Alternative gene expression in type I and type II cells may enable further nuclear changes during conjugation of Blepharisma japonicum

In contrast to most ciliates, meiosis and successive nuclear changes during conjugation occur only in heterotypic pairs in Blepharisma. It has been suggested that homotypic pairs are ready for conjugation, but lack a trigger to initiate the nuclear changes, and the conjugation process is arrested before the onset of meiosis. To explore the possible nature of the trigger, we previously identified the genes BjCdk1 (homologous to cdk1/cdc2), Bj4HPPD (4-hydroxy-phenylpyruvate dioxygenase) and BjCks (cyclin dependent kinase regulatory subunit) whose expression is up-regulated in gamone1-treated type II cells. In this study, we investigated the molecular structures of these three genes, and compared their expression patterns in homotypic and heterotypic pairs, finding remarkable differences. BjCdk1, Bj4HPPD and BjCks were expressed specifically in gamone1-treated type II cells, but not in gamone2-treated type I cells. In heterotypic pairs, the expression of these genes stayed at the same level or gradually decreased throughout the entire process of conjugation, but it rapidly decreased and ceased after 10hours in homotypic pairs. These results indicate that some genes are expressed in a mating-type specific manner. Alternative gene expression in mating type I and type II cells and merging of individual factors in a heterotypic pair may induce nuclear changes including meiosis.     

Propagation of meiosis and other nuclear changes in multicellular complexes of Blepharisma

Meiosis and other nuclear changes in conjugation of Blepharisma japonicum regularly occur when cells of complementary mating types I and II unite (heterotypic union). But no nuclear changes occur if unions are induced between cells of the same mating type (homotypic union). Similarly, chains of homotypically united cells, induced by treating type II cells of a doublet strain (mutant with two attachment points) with gamone of mating type I, do not undergo nuclear changes. However, if a type I cell unites at one end of such a chain, the nuclear changes of conjugation occur not only in the doublet to which the type I cell unites but also in other doublets in the same chain. We examined the mode of propagation of nuclear activation by surgically separating all cells in the chain and observing the subsequent occurrence of nuclear changes in these isolated cells. The nuclear activation began at the site of heterotypic union and propagated from cell-to-cell without skipping. In chains of a given length, the propagation slowed down as it proceeded in the chain. If compared at the corresponding site of the chain, the propagation was slower in longer chains. We conclude that meiosis and other nuclear changes in conjugation are initiated by a substance originating at the site of heterotypic union and transferable to other cells through the united regions of the cells.     

Chemically moderated gamete preferences predict offspring fitness in a broadcast spawning invertebrate

Sperm chemoattraction, where sperm locate unfertilized eggs by following a concentration gradient of egg-derived chemoattractants, has been widely documented across numerous taxa. While marine invertebrates are favoured models for understanding the underlying mechanisms of sperm chemoattraction, the evolutionary forces underpinning the process remain enigmatic. Here, we show that in mussels (Mytilus galloprovincialis), chemically moderated gamete preferences promote assortative fertilizations between genetically compatible gametes. When offered the choice of egg clutches from two females, sperm exhibited consistent but differential ‘preferences’ for chemical cues secreted from conspecific eggs. Critically, our data reveal that the preferences shown by sperm during the egg-choice trials are highly predictive of early embryonic viability when eggs and sperm from the same individuals are mixed during standard (no-choice) fertilization assays. Moreover, we demonstrate that by experimentally separating chemoattractants from eggs, sperm swimming behaviour is differentially regulated by egg-derived chemoattractants, and that these changes in sperm behaviour are highly consistent with observed patterns of gamete preferences, fertilization and larval survival. Together, this integrated series of experiments reveals that the behaviour of sperm is fine-tuned to respond differentially to the chemical signals emitted from different conspecific eggs, and that these choices have measurable fitness benefits.     

