Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila

Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the α-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of α-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as α-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on α-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.     

Extrachromosomal rDNA of Tetrahymena thermophila is not a perfect palindrome

We have determined the restriction-endonuclease-cleavage map and the nucleotide sequence of the central 1.4 kb fragment of the macronuclear extrachromosomal rDNA of Tetrahymena thermophila. These data demonstrate that this molecule is not a perfect palindrome, having a 29 bp AT-rich non-palindromic sequence at its center. This observation is important in determining the mechanism by which a single chromosomally integrated rRNA gene in the micronucleus is rearranged and amplified during sexual development to yield multiple copies of extrachromosomal rDNA in the macronucleus.     

Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of alpha- and beta-tubulin, respectively. Mutants lacking all polyglycylation sites on alpha-tubulin have normal phenotype, whereas similar sites on beta-tubulin are essential. A viable mutant with three mutated sites in beta-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of beta-tubulin. In this double mutant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of beta-tubulin can be transferred to alpha-tubulin, indicating it is the total amount of polyglycylation on both alpha- and beta-tubulin that is essential for survival.     

Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.     

The condensin complex is essential for amitotic segregation of bulk chromosomes, but not nucleoli, in the ciliate Tetrahymena thermophila

The macronucleus of the binucleate ciliate Tetrahymena thermophila contains fragmented and amplified chromosomes that do not have centromeres, eliminating the possibility of mitotic nuclear division. Instead, the macronucleus divides by amitosis with random segregation of these chromosomes without detectable chromatin condensation. This amitotic division provides a special opportunity for studying the roles of mitotic proteins in segregating acentric chromatin. The Smc4 protein is a core component of the condensin complex that plays a role in chromatin condensation and has also been associated with nucleolar segregation, DNA repair, and maintenance of the chromatin scaffold. Mutants of Tetrahymena SMC4 have remarkable characteristics during amitosis. They do not form microtubules inside the macronucleus as normal cells do, and there is little or no bulk DNA segregation during cell division. Nevertheless, segregation of nucleoli to daughter cells still occurs, indicating the independence of this process and bulk DNA segregation in ciliate amitosis.     

Glutathione in Tetrahymena thermophila

In Tetrahymena, glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.     

The CNA1 histone of the ciliate Tetrahymena thermophila is essential for chromosome segregation in the germline micronucleus

Ciliated protozoans present several features of chromosome segregation that are unique among eukaryotes, including their maintenance of two nuclei: a germline micronucleus, which undergoes conventional mitosis and meiosis, and a somatic macronucleus that divides by an amitotic process. To study ciliate chromosome segregation, we have identified the centromeric histone gene in the Tetrahymena thermophila genome (CNA1). CNA1p specifically localizes to peripheral centromeres in the micronucleus but is absent in the macronucleus during vegetative growth. During meiotic prophase of the micronucleus, when chromosomes are stretched to twice the length of the cell, CNA1p is found localized in punctate spots throughout the length of the chromosomes. As conjugation proceeds, CNA1p appears initially diffuse, but quickly reverts to discrete dots in those nuclei destined to become micronuclei, whereas it remains diffuse and is gradually lost in developing macronuclei. In progeny of germline CNA1 knockouts, we see no defects in macronuclear division or viability of the progeny cells immediately following the knockout. However, within a few divisions, progeny show abnormal mitotic segregation of their micronucleus, with most cells eventually losing their micronucleus entirely. This study reveals a strong dependence of the germline micronucleus on centromeric histones for proper chromosome segregation.     

Cdk3, a conjugation-specific cyclin-dependent kinase,is essential for the initiation of meiosis in Tetrahymena thermophila

Meiosis is an important process in sexual reproduction. Meiosis initiation has been found to be highly diverse among species. In yeast, it has been established that cyclin-dependent kinases (Cdks) and cyclins are essential components in the meiosis initiation pathway. In this study, we identified 4 Cdks in the model ciliate, Tetrahymena thermophila, and we found one of them, Cdk3, which is specifically expressed during early conjugation, to be essential for meiosis initiation. Cdk3 deletion led to arrest at the pair formation stage of conjugation. We then confirmed that Cdk3 acts upstream of double-strand break (DSB) formation. Moreover, we detected that Cdk3 is necessary for the expression of many genes involved in early meiotic events. Through proteomic quantification of phosphorylation, co-expression analysis and RNA-Seq analyses, we identified a conjugation-specific cyclin, Cyc2, which most likely partners with Cdk3 to initiate meiosis.     

