydomonas, Tetrahymena, and sea urchins.

Although eukaryotic flagella and cilia all share the basic 9+2 microtubule-organization of their internal axonemes, and are capable of generating bending-motion, the waveforms, amplitudes, and velocities of the bending-motions are quite diverse. To explore the structural basis of this functional diversity of flagella and cilia, we here compare the axonemal structure of three different organisms with widely divergent bending-motions by electron cryo-tomography. We reconstruct the 3D structure of the axoneme of Tetrahymena cilia, and compare it with the axoneme of the flagellum of sea urchin sperm, as well as with the axoneme of Chlamydomonas flagella, which we analyzed previously. This comparative structural analysis defines the diversity of molecular architectures in these organisms, and forms the basis for future correlation with their different bending-motions.

The envelope damage of Tetrahymena in the presence of TiO2 combined with UV light.

The biological consequences of exposure to TiO(2), UV light and their combined effects were studied on the cellular envelopes of Tetrahymena. For Tetrahymena cells treated with TiO(2) or UV light alone, the cell membrane shrunk while still maintaining the original elliptoid shape. Cells treated by TiO(2) under UV light irradiation experienced the most serious damage by peroxidation. A pear-shaped cell was formed due to serious shrinkage and cilia loss. An increase in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene was observed, indicating a significant decrease in membrane fluidity. Quantum dot (QD) labeling revealed that damaged cells could not function properly or absorb extracellular materials selectively. QDs were able to enter the damaged cells or be absorbed on the cell surface. Attenuated total reflection Fourier transform infrared spectra revealed that amide groups and PO(2)(-) of the phospholipid phospho-diester, both in the hydrophobic end exposed to the outer layer, were the easiest to be oxidized. Other groups like CH(2) and CH(3) were also involved, but showed a resistance to photocatalytic peroxidation. The differences can be attributed to the bond energy as well as the ordering and position of the groups.

Tetrahymena sp. infection in guppies, Poecilia reticulata Peters: parasite characterization and pathology of infected fish.

Tetrahymena sp. infection was diagnosed in guppies imported from Singapore. The parasite was isolated (Tet-NI) and optimally cultured in vitro in RM-9 medium. Cytological analyses [silver-staining and scanning electron microscopy (SEM)] revealed a pyriform-shaped, 64 x 41-microm holotrich ciliate without caudal cilium, containing a macro-nucleus (18.25 x 16.83 microm) and micro-nucleus (5.73 x 5.40 microm). Wet-mount examination and histological analyses of fish exposed to the parasite by co-habitation, immersion and infection by i.p. (intra-peritoneal) and i.m. (intra-muscular) injection revealed numerous ciliates on the skin, and in the gill and caudal fin blood vessels. Ciliates surrounded internal organs, the peri-orbital region of the eye, and were observed inside developing guppy embryos. Some muscle necrosis was associated with infection, but little or no inflammatory response. Immersion, co-habitation and i.m. injection caused relatively high infection rates and levels in the skin and tail, and lower infection in the gill blood vessels and internal organs; i.p. injection caused higher infection in the gill blood vessels and internal organs. Co-habited fish had relatively high infection levels in the hind-gut sub-mucosa. This is the first report of controlled systemic infection by Tetrahymena sp.

Nuclear dimorphism: two peas in a pod.

The macro- and micronuclei of Tetrahymena reside in the same cytoplasm but are about as different as night and day. This extreme case of nuclear dimorphism can now be partially attributed to differences in the subunit compositions of their nuclear pore complexes.

TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia.

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer's vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.

Morphological and molecular characterization of renal ciliates infecting farmed snails in Spain.

