Search

The pervasiveness of sex despite its well-known costs is a long-standing puzzle in evolutionary biology. Current explanations for the success of sex in nature largely rely on the adaptive significance of the new or rare genotypes that sex may generate. Less explored is the possibility that sex-underlying molecular mechanisms can enhance fitness and convey benefits to the individuals that bear the immediate costs of sex. Here, we show that the molecular environment associated with self-fertilization can increase stress resistance in the ciliate Paramecium tetraurelia. This advantage is independent of new genetic variation, coupled with a reduced nutritional input, and offers fresh insights into the mechanistic origin of sex. In addition to providing evidence that the molecular underpinnings of sexual reproduction and the stress response are linked in P. tetraurelia, these findings supply an integrative explanation for the persistence of self-fertilization in this ciliate.

This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of Paramecium caudatum is a programmed process conducted by a specific gene and its product protein. In Paramecium tetraurelia, autogamy was revealed to depend on the number of DNA syntheses rather than the number of cell divisions in clonal aging. The cytoplasmic complementarity test established that microinjection of wild-type cytoplasm can correct genetic defects of mutants. The concept of complementarity together with protein chemistry revealed compounds that control membrane excitability. In non-Mendelian inheritance, noncoding small RNAs made from the parental micronucleus regulate the rearrangement of the progeny's macronuclear DNA. The macronucleus has the potential to be used as a factory for genetic engineering. The development and differentiation of progeny's nuclei in mating pairs are controlled by the parental macronucleus. The chemical reaction processes associated with exocytosis have been revealed by microinjection of various enzymes and antibodies. Using the fusion gene of histone H2B and yellow-fluorescence protein, it was revealed that the fusion gene-mRNA is transferred between cells during mating. Experiments with endosymbiotic bacteria and the host shed light on the conditions needed to establish sustainable symbiotic relationships.

Noncoding RNAs (ncRNAs) make up to ∼98% percent of the transcriptome of a given organism. In recent years, one relatively new class of ncRNAs, long noncoding RNAs (lncRNAs), were shown to be more than mere by-products of gene expression and regulation. The unicellular eukaryote Paramecium tetraurelia is a member of the ciliate phylum, an extremely heterogeneous group of organisms found in most bodies of water across the globe. A hallmark of ciliate genetics is nuclear dimorphism and programmed elimination of transposons and transposon-derived DNA elements, the latter of which is essential for the maintenance of the somatic genome. Paramecium and ciliates in general harbor a plethora of different ncRNA species, some of which drive the process of large-scale genome rearrangements, including DNA elimination, during sexual development. Here, we identify and validate the first known functional lncRNAs in ciliates to date. Using deep-sequencing and subsequent bioinformatic processing and experimental validation, we show that Paramecium expresses at least 15 lncRNAs. These candidates were predicted by a highly conservative pipeline, and informatic analyses hint at differential expression during development. Depletion of two lncRNAs, lnc1 and lnc15, resulted in clear phenotypes, decreased survival, morphological impairment, and a global effect on DNA elimination.

The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline and somatic nuclei, massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.

Mutation accumulation (MA) experiments are conventionally employed to study spontaneous germline mutations. However, MA experiments can also shed light on somatic genome plasticity in a habitual and genetic drift-maximizing environment. Here, we revisit an MA experiment that uncovered extraordinary germline genome stability in Paramecium tetraurelia, a single-celled eukaryote with nuclear dimorphism. Our re-examination of isogenic P. tetraurelia MA lines propagated in nutrient-rich medium for >40 sexual cycles reveals that their polyploid somatic genome accrued hundreds of intervening DNA segments (IESs), which are normally eliminated during germline-soma differentiation. These IESs frequently occupy a fraction of the somatic DNA copies of a given locus, producing IES excision/retention polymorphisms, and preferentially fall into a class of epigenetically controlled sequences. Relative to control lines, retained IESs are flanked by stronger cis-acting signals and interrupt an excess of highly expressed coding exons. These findings suggest that P. tetraurelia’s elevated germline DNA replication fidelity is associated with pervasive somatic genome plasticity. They show that MA regimes are powerful tools for investigating the role that developmental plasticity, somatic mutations, and epimutations have in ecology and evolution.

