Ciliate Mating Types and Pheromones

The story of our knowledge of ciliate mating types and their relevant signaling molecules, originally referred to as mating type substances/factors and today usually described as pheromones (to imply a more general function as chemicals used to communicate between individuals of the same species), is inextricably bound to our understanding about ciliate sexual activity. This activity is commonly manifested as conjugation, autogamy in ciliates, like parthenogenesis in animals, being an additional and much more sporadic alternative. Although conjugation has been described in a variety of organisms, ciliate conjugation is a unique phenomenon of cell-cell interaction and reversible union in mating pairs, that has nothing to do with phenomena of gamete-gamete interaction and irreversible union into a synkaryon. The two ciliates (designated as gamonts) which unite into a conjugal, or mating pair are de facto hermaphrodite, vegetatively reproducing cells which will generate two sexually complementary haploid gametic nuclei, one migratory (male) and one stationary (female), only as result of a meiotic process that involves their diploid micronuclei and is triggered by the cell mating union itself

Identification and partial characterization of cAMP-phosphodiesterases in the ciliate Euplotes raikovi.

In the ciliate Euplotes raikovi, two specific isoforms of cAMP- dependent phosphodiesterases were identified, one in the soluble and the other in the particulate fraction of the cell. Their activity was shown to be stimulated by Mg2+, insensitive to Ca2+ and cGMP, and scarcely inhibited by theophylline and 3-isobutyl-1-methyl-xanthine. They appear to be related to some phosphodiesterases of class II of other unicellular organisms in their biochemical features, and their enzymatic activity is up-regulated by elevation of intracellular cAMP level similarly to PDE-4 isoforms of mammals

THE PROTEIN PHEROMONE FAMILY OF E. PETZI, A PSYCHROPHILIC AND EARLY BRANCHING EUPLOTES SPECIES

Euplotes species are valuable for the study of the structural and functional biology of water-borne protein pheromones that cells constitutively synthesize and use in intra-specific chemical communication. We have recently devoted particular attention to the pheromone family of the “cold loving” (psychrophilic) species E. petzi which dwells in the freezing Antarctic and Arctic coastal sea waters, and forms, together with E. sinicus, the earliest branch of the Euplotes phylogenetic tree. From cultures of genetically distinct strains, we have isolated and sequenced four E. petzi pheromones. With respect to the known pheromones from E. raikovi, E. octocarinatus, E. nobilii and E. crassus, the E. petzi pheromones are smaller (32 amino acids) and richer in Cys residues (eight) located in strictly conserved positions. These residues are predicted to form four intra-chain disulfide bridges, which suggests a compact globular fold of the molecules. However, the NMR solution structure determined for one of the E. petzi pheromones challenges this hypothesis. The structure consists of one more extended eight-residue alpha-helix and one smaller four-residue helix, and shows large polypeptide segments devoid of regular secondary structures. Pheromones from other Euplotes species which live in temperate waters and branch later than E. petzi in the Euplotes phylogenetic tree are known to be characterized by a three-helix fold and unstructured regions of comparatively limited dimensions. In the light of this knowledge, we can thus draw two distinct conclusions from our findings. The first, of phylogenetic nature, is that the structural evolution of the Euplotes pheromones involves an increase in size and complexity. This is in line with the smaller and simpler organization that also the macronuclear E. petzi pheromone genes show with respect to their homologues in other Euplotes species. The second conclusion is that the extended unstructured regions of E. petzi pheromones are likely correlated with an increased flexibility of the molecular backbone and, hence, reflect a common feature of protein cold-adaptation. In this regard, further insights will be obtained by ongoing experiments which aim to assess the unfolding and refolding properties of E. petzi pheromones when exposed to increased temperatures and variations of other environmental parameters

