Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: Starved versus nonstarved

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent (“initiated”) cells were compared with those from non-starved, mating-incompetent (“non-initiated”) cells, by gel electro-phoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glyco-conjugate as having a potential role in control of pair formation during mating. © 1992 Wiley-Liss, Inc.     

Inhibition of Pair Formation by Concanavalin A and Concanavalin A-Binding Glycoconjugates in Euplotes octocarinatus

ABSTRACT. Concanavalin A (≥ 50 μg/ml) inhibits pair formation in both of the two complementary mating types of Euplotes octocarinatus studied in this investigation. This effect can be reversed by methyl-α- d -mannose. Concanavalin A is accessible for methyl-α- d -mannose until pairs are formed. Methyl-α- d -mannose as well as methyl-α- d -glucose and 2-acetamido-2-deoxy- d -glucose alone do not inhibit pair formation unless applied in concentrations ≥ 60 mM. The Concanavalin A-sensitive phase of preconjugant interaction starts 2 h after cells are induced to conjugate. Based on these observations we suggest that Concanavalin A might exhibit its action by binding to carbohydrate moieties of preconjugation-specific adhesion molecules and thereby might allosterically block interactions with their counterparts. To identify preconjugation-specific alterations in number or localization of Concanavalin A-binding glycoconjugates, we probed western blots of total cell proteins or fixed cells, respectively, with digoxigenin-labeled Concanavalin A. On Concanavalin A blots 20 different Concanavalin A-binding glycoconjugates were identified in mating-competent cells. Localization of Concanavalin A-binding sites on mating-competent cells by light microscopy resulted in predominant labeling of a comma-shaped structure near the paroral membranelle. During the preconjugation period no changes in number or localization of Con A-binding glycoconjugates were detected. Possible reasons are discussed.     

Cell pairing and methylation in Tetrahymena thermophila are altered by exogenous homocysteine

Homocysteine is causally associated with birth defects such as spina bifida, and with premature vascular disease. We have investigated the effects of homocysteine on a cell-cell interaction in a fundamental eukaryotic system, the free-living ciliate Tetrahymena. Exogenously added homocysteine inhibits cell pairing in a dose-dependent manner. These effects are exacerbated by adenosine, which by itself has little demonstrable influence on pairing. S-adenosylhomocysteine (SAH) is a product of the reaction between adenosine and homocysteine, and is an inhibitor of methyl transferases. We therefore predicted that protein methylation would be significantly inhibited by homocysteine. A direct test of that hypothesis involved a demonstration that incorporation of an isotopically labeled methyl group from methionine into proteins was significantly reduced by homocysteine. The undermethylated proteins are of low molecular weight, and might correspond to known methylatable signaling proteins. We show that vanadate, an inhibitor of protein phosphatase, also inhibits cell pairing, and that the effects of vanadate and homocysteine are additive. This is the first demonstration that methylation and possibly phosphorylation play a regulatory role in cell-cell interactions in ciliates.     

Stochastic developmental variation in the ratio of allelic rDNAs among newly differentiated,heterozygous macronuclei of Tetrahymena thermophila

Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei. The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell. The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle. Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification. Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR. We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC. We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs. Either rDNA type can be in the majority. Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself. The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4–8 at the most, in a MAC anlage. In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA. Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification. We discuss the relevance of this stochastic developmental variability to classical genetic observations of Nanney and their collaborators on other T. thermophila loci. © 1992 Wiley-Liss, Inc.     

Macronuclear development in conjugants of Tetrahymena thermophila, which were artificially separated at meiotic prophase

Conjugant pairs of Tetrahymena thermophila were mechanically separated by vigorous pipetting at the early stages of meiotic prophase. The complete sequence of conjugational nuclear events including the appearance of pronuclei, development of the new macronuclei (postzygotic development), and resorption of the old macronuclei was observed in the separated cells, without pronuclear exchange. The pronuclei in the separated cells were recognised by the presence of components of the extranuclear cytoskeleton, which were labelled with anti-tubulin and anti-fenestrin antibodies in the same way as in undisturbed conjugants. The apical region of the separated conjugants (the post-junction area), corresponding to the junction area of conjugants was labelled with anti-fenestrin antibody and maintained the properties required for the nuclear development. The results of the genetic study were consistent with a hypothesis that cytogamy (pronuclear fusion) was induced in the separated conjugants. Therefore, the lasting cell contact is not necessary for the successful completion of conjugational nuclear events.     

Development in Electrofused Conjugants of Tetrahymena thermophila

Electric shock can create parabiotic fusions of living Tetrahymena cells. In this study, cells were mated and successful pairs were electrofused with either vegetatively growing cells or other mating pairs. In particular, we electrofused pairs from normal [diploid x diploid] matings with vegetatively dividing cells in G- or M-phase of the cell cycle. We also fused [diploid x diploid] conjugants with mating pairs involving an aneuploid partner [diploid x "star"], which typically undergo an abortive conjugal pathway termed genomic exclusion. Using such parabiotic fusions we identified and characterized two developmentally critical landmarks: 1) the "abort" signal, which is initiated in pairs with nuclear defects (this first becomes evident soon after the completion of Meiosis I or the beginning of Meiosis II); and 2) the "terminal commitment point", a developmental stage in normal [diploid x diploid] pairs after which conjugation no longer responds to a parabiotically transmitted abort signal (this correlates with the onset of the second postzygotic nuclear division). Finally we demonstrate that a conjugal-arrest-activity varies with the vegetative cell cycle, reaching its highest level of activity during M-phase and dropping just after cytokinesis.     