Most sRNA biogenesis mechanisms involve either RNAse III cleavage or ping-pong amplification by different Piwi proteins harbouring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from targeted endogenous loci. Our data does not indicate any signatures from ping-pong amplification but Dicer cleavage of long dsRNA. Ptiwi13 and 14 prefer different sub-cellular localizations and different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and show a general preference for uridine-rich sRNAs along the entire sRNA length. In addition, Ptiwi14-loaded siRNAs show a 5´-U signature. Our data indicates both Ptiwis and 2´-O-methylation contributing to strand selection of Dicer cleaved siRNAs. This unexpected function of the two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. We describe an unusual mode of action of Piwi proteins extending not only the great variety of Piwi-associated RNAi pathways but moreover raising the question whether this could have been the primordial one.

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis have been gleaned by assessing the rate of phenotypic assortment. Though powerful, this experimental approach relies on the availability of phenotypic markers. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dualism and a highly polyploid somatic nucleus, we probe the limits of single-cell whole-genome sequencing to study the consequences of amitosis. To this end, we first evaluate the suitability of single-cell sequencing to study the AT-rich genome of P.&nbsp, tetraurelia, focusing on common sources of genome representation bias. We then asked: can alternative rearrangements of a given locus eventually assort after a number of amitotic divisions? To address this question, we track somatic assortment of developmentally acquired Internal Eliminated Sequences (IESs) up to 50 amitotic divisions post self-fertilization. To further strengthen our observations, we contrast empirical estimates of IES retention levels with in silico predictions obtained through mathematical modeling. In agreement with theoretical expectations, our empirical findings are consistent with a mild increase in variation of IES retention levels across successive amitotic divisions of the macronucleus. The modest levels of somatic assortment in P.&nbsp, tetraurelia suggest that IESs retention levels are largely sculpted at the time of macronuclear development, and remain fairly stable during vegetative growth. In forgoing the requirement for phenotypic assortment, our approach can be applied to a wide variety of amitotic species and could facilitate the identification of environmental and genetic factors affecting amitosis.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the >90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur co-transcriptionally and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We showed that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, amongst the smallest periodicities reported. Our analysis revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint to locate introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for the nonsense-mediated decay effectors, we showed that introns located at the edge of nucleosomes display higher splicing efficiency than those at the centre. However, multiple regression analysis indicated that the GC content, rather than nucleosome positioning, directly contributes to intron splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning and intron evolution in Paramecium.

Motivation: Long-read sequencing technologies can be employed to detect and map DNA modifications at the nucleotide resolution on a genome-wide scale. However, published software packages neglect the integration of genomic annotation and comprehensive filtering when analyzing patterns of modified bases detected using Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) data. Here, we present DNA Modification Annotation (DNAModAnnot), a R package designed for the global analysis of DNA modification patterns using adapted filtering and visualization tools. Results: We tested our package using PacBio sequencing data to analyze patterns of the 6-methyladenine (6mA) in the ciliate Paramecium tetraurelia, in which high 6mA amounts were previously reported. We found P. tetraurelia 6mA genome-wide distribution to be similar to other ciliates. We also performed 5-methylcytosine (5mC) analysis in human lymphoblastoid cells using ONT data and confirmed previously known patterns of 5mC. DNAModAnnot provides a toolbox for the genome-wide analysis of different DNA modifications using PacBio and ONT long-read sequencing data., SCOPUS: ar.j, info:eu-repo/semantics/published

Can ecological changes impact somatic genome development? Efforts to resolve this question could reveal a direct link between environmental changes and somatic variability, potentially illuminating our understanding of how variation can surface from a single genotype under stress. Here, we tackle this question by leveraging the biological properties of ciliates. When Paramecium tetraurelia reproduces sexually, its polyploid somatic genome regenerates from the germline genome through a developmental process that involves the removal of thousands of ORF-interrupting sequences known as internal eliminated sequences (IESs). We show that exposure to nonstandard culture temperatures impacts the efficiency of this process of programmed DNA elimination, prompting the emergence of hundreds of incompletely excised IESs in the newly developed somatic genome. These alternative DNA isoforms display a patterned genomic topography, impact gene expression, and might be inherited transgenerationally. On this basis, we conclude that environmentally induced developmental thermoplasticity contributes to genotypic diversification in Paramecium.