Euplotes pheromones

Species of Euplotes secrete protein signals (pheromones) in relation with their mating-type mechanisms of self-nonself recognition. Significant numbers of these pheromones have been characterized to varied extents of structural complexity in E. raikovi, E. nobilii, and E. octocarinatus. In every case, they form species-specific families of structurally homologous, cysteine-rich proteins. In E. raikovi, these proteins have been shown to share a common architecture based on a three-helix bundle, that permits them to bind to cells in competition with one another and elicit varied, context-dependent responses

Characterization and Expression of the Gene Encoding En-MAPK1, an Intestinal Cell Kinase (ICK)-like Kinase Activated by the Autocrine Pheromone-Signaling Loop in the Polar Ciliate, Euplotes nobilii

In the protozoan ciliate Euplotes, a transduction pathway resulting in a mitogenic cell growth response is activated by autocrine receptor binding of cell type-specific, water-borne signaling protein pheromones. In Euplotes raikovi, a marine species of temperate waters, this transduction pathway was previously shown to involve the phosphorylation of a nuclear protein kinase structurally similar to the intestinal-cell and male germ cell-associated kinases described in mammals. In E. nobilii, which is phylogenetically closely related to E. raikovi but inhabits Antarctic and Arctic waters, we have now characterized a gene encoding a structurally homologous kinase. The expression of this gene requires +1 translational frameshifting and a process of intron splicing for the production of the active protein, designated En-MAPK1, which contains amino acid substitutions of potential significance for cold-adaptation

Cross-talk between the autocrine (mitogenic) pheromone loop of the ciliate Euplotes raikovi and the intracellular cyclic AMP concentration

Cell type-specific protein signals, called pheromones, are constitutively secreted by Euplotes raikovi and bound back in autocrine fashion, with a positive effect on the vegetative (mitotic) cell growth. In cells growing suspended with their secreted pheromone, it was found that any interruption of this autocrine signaling loop was immediately followed by an effective enhancement of the basal intracellular cyclic AMP (cAMP) level. To establish a cause-effect relationship between these pheromone-induced variations in the cytoplasmic cAMP level and cell growth, cells ready to pass from a resting stage to a new growth cycle were conditioned either to incorporate a cAMP analog resistant to phosphodiesterase degradation, or to utilize cAMP released (following cell irradiation) from incorporated “caged” cAMP. Cells responded at every induced increase in their basal cAMP level by markedly decreasing their commitment to start a new growth cycle. It was deduced that the autocrine signaling of E. raikovi pheromones involves cAMP as inhibitor of its mitogenic activity

Genetic relationships in bipolar species of the protist ciliate, Euplotes

Protists thrive in polar oceans, where they represent a major driving force for globally important biogeochemical cycles and a key food-web component. Their biogeography is frequently associated to bipolar patterns of distribution. Although conceptually well supported by apparently unrestricted migration rates, the experimental certification of these patterns copes with the protist paucity of morphological characters with taxonomic value and difficulties in applying conventional species concepts. We studied three marine species of the ciliate Euplotes, E. euryhalinus, E. nobilii, and E. petzi, for their bipolar distribution by comparing the SSU-rRNA gene sequences and mating interactions of Antarctic, Patagonian, and Arctic strains. Each species was analogously found not to carry significantly varied SSU-rRNA gene sequences, implying a common occurrence of trans-equatorial genetic mixing. However, mating analyses revealed significant inter-species differences. Scarce Antarctic x Arctic strain mating compatibility distinguished E. petzi from E. euryhalinus and E. nobilii, in which mating pairs between Antarctic and Arctic strains were successfully induced. Yet, E. nobilii was the only one of the two species to show cross-fertilizing and fertile mating pairs. Taking the biological concept of species as discriminatory, it was thus concluded that only E. nobilii warrants the definition of genuine bipolar species

Homo- and hetero-oligomeric protein–protein associations explain autocrine and heterologous pheromone-cell interactions in Euplotes