ConcanavalinA receptors and the chemosensory behaviour of Tetrahymena thermophila

The relationship between concanavalin A (ConA) receptors and the chemosensory behaviour of the ciliated protozoan Tetrahymena thermophila was studied using the peptide chemoattractants proteose peptone and fibroblast growth factor. Studies on the chemosensory behaviour in semisolid methylcellulose showed that 50 μg/ml ConA selectively inhibited the persistent element of swimming behaviour by reducing time runs of cells responding to proteose peptone from 12.2±4.5 min to 0.8±0.3 min. Methyl-alpha-D-mannoside, but not methyl-alpha-D-galactoside, abolished the inhibitory effect of ConA, suggesting that mannoside-containing ConA receptors are involved in maintaining a persistent swimming behaviour. Control experiments, carried out in liquids where persistent swimming is less important for cellular behaviour, showed that ConA did not affect proteose-peptone-induced chemoattraction under these conditions as measured by a two-phase assay for chemoattraction. Also, no inhibitory effect of ConA could be found on swimming rates when individual velocities of ConA-treated cells were determined. When tested in liquid chemoattraction assays, ConA was found to be a weak but significant chemoattractant. Studies of the cellular location of ConA receptors on the plasma membrane of starved cells showed an unequal distribution. A preferential clustering of receptors at the anterior end of the cell was observed when determined at high concentrations (100 μg/ml) of fluorescent ConA. Methyl-alpha-D-mannoside but not methyl-alpha-D-galactoside abolished the fluorescent ConA labelling, indicating a preferential clustering of these mannoside-containing receptors at the anterior part of the plasma membrane and cilia. At lower concentrations (25 μg/ml), FITC-ConA produced more general labelling of the entire cell membrane. The results suggest that ConA receptors are necessary for the persistent element of swimming and that binding of ConA to its receptors interferes with processes related to signal transduction rather than by limiting the free movement of cilia required for locomotion. The gradient of receptors seen at high FITC-ConA concentrations may be important for a putative spatial chemosensory mechanism, i.e. chemotaxis.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Gamone Production in Large-Scale Cultures of Euplotes octocarinatus1

We report a method that allows us to grow and maintain the freshwater ciliate Euplotes octocarinatus in large quantities. Frequent exchange of culture fluid proved more effective than aeration in obtaining high cell densities (4200 cells/ml) and reasonable doubling times in large-scale cultures. For harvesting gamone 1, the cell density was raised to 10,000 cells/ml. Under these conditions, the cells continued to produce and secrete gamone; they were slightly starved, but they no longer divided. Cell-free fluid with a steady and relatively high yield of gamone was obtained from two such cultures over a period of five months. We isolated gamone 1 also from cell homogenates and compared it with secreted gamone 1, but found no differences in the gamones from these two sources.     

A surface glycoprotein indispensable for gamete fusion in the social amoeba Dictyostelium discoideum

Sexual reproduction is essential for the maintenance of species in a wide variety of multicellular organisms, and even unicellular organisms that normally proliferate asexually possess a sexual cycle because of its contribution to increased genetic diversity. Information concerning the molecules involved in fertilization is accumulating for many species of the metazoan, plant, and fungal lineages, and the evolutionary consideration of sexual reproduction systems is now an interesting issue. Macrocyst formation in the social amoeba Dictyostelium discoideum is a sexual process in which cells become sexually mature under dark and submerged conditions and fuse with complementary mating-type cells. In the present study, we isolated D. discoideum insertional mutants defective in sexual cell fusion and identified the relevant gene, macA, which encodes a highly glycosylated, 2,041-amino-acid membrane protein (MacA). Although its overall similarity is restricted to proteins of unknown function within dictyostelids, it contains LamGL and discoidin domains, which are implicated in cell adhesion. The growth and development of macA-null mutants were indistinguishable from those of the parental strain. The overexpression of macA using the V18 promoter in a macA-null mutant completely restored its sexual defects. Although the macA gene encoded exactly the same protein in a complementary mating-type strain, it was expressed at a much lower level. These results suggest that MacA is indispensable for gamete interactions in D. discoideum, probably via cell adhesion. There is a possibility that it is controlled in a mating-type-dependent manner.     