In animals and plants, the H3K9me3 and H3K27me3 chromatin silencing marks are deposited by different protein machineries. H3K9me3 is catalyzed by the SET-domain SU(VAR)3–9 enzymes, while H3K27me3 is catalyzed by the SET-domain Enhancer-of-zeste enzymes, which are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). Here, we show that the Enhancer-of-zeste-like protein Ezl1 from the unicellular eukaryote Paramecium tetraurelia, which exhibits significant sequence and structural similarities with human EZH2, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27. We find that H3K9me3 and H3K27me3 co-occur at multiple families of transposable elements in an Ezl1-dependent manner. We demonstrate that loss of these histone marks results in global transcriptional hyperactivation of transposable elements with modest effects on protein-coding gene expression. Our study suggests that although often considered functionally distinct, H3K9me3 and H3K27me3 may share a common evolutionary history as well as a common ancestral role in silencing transposable elements., H3K9me3 and H3K27me3 chromatin silencing marks are usually deposited by different SET-domain proteins. Here the authors show that the Enhancer-of-zeste-like protein Ezl1, from the unicellular eukaryote Paramecium tetraurelia, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27, and controls the repression of transposable elements.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression., A comparative genomics study of nine Paramecium species reveals successful invasion of genes by transposable elements in their germline genomes, showing that the internal eliminated sequences (IESs) followed an evolutionary trajectory remarkably similar to that of spliceosomal introns.

Strict vertical (parent-to-offspring) transmission of symbionts may be associated with benevolence and mutualism, because host reproduction and symbiont transmission are linked. Nonetheless, many such symbionts do reduce host fitness costs and the factors driving their evolution are unclear. Using microcosm populations ofCaedibacter taeniospiralis, a vertically transmitted bacterial symbiont of the protozoanParamecium tetraurelia, we investigated evolutionary change under permissive conditions (26°C) and under heat stress (32°C), over 150 host generations. We found evidence for symbiont adaptation to heat stress, involving increased infection persistence and, consequently, higher rates of vertical transmission. For certain genetic backgrounds, heat-stress adaptation was associated with higher proliferation of infected lines at 26°C, suggesting cost-free evolution of super-generalists. Despite a general decrease in symbiont virulence relative to ancestral strains, infected evolved lines still showed substantial reductions (>20%) in population growth. There was no sign of a temperature-specific reduction in virulence or protection of the host against heat stress. Our results show that obligately vertically transmitted symbionts can still qualify as ‘parasites’ and evolve adaptations that do not help their hosts. Long-term infection persistence might require additional mechanisms, such as the symbiont-mediated ’killer trait’ in this system, allowing the selective elimination of uninfected individuals in the population.

Calcium ions (Ca2+) entering cilia through the ciliary voltage-gated calcium channels (CaV) during the action potential causes reversal of the ciliary power stroke and backward swimming in Paramecium tetraurelia. How calcium is returned to the resting level is not yet clear. Our focus is on calcium pumps as a possible mechanism. There are 23 P. tetraurelia genes for calcium pumps that are members of the family of plasma membrane Ca2+ ATPases (PMCAs). They have domains homologous to those found in mammalian PMCAs. Of the 13 pump proteins previously identified in cilia, ptPMCA2a and ptPMCA2b are most abundant in the cilia. We used RNAi to examine which PMCA might be involved in regulating intraciliary Ca2+ after the action potential. RNAi for only ptPMCA2a and ptPMCA2b causes cells to significantly prolong their backward swimming, which indicates that Ca2+ extrusion in the cilia is impaired when these PMCAs are depleted. We used immunoprecipitations (IP) to find that ptPMCA2a and ptPMCA2b are co-immunoprecipitated with the CaV channel α1 subunits that are found only in the cilia. We used iodixanol (OptiPrep) density gradients to show that ptPMCA2a and ptPMCA2b and CaV1c are found in the same density fractions. These results suggest that ptPMCA2a and ptPMCA2b are located in the proximity of ciliary CaV channels.

The centriole has a helical inner scaffold that imparts microtubule triplet cohesion and centriole geometry., The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo–electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

