Two Tetrahymena kinesin-9 family members exhibit slow plus-end-directed motility in vitro.

The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family.

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.

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.

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.

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.

A Hexameric Ribozyme Nanostructure Formed by Double-Decker Assembly of a Pair of Triangular Ribozyme Trimers.

The modular architecture of naturally occurring ribozymes makes them a promising class of structural platform for the design and assembly of three-dimensional (3D) RNA nanostructures, into which the catalytic ability of the platform ribozyme can be installed. We have constructed and analyzed RNA nanostructures with polygonal-shaped (closed) ribozyme oligomers by assembling unit RNAs derived from the Tetrahymena group I intron with a typical modular architecture. In this study, we dimerized ribozyme trimers with a triangular shape by introducing three pillar units. The resulting double-decker nanostructures containing six ribozyme units were characterized biochemically and their structures were observed by atomic force microscopy. The double-decker hexamers exhibited higher catalytic activity than the parent ribozyme trimers.

How to Kinetically Dissect an RNA Machine.

RNA-based machines are ubiquitous in Nature and increasingly important for medicines. They fold into complex, dynamic structures that process information and catalyze reactions, including reactions that generate new RNAs and proteins across biology. What are the experimental strategies and steps that are necessary to understand how these complex machines work? Two 1990 papers from Herschlag and Cech on "Catalysis of RNA Cleavage by the Tetrahymena thermophila Ribozyme" provide a master class in dissecting an RNA machine through kinetics approaches. By showing how to propose a kinetic framework, fill in the numbers, do cross-checks, and make comparisons across mutants and different RNA systems, the papers illustrate how to take a mechanistic approach and distill the results into general insights that are difficult to attain through other means.

Effects of chain length of polyethylene glycol molecular crowders on a mutant Tetrahymena group I ribozyme lacking large peripheral module.

While current group I ribozymes use several distinct strategies to function under conditions of low Mg2+ concentration (≤ 3 mM), a deletion mutant of the Tetrahymena ribozyme (ΔP5 ribozyme) is virtually inactive with 3 mM Mg2+ due to removal of the large peripheral module, P5abc, supporting the active conformation of the core module. We investigated the molecular crowding effects of synthetic polyethylene glycols (PEGs) on the activity of the ΔP5 ribozyme. Among PEG molecules with different chain lengths, PEG600 improved the activity of the ΔP5 ribozyme most effectively in the presence of 3 mM Mg2+.

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs.

The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of microtubule inner proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

Ultra low doses and biological amplification: Approaching Avogadro's number.

This paper describes evidence establishing that ultra-low doses of diverse chemical agents at concentrations from 10-18 to 10-24 M (e.g., approaching and/or less than 1 atom or molecule of a substance/cell based on Avogadro's constant - 6.022×1023/mole) are capable of engaging receptor and intracellular signaling systems to elicit reproducible effects in a variety of species, from unicellular organisms to humans. Multiple experimental studies have shown that only one or very few molecules are needed to activate a cell and/or entire organism via cascade(s) of amplification mechanisms and processes. For example, ultra-low dose ligand exposure was able to activate both an individual cell, and ~3000 to 25,000 neighboring cells on average, by about 50%. Such activation of cells and whole organisms typically displayed hormetic-biphasic dose responses. These findings indicate that numerous, diverse phylogenetic systems have evolved highly sensitive detection and signaling mechanisms to enhance survival functions, such as defense against infectious agents, responses to diverse types of pheromone communications (e.g., alarm, sexual attraction), and development of several types of cellular protection/resilience processes. This suggests that ultra-low dose effects may be far more common than have been recognized to date. We posit that such findings have important implications for evolutionary theory, ecological and systems biology, and clinical medicine.

STORM imaging reveals the spatial arrangement of transition zone components and IFT particles at the ciliary base in Tetrahymena.