Unidirectional mating-type switching confers self-fertility to Thielaviopsis cerberus,the only homothallic species in the genus

Sexual reproduction is ubiquitous in nature, and nowhere is this more so than in the fungi. Heterothallic behaviour is observed when there is a strict requirement of contact between two individuals of opposite mating type for sexual reproduction to occur. In contrast, a homothallic species can complete the entire sexual cycle in isolation, although several genetic mechanisms underpin this self-fertility. These can be inferred by characterising the structure and gene-content of the mating-type locus, which contains genes that are involved in the regulation of sexual reproduction. In this study, the genetic basis of homothallism in Thielaviopsis cerberus was investigated, the only known self-fertile species within this genus. Using genome sequencing and conventional molecular techniques, two versions of the mating-type locus were identified in this species. This is typical of species that have a unidirectional mating-type switching reproductive strategy. The first version was a self-fertile locus that contained four known mating-type genes, while the second was a self-sterile version with a single mating-type gene. The conversion from a self-fertile to a self-sterile locus is likely mediated by a homologous recombination event at two direct repeats present in the self-fertile locus, resulting in the deletion of three mating-type genes and one of the repeats. Both locus versions were present in isolates that were self-fertile, while self-sterility was caused by the presence of only a switched locus. This study provides a clear example of the architectural fluidity in the mating-type loci that is common among even closely related fungal species.     

Species-specific aggregation factor in sponges. IV. Inactivation of the aggregation factor by mucoid cells from another species.

The role of protein synthesis in the cell union was investigated in conjugation of Blepharisma intermedium. In order to avoid possible complications due to the occurrence of other processes in conjugation, the homotypic cell union, in which conjugation is arrested at the stage of cell union without further changes, was used. Such unions were induced by treating cells of one mating type with the gamone of the other mating type for about 2 h. The induction of cell union was regularly accompanied by increased protein synthesis, which started 5 min after the beginning of the gamone treatment and continued for about 2 h. When protein synthesis was inhibited by cycloheximide, cell union was also inhibited. The extent of the two inhibitions were closely correlated. We concluded that gamone induces proteins and that protein synthesis is essential for cell union. Proteins synthesized in gamone-treated and non-treated cells were also separated and compared. Consideration of these results leads to a hypothesis that most of the gamone-induced proteins are membrane proteins normally synthesized, though in lesser amount, in non-conjugating cells and that cells gain the capacity to unite when these proteins are accumulated at a restricted area on the cell surface by another gamone-controlled mechanism.     

A newly identified chemotactic sexual pheromone from Closterium ehrenbergii

 In sexual reproduction of Closterium ehrenbergii, pairing with the sexual partner cells is the first process observed. A cell migration-inducing activity, specific for mating-type plus (mt⁺; NIES-228) cells, was detected in the culture medium of mating-type minus (mt^–; NIES-229) cells. Light was necessary for production of the active substance by mt^– cells and for migration of mt⁺ cells. The active substance was heat-labile and had an apparent molecular mass of 20 kDa, as determined by gel filtration. A protein of 20 kDa was detected in the active fraction of gel filtration after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on these results, it is proposed that a chemotactic sexual pheromone involved in the formation of sexual pairs of cells is secreted by mt^– cells of C. ehrenbergii and is proteinaceous, like other sexual pheromones secreted by Closterium species. Received: 31 July 1997 / Revision accepted: 25 November 1997     

Isolation and partial characterization of gamone 1 of Euplotes octocarinatus

A polypeptide, termed gamone 1, was isolated and purified to homogeneity from culture filtrates of mating type VII of the freshwater ciliate Euplotes octocarinatus. The gamone induces intraclonal conjugation in cells of certain other mating types. The isolation and purification of the gamone was carried out by a combination of two chromatographic steps. The purified gamone was found to be still effective in a concentration of approx. 10⁻¹⁵M.     

Cell surface control in Blepharisma intermedium (Protozoa ciliata). Blocking of gamone II-induced homotypic pairing by different membrane interacting ligands

PHA, Con-A, or anti-tubulin antibodies inhibit homotypic pair formation, in B. intermedium mating type-I cells in the presence of suboptimal concentrations of gamone II. The inhibition is dependent on the dose of gamone added; the structural conformation and the relative concentration of the inhibitor; and the time of addition of the inhibitor. The block can be selectively prevented by competitive inhibitors of each ligand. The receptors for the inhibitors are distinctive and there is no cross-reaction between the ligands. It is concluded that ligand binding and subsequent receptor-ligand aggregation must induce a change within the cell-surface membrane, which distorts the distribution and/or affects an optimal conformational aspect of a specific membrane-receptor system for the gamone, a prerequisite for cell pair formation.     