Renal infections by parasitic ciliates were studied in adult snails of Helix aspersa aspersa and Helix aspersa maxima collected from 2 mixed rearing system-based heliciculture farms located in Galicia (NW Spain). The occurrence of ciliates was also examined in slugs (Deroceras reticulatum) invading the greenhouses where first growing and fattening of snails is carried out. Histological examinations revealed a severe destruction of the renal epithelium in heavily infected hosts. Three ciliate isolates, one from each host species, were obtained and grown in axenic cultures. Cultured and parasitic ciliates were characterized morphologically and morphometrically. In addition, the encystment behaviour, the occurrence of autogamy, and the sequences of the mitochondrial cytochrome-c oxidase subunit 1 (cox1) and the small subunit ribosomal RNA (SSU rRNA) genes were also studied in the 3 isolates. A polymorphic life cycle involving resting and reproductive cysts, together with the morphological and morphometrical characteristics and the confirmation that autogamy occurs within cysts, demonstrate that our ciliates belong to the species Tetrahymena rostrata (Kahl, 1926) Corliss, 1952. The 3 isolates formed a well-supported clade using both genetic markers, and were clearly separate from the strain ATCC(R) 30770, which has been identified as Tetrahymena rostrata. We argue that our Spanish isolates should be regarded as Tetrahymena rostrata, and that the ATCC isolate should be regarded as a misidentification as neither cytological nor cytogenetical support for its identity has been presented.

Basal body assembly in ciliates: the power of numbers.

Centrioles perform the dual functions of organizing both centrosomes and cilia. The biogenesis of nascent centrioles is an essential cellular event that is tightly coupled to the cell cycle so that each cell contains only two or four centrioles at any given point in the cell cycle. The assembly of centrioles and their analogs, basal bodies, is well characterized at the ultrastructural level whereby structural modules are built into a functional organelle. Genetic studies in model organisms combined with proteomic, bioinformatic and identifying ciliary disease gene orthologs have revealed a wealth of molecules requiring further analysis to determine their roles in centriole duplication, assembly and function. Nonetheless, at this stage, our understanding of how molecular components interact to build new centrioles and basal bodies is limited. The ciliates, Tetrahymena and Paramecium, historically have been the subject of cytological and genetic study of basal bodies. Recent advances in the ciliate genetic and molecular toolkit have placed these model organisms in a favorable position to study the molecular mechanisms of centriole and basal body assembly.

Passage through Tetrahymena tropicalis triggers a rapid morphological differentiation in Legionella pneumophila.

The intracellular bacterial pathogen Legionella pneumophila follows a developmental cycle in which replicative forms (RFs) differentiate into infectious stationary-phase forms (SPFs) in vitro and in vivo into highly infectious mature intracellular forms (MIFs). The potential relationships between SPFs and MIFs remain uncharacterized. Previously we determined that L. pneumophila survives, but does not replicate, while it transiently resides (for 1 to 2 h) in food vacuoles of the freshwater ciliate Tetrahymena tropicalis before being expelled as legionellae-laden pellets. We report here that SPFs have the ability to rapidly (<1 h) and directly (in the absence of bacterial replication) differentiate into MIFs while in transit through T. tropicalis, indicating that SPFs and MIFs constitute a differentiation continuum. Mutant RFs lacking the sigma factor gene rpoS, or the response regulator gene letA, were unable to produce normal SPFs in vitro and did not fully differentiate into MIFs in vivo, further supporting the existence of a common mechanism of differentiation shared by SPFs and MIFs. Mutants with a defective Dot/Icm system morphologically differentiated into MIFs while in transit through T. tropicalis. Therefore, T. tropicalis has allowed us to unequivocally conclude that SPFs can directly differentiate into MIFs and that the Dot/Icm system is not required for differentiation, two events that could not be experimentally addressed before. The Tetrahymena model can now be exploited to study the signals that trigger MIF development in vivo and is the only replication-independent model reported to date that allows the differentiation of Dot/Icm mutants into MIFs.

Ingestion and inactivation of bacteriophages by Tetrahymena.

Abiotic factors are thought to be primarily responsible for the loss of bacteriophages from the environment, but ingestion of phages by heterotrophs may also play a role in their elimination. Tetrahymena thermophila has been shown to ingest and inactivate bacteriophage T4 in co-incubation experiments. In this study, other Tetrahymena species were co-incubated with T4 with similar results. In addition, T. thermophila was shown to inactivate phages T5 and lambda in co-incubations. Several approaches, including direct visualization by electron microscopy, demonstrated that ingestion is required for T4 inactivation. Mucocysts were shown to have no role in the ingestion of T4. When (35)S-labeled T4 were fed to T. thermophila in a pulse-chase experiment, the degradation of two putative capsid proteins, gp23(*) and hoc, was observed. In addition, a polypeptide with the apparent molecular mass of 52 kDa was synthesized. This suggests that Tetrahymena can use phages as a minor nutrient source in the absence of bacteria.