The base of the cilium comprising the transition zone (TZ) and transition fibers (TF) acts as a selecting gate to regulate the intraflagellar transport (IFT)-dependent trafficking of proteins to and from cilia. Before entering the ciliary compartment, IFT complexes and transported cargoes accumulate at or near the base of the cilium. The spatial organization of IFT proteins at the cilia base is key for understanding cilia formation and function. Using stochastic optical reconstruction microscopy (STORM) and computational averaging, we show that seven TZ, nine IFT, three Bardet-Biedl syndrome (BBS), and one centrosomal protein, form 9-clustered rings at the cilium base of a ciliate Tetrahymena thermophila. In the axial dimension, analyzed TZ proteins localize to a narrow region of about 30 nm while IFT proteins dock approximately 80 nm proximal to TZ. Moreover, the IFT-A subcomplex is positioned peripheral to the IFT-B subcomplex and the investigated BBS proteins localize near the ciliary membrane. The positioning of the HA-tagged N- and C-termini of the selected proteins enabled the prediction of the spatial orientation of protein particles and likely cargo interaction sites. Based on the obtained data, we built a comprehensive 3D-model showing the arrangement of the investigated ciliary proteins.

Uptake, excretion and toxicity of titanate nanotubes in three stains of free-living ciliates of the genus Tetrahymena.

The potential exposure of titanate nanotubes (TNTs) to wildlife and humans may occur as a result of increased use and application as functional nanomaterials. However, there is a dearth of knowledge regarding the pathways of uptake and excretion of TNTs and their toxicity in cells. In this study, three strains of the Tetrahymena genus of free-living ciliates, including a wild type strain (SB210) and two mutant strains (SB255: mucocyst-deficient; NP1: temperature-sensitive "mouthless''), were used to study the pathways of uptake and excretion and evaluate the cytotoxicity of TNTs. The three Tetrahymena strains were separately exposed to 0, 0.01, 0.1, 1 or 10 mg/L of TNTs, and cells were collected at different time points for quantification of intracellular TNTs (e.g., 5, 10, 20, 40, 60, 90 and 120 min) and evaluation of cytotoxicity (12 and 24 h). TNT contents in NP1 and SB255 were greater or comparable to the contents in SB210 while exposure to 10 mg/L TNTs in 120 min. Furthermore, exposure to 10 mg/L TNTs for 24 h caused greater decreases in cell density of NP1 (38.2 %) and SB255 (36.8 %) compared with SB210 (26.5 %) and upregulated the expression of caspase 15 in SB210. Taken together, our results suggested that TNT uptake by pinocytosis and excretion by exocytosis in Tetrahymena, and the exposure could cause cytotoxicity which can offer novel insights into the accumulation kinetics of nanotubes and even nanomaterials in single cell.

Pyridine Nucleotide-Linked Lactate Dehydrogenase of Tetrahymena: Evidence for D- and L-Enzymes in the Mitochondria.

An NAD-linked lactate dehydrogenase (LDH) in a crude mitochondrial fraction obtained from Tetrahymena homogenates was previously reported by this laboratory. This fraction contains the NADH and succinate oxidase system as well as the mitochondrial cytochromes and carries out oxidative phosphorylation. The preparation catalyzes the oxidation of D- and L-lactate linked only to certain analogs of NAD; it has not been possible to demonstrate NAD-dependent D- or L-lactate oxidation nor is there any evidence that either of these enzymes is a flavoprotein as indicated by their inability to reduce directly certain artificial electron acceptors. A lactate racemase is not present.

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms.

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER) and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent secretory cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

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.

Development of an expression system in Tetrahymena inner arm dyneins and motile properties of the single-headed subspecies (Dyh8p and Dyh12p).