Odd mating-type substances may work as precursor molecules of even mating-type substances in Paramecium caudatum

Mating-type substances are key molecules in the sexual recognition of the odd (O) and even (E) complementary mating-type cells in Paramecium caudatum. Indirect evidence suggested that the substances were proteins and were located on ventral surface cilia. Monoclonal antibodies inhibiting the mating reactivity of the O cells have been obtained. Using these antibodies, we tried to detect antigen molecules as dot-blot signals. Strong dot-blot signals of antigens were only detected from the mating reactive cells, but they were not detected from the well-fed and starved cells without mating reactivity. In addition to identifying the antigen on cilia and cytoplasm of the O cell, the antigen was detected from the cytoplasm of the E cells but never from their cilia. Furthermore, extracts of the E cells induced mating reaction with the living E cells but not with O cells. Thus, the O mating-type substances exist in the cytoplasm of the E mating-type cells, supporting strongly the hypothesis that O mating-type substances are precursor molecules of the E mating-type substances.     

DNA methylation in vegetative and conjugating cells of a protozoan ciliate: Blepharisma japonicum

We report here the presence of N⁶-methyladenine (MeAde) in the macronuclear DNA (maDNA) of Blepharisma japonicum vegetative cells. We have further investigated the relationship between DNA methylation and cell union in cells activated for conjugation. Such activation was induced by treating cells of mating type I with complementary gamone 2. We found a reduction of about 24% of MeAde content in gamone-treated cells ready for cell union. First indications of the presence and reduction of MeAde content came from electrophoresis of maDNA digested by appropriate restriction endonucleases. Chromatographic determination of the amount of methylated base by HPLC substantiated these observations. In vegetative cells, 1.576 ± 0.02% of total adenine was found to be methylated as opposed to 1.193 ± 0.04% in activated cells. The HPLC analysis of maDNA also revealed a peak with a retention time corresponding to that of 5-hydroxymethyluracil, already found in some species of dinoflagellates. In that gamone treatment is correlated with a differential gene expression (indicated by a differential RNA and protein synthesis), our results suggest that there is a relationship between macronuclear genome activation and demethylation of maDNA. This is the first report of a correlation between gene activation and adenine demethylation in a eukaryotic organism.     

Entropy-driven motility of Sinorhizobium meliloti on a semi-solid surface

Sinorhizobium meliloti growing on soft agar can exhibit an unusual surface spreading behaviour that differs from other bacterial surface motilities. Bacteria in the colony secrete an exopolysaccharide-rich mucoid fluid that expands outward on the surface, carrying within it a suspension of actively dividing cells. The moving slime disperses the cells in complex and dynamic patterns indicative of simultaneous bacterial growth, swimming and aggregation. We find that while flagellar swimming is required to maintain the cells in suspension, the spreading and the associated pattern formation are primarily driven by the secreted exopolysaccharide EPS II, which creates two entropy-increasing effects: an osmotic flow of water from the agar to the mucoid fluid and a crowding or depletion attraction between the cells. Activation of these physical/chemical phenomena may be a useful function for the high molecular weight EPS II, a galactoglucan whose biosynthesis is tightly regulated by the ExpR/SinI/SinR quorum-sensing system: unlike bacterial colonies that spread via bacterium-generated, physical propulsive forces, S. meliloti under quorum conditions may use EPS II to activate purely entropic forces within its environment, so that it can disperse by passively ‘surfing’ on those forces.     

