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

Comparative Studies on the Polymorphism and Copy Number Variation of mtSSU rDNA in Ciliates (Protista, Ciliophora): Implications for Phylogenetic, Environmental, and Ecological Research

While nuclear small subunit ribosomal DNA (nSSU rDNA) is the most commonly‐used gene marker in studying phylogeny, ecology, abundance, and biodiversity of microbial eukaryotes, mitochondrial small subunit ribosomal DNA (mtSSU rDNA) provides an alternative. Recently, both copy number variation and sequence variation of nSSU rDNA have been demonstrated for diverse organisms, which can contribute to misinterpretation of microbiome data. Given this, we explore patterns for mtSSU rDNA among 13 selected ciliates (representing five classes), a major component of microbial eukaryotes, estimating copy number and sequence variation and comparing to that of nSSU rDNA. Our study reveals: (1) mtSSU rDNA copy number variation is substantially lower than that for nSSU rDNA; (2) mtSSU rDNA copy number ranges from 1.0 × 10 to 8.1 × 10 ; (3) a most common sequence of mtSSU rDNA is also found in each cell; (4) the sequence variation of mtSSU rDNA are mainly indels in poly A/T regions, and only half of species have sequence variation, which is fewer than that for nSSU rDNA; and (5) the polymorphisms between haplotypes of mtSSU rDNA would not influence the phylogenetic topology. Together, these data provide more insights into mtSSU rDNA as a powerful marker especially for microbial ecology studies. 4

Timing and characteristics of nuclear events during conjugation and genomic exclusion in Paramecium multimicronucleatum

Ciliated protists are ideal material for studying the origin and evolution of sex, because of their nuclear dimorphism (containing both germline micronucleus and somatic macronucleus in the same cytoplasm), special sexual processes (conjugation and autogamy), and high diversity of mating-type systems. However, the study of sexual process is limited to only a few species, due to the difficulties in inducing or observing conjugation. In the present study, we investigate the conjugation process in Paramecium multimicronucleatum: (1) of the three prezygotic divisions, all micronuclei undergo the first two divisions (meiosis I, II), while a variable number of nuclei undergo the third division (mitosis); (2) the synkaryon divides three times after fertilization, giving rise to eight products that differentiate into four macronuclear anlagen and four micronuclei; (3) cells restore the vegetative stage after two successive cell fissions during which the macronuclear anlagen are distributed into daughter cells without division, while micronuclei divide mitotically; (4) the parental macronucleus begins to fragment following the first meiotic division and finally degenerates completely; (5) the entire process takes about 110 h, of which about 85 h are required for macronuclear development. In addition, we describe for the first time the process of genomic exclusion occurring between amicronucleate and micronucleate cells of P. multimicronucleatum, during which the micronucleate cell contributes a pronucleus to the amicronucleate cell, resulting in both exconjugants being homozygotes. These results provide new insights into the diversity of sexual processes and lay an important cytological basis for future in-depth studies of mating systems in ciliates

Genome Analyses of the New Model Protist \u3ci\u3eEuplotes vannus\u3c/i\u3e Focusing on Genome Rearrangement and Resistance to Environmental Stressors

As a model organism for studies of cell and environmental biology, the free-living and cosmopolitan ciliate Euplotes vannus shows intriguing features like dual genome architecture (i.e., separate germline and somatic nuclei in each cell/organism), “gene-sized” chromosomes, stop codon reassignment, programmed ribosomal frameshifting (PRF) and strong resistance to environmental stressors. However, the molecular mechanisms that account for these remarkable traits remain largely unknown. Here we report a combined analysis of de novo assembled high-quality macronuclear (MAC; i.e., somatic) and partial micronuclear (MIC; i.e., germline) genome sequences for E. vannus, and transcriptome profiling data under varying conditions. The results demonstrate that: (a) the MAC genome contains more than 25,000 complete “gene-sized” nanochromosomes (~85 Mb haploid genome size) with the N50 ~2.7 kb; (b) although there is a high frequency of frameshifting at stop codons UAA and UAG, we did not observe impaired transcript abundance as a result of PRF in this species as has been reported for other euplotids; (c) the sequence motif 5′-TA-3′ is conserved at nearly all internally-eliminated sequence (IES) boundaries in the MIC genome, and chromosome breakage sites (CBSs) are duplicated and retained in the MAC genome; (d) by profiling the weighted correlation network of genes in the MAC under different environmental stressors, including nutrient scarcity, extreme temperature, salinity and the presence of ammonia, we identified gene clusters that respond to these external physical or chemical stimulations, and (e) we observed a dramatic increase in HSP70 gene transcription under salinity and chemical stresses but surprisingly, not under temperature changes; we link this temperature-resistance to the evolved loss of temperature stress-sensitive elements in regulatory regions. Together with the genome resources generated in this study, which are available online at Euplotes vannus Genome Database (http://evan.ciliate.org), these data provide molecular evidence for understanding the unique biology of highly adaptable microorganisms

Fluorescent sensing of anions based on excited state intramolecular proton transfer in N-(3-hydroxy-2-naphthamido)-N'-phenylthiourea