The role of cortical geometry in the nuclear development of Tetrahymena thermophila

Vegetative cells were subjected to electrofusion and the resulting heteropolar doublets were then mated to normal single cells and followed throughout conjugation using cytological and genetic techniques. The unique cyto-geometry created in a heteropolar doublet--a continuous cytoplasmic compartment bounded by two anterior poles and sharing a fused posterior pole at midbody, and the potential for two conjugal exchange junctions--resulted in instructive perturbations of nuclear behavior. Our results indicate that the course of nuclear development is strongly dependent on the cortical geometry of conjugating cells. Specifically, 1) continuation of development after meiosis requires an established conjugal junction; 2) after pronuclear exchange, pronuclei are subjected to attractive forces; and 3) products of the second postzygotic division are actively positioned near the posterior region of the cell cortex where they develop into micronuclei.     

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.     

Interactions between janus and bcd cortical pattern mutants in Tetrahymena thermophila

Summary An analysis of bcd, janA; bcd, janB; and bcd, janC double-mutant phenotypes in Tetrahymena thermophila has allowed us to examine patterning processes affected by two different classes of mutations. bcd brings about a broadening of the oral and contractile vacuole pore domains in the ciliate cortex, while the janus mutations generate a mirror-image duplication of the ventral cortical pattern. We observed both bcd and janus characteristics expressed in the double mutants, as well as features unique to the double-mutant. Temperature-shift experiments employing the temperature-sensitive janB mutation in a double-mutant (bcd, janB) combination allowed us to observe the changes in pattern as a mirror-image geometry was brought into expression and subsequently removed within the bcd, janB double homozygote. These experiments suggest that there are multiple pattern-mechanisms at work with differing kinetics of expression in the ciliate cortex. We discuss how the bcd mutation could influance expression of the janus mutations in light of a model previously proposed to account for the janus phenotype.     

Isolation of a Tetrahymena thermophila strain which induced metaphase I meiotic arrest: new pathway of abortive conjugation

A hypodiploid strain of Tetrahymena thermophila has been obtained that shows arrest at the stage of condensed nuclei, corresponding to metaphase I of normal conjugants and induced arrest at meiotic metaphase I (i.e. at the stage of condensed, bivalent chromosomes) in its wt partner mate. The metaphase I arrested conjugants retained their old macronuclei and most of them underwent cell fusion, instead of separation of exconjugants. The doublets were viable and cortically integrated. When the arrest inducing strain was crossed to the haploid tester strain, the haploid micronuclei were arrested in the meiotic metaphase I as the diploid ones had been; the monovalent, chromosomes were condensed, the arms of sister chromatids were not separated, and they were not segregated. Separation of the arms of sister chromatids and disjunction of bivalent chromosomes were not prerequisite for the formation of microtubular spindles in those cells that were arrested in meiotic metaphase I. After re-feeding, the doublet cells resumed cell divisions, segregating two macronuclei and micronuclei at random. One macronucleus was derived from the arrest inducing strain and the other from the tester strain. Heterokaryon strains with macronuclei derived from the parental arrest inducing strain and with the micronucleus derived from the parental wt tester strain were obtained. Surprisingly, these heterokaryons did not induce meiotic arrest. Thus, the arrest in the melotic metaphase I was induced by the micronucleus and not by the macronucleus of the arrest inducing strain.     

Identification of Tetrahymena Ciliary Surface Proteins Labeled with Sulfosuccinimidyl 6-(Biotinamido) Hexanoate and Concanavalin A and Fractionated with Triton X-114

ABSTRACT. Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from >350 kDa to <20 kDa, were resolved. The major surface 50–60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg²⁺-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: Early expression of the zygotic genotype

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1–2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macro-nuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time. © 1992 Wiley-Liss, Inc.     

Genetic and environmental factors affecting mating type frequency in natural isolates of Tetrahymena thermophila

In Tetrahymena thermophila mating type alleles specify temperature sensitive frequency distributions of multiple mating types. A-like alleles specify mating types I, II, III, V and VI, whereas B-like alleles specify mating types II through VII. We have characterized the mating type distributions specified by several A- and B-like genotypes segregated by genomic exclusion from cells isolated from a pond in northwestern Pennsylvania. The B-like genotypes are alike in specifying very low frequencies of mating type III, but differ with respect to the frequencies of other mating types, particularly II and VII. An A-like genotype specifies a high frequency of mating type III and is unstable in successive generations for the expression of mating type II, suggesting a possible modifier. Inter se crosses performed at 18 degrees C, 28 degrees C and 34 degrees C showed that each genotype specifies a frequency distribution that is uniquely affected by temperature. No mating type was affected the same way by temperature in all genotypes. In A/B heterozygotes, the B-like genotype exhibited partial dominance. The genotypes described here differ significantly from previously described genotypes from the same pond, indicating that there are numerous mating type alleles. For frequency-dependent selection to equalize mating type frequencies, it must act not only on complex multiple alleles but also on the response of mating type alleles to temperature.     