Oligomeric Structure of Type I and Type II Transforming Growth Factor β Receptors: Homodimers Form in the ER and Persist at the Plasma Membrane

Abstract. Transforming growth factor β (TGF-β) signaling involves interactions of at least two different receptors, types I (TβRI) and II (TβRII), which form ligand-mediated heteromeric complexes. Although we have shown in the past that TβRII in the absence of ligand is a homodimer on the cell surface, TβRI has not been similarly investigated, and the site of complex formation is not known for either receptor. Several studies have indicated that homomeric interactions are involved in TGF-β signaling and regulation, emphasizing the importance of a detailed understanding of the homooligomerization of TβRI or TβRII. Here we have combined complementary approaches to study these homomeric interactions in both naturally expressing cell lines and cells cotransfected with various combinations of epitope-tagged type I or type II receptors. We used sedimentation velocity of metabolically labeled receptors on sucrose gradients to show that both TβRI and TβRII form homodimer-sized complexes in the endoplasmic reticulum, and we used coimmunoprecipitation studies to demonstrate the existence of type I homooligomers. Using a technique based on antibody-mediated immunofluorescence copatching of receptors carrying different epitope tags, we have demonstrated ligand-independent homodimers of TβRI on the surface of live cells. Soluble forms of both receptors are secreted as monomers, indicating that the ectodomains are not sufficient to mediate homodimerization, although TGF-β1 is able to promote dimerization of the type II receptor ectodomain. These findings may have important implications for the regulation of TGF-β signaling.     

Cell-to-cell contact by locally differentiated surfaces in conjugation of Blepharisma

Local functional differences of the cell surface were investigated in the formation of cell unions in conjugation of Blepharisma intermedium. When cells of this ciliate are treated by the gamone (the conjugation-inducing substance) of the complementary mating type, they first unite by cilia and then more intimately by the direct contact of cell bodies. The ciliary union was found to be formed by a specific contact between two morphologically distinguishable surfaces, the adoral zone of membranelles (AZM) of one cell and the anterior extension of the undulating membrane (extUM) of the other. The AZM gains the capacity to unite first and the extUM next. The extUM of gamone-treated cells sticks not only to the AZM but also to glass and some other artificial substrata under some conditions. These stickings are inhibited by cycloheximide which also inhibits the ciliary union. The role of specific contact between AZM and extUM in the cell recognition is discussed.     

Distal and Proximal Actions of Peptide Pheromone M-Factor Control Different Conjugation Steps in Fission Yeast

Mating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in Schizosaccharomyces pombe. A peptide pheromone, M-factor, produced by M-cells is recognized by the receptor of P-cells. An M-factor-less mutant, in which the M-factor-encoding genes are deleted, is completely sterile. In liquid culture, sexual agglutination was not observed in the mutant, but it could be recovered by adding exogenous synthetic M-factor, which stimulated expression of the P-type-specific cell adhesion protein, Map4. Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant. When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed. Notably, M-factor-less mutant cells were also incorporated in these aggregates. In this mixed culture, P-cells conjugated preferentially with M-cells secreting M-factor, and rarely with M-factor-less M-cells. The kinetics of mating parameters in liquid culture revealed that polarized growth commenced from the contact region of opposite mating-type cells. Taken together, these findings indicate that M-factor at a low concentration induces adhesin expression, leading to initial cell-cell adhesion in a type of “distal pheromone action”, but M-factor that is secreted directly in the proximity of the adhered P-cells may be necessary for cell fusion in a type of “proximal pheromone action”.     

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.     

Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, α and a. The α and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The α and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.     

Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones

In the yeast Saccharomyces cerevisiae, sexual conjugation between haploid cells of opposite mating type results in the formation of a diploid zygote. When treated with fluorescently labeled concanavalin A, a zygote stains nonuniformly, with the greatest fluorescence occurring at the conjugation bridge between the two haploid parents. In the mating mixture, unconjugated haploid cells often elongate to pear-shaped forms ("shmoos") which likewise exhibit asymmetric staining with the most intense fluorescence at the growing end. Shmoo formation can be induced in cells of one mating type by the addition of a hormone secreted by cells of the opposite mating type; such shmoos also stain asymmetrically. In nearly all cases, the nonmating mutants that were examined stained uniformly after incubation with the appropriate hormone. Asymmetric staining is not observed with vegetative cells, even those that are budded. These results suggest that, before and during conjugation, localized cell surface changes occur in cells of both mating types; the surface alterations facilitate fusion and are apparently mediated by the hormones in a manner that is mating-type specific.