Cyc17, a meiosis-specific cyclin,is essential for anaphase initiation and chromosome segregation in Tetrahymena thermophila

Although the role of cyclins in controlling nuclear division is well established, their function in ciliate meiosis remains unknown. In ciliates, the cyclin family has undergone massive expansion which suggests that diverse cell cycle systems exist, and this warrants further investigation. A screen for cyclins in the model ciliate Tetrahymena thermophila showed that there are 34 cyclins in this organism. Only 1 cyclin, Cyc17, contains the complete cyclin core and is specifically expressed during meiosis. Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings. Further investigation suggested that Cyc17 is involved in regulating spindle pole attachment, and is thus essential for chromosome segregation at meiosis. These findings suggest a simple model in which chromosome segregation is influenced by Cyc17.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

Centromeric histone H3 is essential for vegetative cell division and for DNA elimination during conjugation in Tetrahymena thermophila

The Tetrahymena thermophila CNA1 gene encodes the centromeric H3, Cna1p. Green fluorescent protein (GFP)-tagged Cna1p localizes in micronuclei in dots whose number and behavior during mitosis and conjugation are consistent with centromeres. During interphase, Cna1p-GFP localizes in peripheral dots, suggesting centromeres are associated with the nuclear envelope. Newly synthesized Cna1p-GFP enters micronuclei in mitosis and accumulates in the nucleoplasm. Its deposition at centromeres starts at early S phase and continues through most of S phase. CNA1 is required for vegetative cell growth. Knockdown of CNA1 genes in the somatic macronucleus results in micronuclear DNA loss and delayed chromosome segregation during mitosis. During conjugation, Cna1p-GFP disappears from the centromeres in the developing macronucleus, consistent with centromeric sequences being internal eliminated sequences. Surprisingly, zygotic CNA1 is required for efficient elimination of germ line-specific sequences during development of the new macronuclei but not for the RNA interference pathway, through which sequences are targeted for elimination. Zygotically expressed Cna1p localizes in the spherical structures in which the later stages of DNA elimination occur, and these structures cannot be formed in the absence of zygotic CNA1, suggesting that, in addition to functioning in centromeres, Cna1p may also play a role in organizing the formation of the DNA elimination structures.     

Oral Assembly in Left-Handed Tetrahymena thermophila

We have investigated oral development in a non-genically derived left-handed (LH) form of Tetrahymena thermophila , in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells'anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a sculptured appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180° rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama [36, 37]. We present a model (Fig. 37) for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for secondary OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

Septins stabilize mitochondria in Tetrahymena thermophila

We describe phylogenetic and functional studies of three septins in the free-living ciliate Tetrahymena thermophila. Both deletion and overproduction of septins led to vacuolization of mitochondria, destabilization of the nuclear envelope, and increased autophagy. All three green fluorescent protein-tagged septins localized to mitochondria. Specific septins localized to the outer mitochondrial membrane, to septa formed during mitochondrial scission, or to the mitochondrion-associated endoplasmic reticulum. The only other septins known to localize to mitochondria are human ARTS and murine M-septin, both alternatively spliced forms of Sep4 (S. Larisch, Cell Cycle 3:1021-1023, 2004; S. Takahashi, R. Inatome, H. Yamamura, and S. Yanagi, Genes Cells 8:81-93, 2003). It therefore appears that septins have been recruited to mitochondrial functions independently in at least two eukaryotic lineages and in both cases are involved in apoptotic events.     

Rate of phenotypic assortment in Tetrahymena thermophila

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf/ the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published R_f values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of R_f. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with ～45 assorting units, as has been asserted. © 1992 Wiley-Liss, Inc.     

Histone methyltransferase TXRl is required for both H3 and H3.3lysine 27 methylation in the well-known ciliated protist Tetrahymena thermophila

DNA replication elongation is tightly controlled by histone-modifying enzymes.Our previous studies showed that the histone methytransferase TXRl(Tetrahymena Trithorax related protein 1) specifically catalyzes H3K27 monomethylation and affects DNA replication elongation in Tetrahymena thermophila.In this study,we investigated whether TXRl has a substrate preference to the canonical H3 over the replacement variant H3.3.We demonstrated by histone mutagenesis that K27 Q mutation in H3.3further aggravated the replication stress phenotype of K27 Q mutation in canonical H3,supporting H3.3 as a physiologically relevant substrate of TXRl.This result is in apparent contrast to the strong preference for canonical H3 recently reported in Arabidopsis homologues ATXR5 and ATXR6,and further corroborates the role of TXRl in DNA replication.     

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila

In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming. The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known. Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli. We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+. In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming. These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal. The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal. An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus. Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+.