In Euplotes, protein pheromones regulate cell reproduction and mating by binding cells in autocrine or heterologous fashion, respectively. Pheromone binding sites (receptors) are identified with membrane-bound pheromone isoforms determined by the same genes specifying the soluble forms, establishing a structural equivalence in each cell type between the two twin proteins. Based on this equivalence, autocrine and heterologous pheromone/receptor interactions were investigated analyzing how native molecules of pheromones Er-1 and Er-13, distinctive of mating compatible E. raikovi cell types, associate into crystals. Er-1 and Er-13 crystals are equally ormed by molecules that associate cooperatively into oligomeric chains rigorously taking a mutually opposite orientation, and each burying two interfaces. A minor interface is pheromone-specific, while a major one is common in Er-1 and Er-13 crystals. A close structural inspection of this interface suggests that it may be used by Er-1 and Er-13 to associate into heterodimers, yet inapt to further associate into higher complexes. Pheromonemolecule homo-oligomerization into chains accounts for clustering and internalization of autocrine pheromone/receptor complexes in growing cells, while the heterodimer unsuitability to oligomerize may explain why heterologous pheromone/receptor complexes fail clustering and internalization. Remaining on the cell surface, they are credited with a key role in cell–cell mating adhesion

Evidence for methionine-sulfoxide-reductase gene transfer from Alphaproteobacteria to the transcriptionally active (macro)nucleus of the ciliate, Euplotes raikovi.

Background: Deleterious phenomena of protein oxidation affect every aerobic organism and methionine residues are their elective targets. The reduction of methionine sulfoxides back to methionines is catalyzed by methionine-sulfoxide reductases (Msrs), enzymes which are particularly active in microorganisms because of their unique nature of individual cells directly exposed to environmental oxidation. Results: From the transcriptionally active somatic genome of a common free-living marine protist ciliate, Euplotes raikovi, we cloned multiple gene isoforms encoding Msr of type A (MsrA) committed to repair methionine-S-sulfoxides. One of these isoforms, in addition to including a MsrA-specific nucleotide sequence, included also a sequence specific for a Msr of type B (MsrB) committed to repair methionine-R-sulfoxides. Analyzed for its structural relationships with MsrA and MsrB coding sequences of other organisms, the coding region of this gene (named msrAB) showed much more significant relationships with Msr gene coding sequences of Rhodobacterales and Rhizobiales (Alphaproteobacteria), than of other eukaryotic organisms. Conclusions: Based on the fact that the msrAB gene is delimited by Euplotes-specific regulatory 5′ and 3′ regions and telomeric C4A4/G4T4 repeats, it was concluded that E. raikovi inherited the coding region of this gene through a phenomenon of horizontal gene transfer from species of Alphaproteobacteria with which it coexists in nature and on which it likely feeds

Evolution of the intracellular transport mechanisms in eukaryotes: ciliates and mammals use the same translocation and nuclear localization signals

In the ciliate E. raikovi, self/non-self recognition phenomena are controlled by cell type-specific, water-borne signal proteins (pheromones) by their binding to target cell-surface receptors. The downstream signal transduction pathway activated by the pheromone-receptor interactions of self type (that promote the vegetative, mitogenic cell growth) involves the phosphorylation of a nuclear protein kinase, designated Er-MAPK1, which is structurally similar to the "intestinal-cell kinase" and "male germ cell-associated kinase" described in mammals. To identify the sequence segments responsible for Er-MAPK1 nuclear localization, mouse fibroblasts were transfected with plasmids containing the reporter gene for the "Green-Fluorescent Protein" (GFP) associated to different fragments of the Er-MAPK1 coding sequence. By expressing GFP-tagged protein constructs in mammalian cells, in the C-terminal domain of Er-MAPK1 it was effectively possible to identify an Arg/Lys-rich motif that is required for the nuclear entry of GFP-fused constructs. These results provide evidence that distant related organisms such as ciliates and mammals use the same molecular language for the nuclear translocation and localization of proteins, thus suggesting that this language arose early in the evolution of the eukaryotic cell