A neutral N-amidothiourea-based excited state intramolecular proton transfer (ESIPT) anion receptor bearing an o-hydroxynaphthamide fluorophore and a thiourea binding site, N-(3-hydroxy-2-naphthamide)-N'-phenylthiourea (1a), was designed and synthesized. Fluorescence and absorption response of 1a toward anions were assessed in acetonitrile. IR and NMR experiments indicated that the "OHa <-O=C" intramolecular hydrogen bond (IHB) in 1a was weak so that it only exhibited the short-wavelength normal emission other than ESIPT fluorescence. Due to the high anion binding affinity of the N-amidothiourea binding site and the formation of a hydrogen binding network in the 1a-anion complex, 1a underwent structural change upon anion binding that strengthens the "OHa <-O=C" IHB, leading to the ESIPT and the observation of the long-wavelength ESIPT emission whereas the normal fluorescence is quenched. On the basis of NMR and fluorescence titrations and control experiments with model compounds, a sensing mechanism of the anion-binding-induced ESIPT was proposed

Primary Structure and Coding Genes of Two Pheromones from the Antarctic Psychrophilic Ciliate, <i>Euplotes focardii</i>

In ciliates, diffusible cell type-specific pheromones regulate cell growth and mating phenomena acting competitively in both autocrine and heterologous fashion. In Euplotes species, these signaling molecules are represented by species-specific families of structurally homologous small, disulfide-rich proteins, each specified by one of a series of multiple alleles that are inherited without relationships of dominance at the mat-genetic locus of the germinal micronuclear genome, and expressed as individual gene-sized molecules in the somatic macronuclear genome. Here we report the 85-amino acid sequences and the full-length macronuclear nucleotide coding sequences of two pheromones, designated Ef-1 and Ef-2, isolated from the supernatant of a wild-type strain of a psychrophilic species of Euplotes, E. focardii, endemic to Antarctic coastal waters. An overall comparison of the determined E. focardii pheromone and pheromone-gene structures with their homologs from congeneric species provides an initial picture of how an evolutionary increase in the complexity of these structures accompanies Euplotes speciation

Time-course analysis of nuclear events during conjugation in the marine ciliate Euplotes vannus and comparison with other ciliates (Protozoa, Ciliophora)

Ciliates represent a morphologically and genetically distinct group of single-celled eukaryotes that segregate germline and somatic functions into two types of nuclei and exhibit complex cytogenetic events during the sexual process of conjugation, which is under the control of the so-called ‘mating type systems’. Studying conjugation in ciliates may provide insight lead to major advances into our understanding of the origins and evolution of sex and fertilization. In the present work, we studied in detail the sexual process of conjugation using the model species Euplotes vannus, and compared these nuclear events with those occurring in other ciliates. Our results indicate that in E. vannus: 1) conjugation requires about 75 hours to complete: the longest step is the development of the new macronucleus (ca. 64h), followed by the nuclear division of meiosis I (5h); the mitotic divisions usually take only 2h; 2) there are three prezygotic divisions (mitosis and meiosis I and II), and two of the eight resulting nuclei become pronuclei; 3) after the exchange and fusion of the pronuclei, two postzygotic divisions occur; two of the four products differentiate into the new micronucleus and macronucleus, respectively, and the parental macronucleus degenerates completely; 4) comparison of the nuclear events during conjugation in different ciliates reveals that there are generally three prezygotic divisions while the number of postzygotic divisions is highly variable. These results can serve as reference to investigate the mating type system operating in this species and to analyze genes involved in the different steps of the sexual process

Conjugation in Euplotes raikovi (Protista, Ciliophora): New Insights into Nuclear Events and Macronuclear Development from Micronucleate and Amicronucleate Cells

Ciliates form a distinct group of single‐celled eukaryotes that host two types of nuclei (micro and macronucleus) in the same cytoplasm and have a special sexual process known as conjugation, which involves mitosis, meiosis, fertilization, nuclear differentiation, and development. Due to their high species diversity, ciliates have evolved different patterns of nuclear events during conjugation. In the present study, we investigate these events in detail in the marine species Euplotes raikovi. Our results indicate that: (i) conjugation lasts for about 50 hours, the longest stage being the development of the new macronucleus (ca. 36 hours); (ii) there are three prezygotic micronuclear divisions (mitosis and meiosis I and II) and two postzygotic synkaryon divisions; and (iii) a fragment of the parental macronucleus fuses with the new developing macronucleus. In addition, we describe for the first time conjugation in amicronucleate E. raikovi cells. When two amicronucleate cells mate, they separate after about 4 hours without evident nuclear changes; when one amicronucleate cell mates with a micronucleate cell, the micronucleus undergoes regular prezygotic divisions to form migratory and stationary pronuclei, but the two pronuclei fuse in the same cell. In the amicronucleate cell, the parental macronucleus breaks into fragments, which are then recovered to form a new functional macronucleus. These results add new information on the process of conjugation in both micronucleate and amicronucleate Euplotes cells