Expression of GFP-actin leads to failure of nuclear elongation and cytokinesis in Tetrahymena thermophila

Green fluorescent protein (GFP)-tagged actin was used to investigate the distribution and function of actin in Tetrahymena. A strain that expresses both GFP-actin and endogenous actin was developed by transformation of Tetrahymena thermophila with a ribosomal DNA-based replicative vector. Confocal microscopy of living cells and immunogold electron microscopy confirmed localization of GFP-actin to basal bodies and the contractile ring. Incorporation of the fusion protein into these and other actin-related structures correlated with severe impairment of macronuclear elongation and cytokinesis. At 30 degrees C macronuclear elongation failed to occur in 25% of the transformants despite completion of micronuclear division. At 20 degrees C macronuclear elongation failed to occur in 2% of the population. Arrest of cytokinesis coincided with failure of macronuclear elongation. Arrested cells developed into homopolar doublets with two sets of oral structures. This study indicates a requirement for actin in nuclear elongation and cytokinesis. Although GFP-actin can interfere with the functioning of actin-containing structures, the GFP-actin transformant strain can be used to monitor actin distribution and dynamics and is therefore an important new tool for further studies of Tetrahymena actin.     

The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila

During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35°C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains). In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity. © 1992 Wiley-Liss, Inc.     

Interdependence of Cell Cycle Events in Tetrahymena thermophila: Formation of Contractile Vacuole Pore and Fission Gap in Ciliary Meridians*

SYNOPSIS The formation of the contractile vacuole pore (CVP) in Tetrahymena thermophila. genotype mol^b/mol^b is temporally and spatially associated with the formation of the fission gap in the CVP meridian. New CVPs arise when fission gaps appear in CVP meridians, the new pores being found anterior to the gaps. When, however, CVP meridians are rotated 180°, the fission gaps develop late. In more than 1/3 of the 180°-rotated CVP meridians, the new CVPs are formed before the appearance of the fission gap. Evidently, the appearance of the gap is not a prerequisite for CVP formation. Nevertheless, mutants exist in which the absence of fission gap and CVP are correlated in some cases and in which the presence of supernumerary fission gap and CVP are correlated in other instances. It is suggested that the 2 developmental events, although not causally related to each other, may be controlled by a common morpho-genetic signal. This commits a certain site (mid-body) along a ciliary meridian to develop the fission gap as well as the CVP; however, after this step of commitment, the appearance of the fission gap is delayed in 180°-rotated CVP meridians.     

Genome analysis of sphingolipid metabolism-related genes in Tetrahymena thermophila and identification of a fatty acid 2-hydroxylase involved in the sexual stage of conjugation

Sphingolipids are bioactive lipids present in all eukaryotes. Tetrahymena thermophila is a ciliate that displays remarkable sphingolipid moieties, that is, the unusual phosphonate-linked headgroup ceramides, present in membranes. To date, no identification has been made in this organism of the functions or related genes implicated in sphingolipid metabolism. By gathering information from the T. thermophila genome database together with sphingolipid moieties and enzymatic activities reported in other Tetrahymena species, we were able to reconstruct the putative de novo sphingolipid metabolic pathway in T. thermophila. Orthologous genes of 11 enzymatic steps involved in the biosynthesis and degradation pathways were retrieved. No genes related to glycosphingolipid or phosphonosphingolipid headgroup transfer were found, suggesting that both conserved and innovative mechanisms are used in ciliate. The knockout of gene TTHERM_00463850 allowed to identify the gene encoding a putative fatty acid 2-hydroxylase, which is involved in the biosynthesis pathway. Knockout cells have shown several impairments in the sexual stage of conjugation since different mating types of knockout strains failed to form cell pairs and complete the conjugation process. This fatty acid 2-hydroxylase gene is the first gene of a sphingolipid metabolic pathway to be identified in ciliates and have a critical role in their sexual stage.     

Evidence for Gene Duplication and Allelic Codominance (not Hierarchical Dominance) at the Mating‐Type Locus of the Ciliate,Euplotes crassus

The high‐multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating‐type substance (pheromone). In strain L‐2D, now known to be homozygous at the mating‐type locus, we previously identified two pheromones (Ec‐α and Ec‐1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR‐73 characterized by a heterozygous genotype and strong mating compatibility with L‐2D strain. It was found that POR‐73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec‐α pheromone of L‐2D cells. The other two pheromones were shown to be new and were designated Ec‐2 and Ec‐3 to denote their structural homology with the Ec‐1 pheromone of L‐2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating‐type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.