Diverse inner arm dyneins cooperate with outer arm dyneins to produce ciliary beating. This study demonstrates an expression system for inner arm dyneins in Tetrahymena. The motor domain of inner arm dynein (Dyh8p or Dyh12p) was fused with the tail of outer arm dynein (Dyh3p) and expressed in viable DYH3-knockout (vKO-DYH3) cells. The chimeric dyneins were observed in the oral apparatus and cilia on the cell bodies, and did not change the swimming speed of vKO-DYH3 cells. In a gliding assay, the motor domains of Dyh8p and Dyh12p moved toward the minus ends of microtubules at 0.8 and 0.3 µm/s, respectively. The gliding velocities of Dyh8p and Dyh12p were decreased in 5 mM ATP but not increased in 0.1 or 0.5 mM ADP. This expression system will be useful for molecular studies on diverse inner arm dyneins.

LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena.

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.

Hidden genomic evolution in a morphospecies-The landscape of rapidly evolving genes in Tetrahymena.

A morphospecies is defined as a taxonomic species based wholly on morphology, but often morphospecies consist of clusters of cryptic species that can be identified genetically or molecularly. The nature of the evolutionary novelty that accompanies speciation in a morphospecies is an intriguing question. Morphospecies are particularly common among ciliates, a group of unicellular eukaryotes that separates 2 kinds of nuclei-the silenced germline nucleus (micronucleus [MIC]) and the actively expressed somatic nucleus (macronucleus [MAC])-within a common cytoplasm. Because of their very similar morphologies, members of the Tetrahymena genus are considered a morphospecies. We explored the hidden genomic evolution within this genus by performing a comprehensive comparative analysis of the somatic genomes of 10 species and the germline genomes of 2 species of Tetrahymena. These species show high genetic divergence; phylogenomic analysis suggests that the genus originated about 300 million years ago (Mya). Seven universal protein domains are preferentially included among the species-specific (i.e., the youngest) Tetrahymena genes. In particular, leucine-rich repeat (LRR) genes make the largest contribution to the high level of genome divergence of the 10 species. LRR genes can be sorted into 3 different age groups. Parallel evolutionary trajectories have independently occurred among LRR genes in the different Tetrahymena species. Thousands of young LRR genes contain tandem arrays of exactly 90-bp exons. The introns separating these exons show a unique, extreme phase 2 bias, suggesting a clonal origin and successive expansions of 90-bp-exon LRR genes. Identifying LRR gene age groups allowed us to document a Tetrahymena intron length cycle. The youngest 90-bp exon LRR genes in T. thermophila are concentrated in pericentromeric and subtelomeric regions of the 5 micronuclear chromosomes, suggesting that these regions act as genome innovation centers. Copies of a Tetrahymena Long interspersed element (LINE)-like retrotransposon are very frequently found physically adjacent to 90-bp exon/intron repeat units of the youngest LRR genes. We propose that Tetrahymena species have used a massive exon-shuffling mechanism, involving unequal crossing over possibly in concert with retrotransposition, to create the unique 90-bp exon array LRR genes.

Two Antagonistic Hippo Signaling Circuits Set the Division Plane at the Medial Position in the Ciliate Tetrahymena.

In a single cell, ciliates maintain a complex pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes. The underlying molecular mechanisms of intracellular pattern formation in ciliates are largely unknown. Ciliates divide by tandem duplication, a process that remodels the parental cell into two daughters aligned head-to-tail. In the elo1-1 mutant of Tetrahymena thermophila, the segmentation boundary/division plane forms too close to the posterior end of the parental cell, producing a large anterior and a small posterior daughter cell, respectively. We show that ELO1 encodes a Lats/NDR kinase that marks the posterior segment of the cell cortex, where the division plane does not form in the wild-type. Elo1 acts independently of CdaI, a Hippo/Mst kinase that marks the anterior half of the parental cell, and whose loss shifts the division plane anteriorly. We propose that, in Tetrahymena, two antagonistic Hippo circuits focus the segmentation boundary/division plane at the equatorial position, by excluding divisional morphogenesis from the cortical areas that are too close to cell ends.