Ciliary shedding occurs from unicellular organisms to metazoans. Although required during the cell cycle and during neurogenesis, the process remains poorly understood. In all cellular models, this phenomenon occurs distal to the transition zone (TZ), suggesting conserved molecular mechanisms. The TZ module proteins (Meckel Gruber syndrome [MKS]/Nephronophtysis [NPHP]/Centrosomal protein of 290 kDa [CEP290]/Retinitis pigmentosa GTPase regulator-Interacting Protein 1-Like Protein [RPGRIP1L]) are known to cooperate to establish TZ formation and function. To determine whether they control deciliation, we studied the function of 5 of them (Transmembrane protein 107 [TMEM107], Transmembrane protein 216 [TMEM216], CEP290, RPGRIP1L, and NPHP4) in Paramecium. All proteins are recruited to the TZ of growing cilia and localize with 9-fold symmetry at the level of the most distal part of the TZ. We demonstrate that depletion of the MKS2/TMEM216 and TMEM107 proteins induces constant deciliation of some cilia, while depletion of either NPHP4, CEP290, or RPGRIP1L prevents Ca2+/EtOH deciliation. Our results constitute the first evidence for a role of conserved TZ proteins in deciliation and open new directions for understanding motile cilia physiology., Functional analysis and subcellular localisation of the conserved transition zone proteins in the ciliate Paramecium tetraurelia demonstrates their involvement in the ciliary shedding process, opening new avenues fir understanding the molecular mechanism of deciliation.

The pervasiveness of sex despite its well-known costs is a long-standing puzzle in evolutionary biology. Current explanations for the success of sex in nature largely rely on the adaptive significance of the new or rare genotypes that sex may generate. Less explored is the possibility that sex-underlying molecular mechanisms can enhance fitness and convey benefits to the individuals that bear the immediate costs of sex. Here we show that self-fertilization can increase stress resistance in the ciliateParamecium tetraurelia. This advantage is independent of new genetic variation, coupled with a reduced nutritional input, and offers fresh insights into the mechanistic origin of sex. In addition to providing evidence that the molecular underpinnings of sexual reproduction and the stress response are linked inP. tetraurelia, these findings supply an explanation for the persistence of self-fertilization in this ciliate.

A conspicuous cell-shape phenotype known as &ldquo, screwy&rdquo, was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5&ndash, 6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.

A human ciliopathy gene codes for Polycystin-2 (Pkd2), a non-selective cation channel. Here, the Pkd2 channel was explored in the ciliate Paramecium tetraurelia using combinations of RNA interference, over-expression, and epitope-tagging, in a search for function and novel interacting partners. Upon depletion of Pkd2, cells exhibited a phenotype similar to eccentric (XntA1), a Paramecium mutant lacking the inward Ca2+-dependent Mg2+ conductance. Further investigation showed both Pkd2 and XntA localize to the cilia and cell membrane, but do not require one another for trafficking. The XntA-myc protein co-immunoprecipitates Pkd2-FLAG, but not vice versa, suggesting two populations of Pkd2-FLAG, one of which interacts with XntA. Electrophysiology data showed that depletion and over-expression of Pkd2 led to smaller and larger depolarizations in Mg2+ solutions, respectively. Over-expression of Pkd2-FLAG in the XntA1 mutant caused slower swimming, supporting an increase in Mg2+ permeability, in agreement with the electrophysiology data. We propose that Pkd2 in P. tetraurelia collaborates with XntA for Mg2+-induced behavior. Our data suggest Pkd2 is sufficient and necessary for Mg2+ conductance and membrane permeability to Mg2+, and that Pkd2 is potentially a Mg2+-permeable channel.

Whole-Genome Duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages. The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods and the relative contributions of different selective pressures to their maintenance is still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliateParamecium tetraureliaand two of its sister species from theP. aureliacomplex. Here, we report the genome sequence and analysis of 10 additionalP. aureliaspecies and one additional outgroup, allowing us to track post-WGD evolution in 13 species that share a common ancestral WGD. We found similar biases in gene retention compatible with dosage constraints playing a major role opposing post-WGD gene loss across all 13 species. Interestingly we found that post-WGD gene loss was slower inParameciumthan in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong inParamecium. We also report a lack of recent segmental duplications inParamecium, which we interpret as additional evidence for strong selective pressures against individual genes dosage changes. Finally, we hope that this exceptional dataset of 13 species sharing an ancestral WGD and two closely related outgroup species will be a useful resource for future studies and will help establishParameciumas a major model organism in the study of post-WGD evolution.

The transition from unicellular to multicellular life was one of a few major events in the history of life that created new opportunities for more complex biological systems to evolve. Predation is hypothesized as one selective pressure that may have driven the evolution of multicellularity. Here we show that de novo origins of simple multicellularity can evolve in response to predation. We subjected outcrossed populations of the unicellular green alga Chlamydomonas reinhardtii to selection by the filter-feeding predator Paramecium tetraurelia. Two of five experimental populations evolved multicellular structures not observed in unselected control populations within ~750 asexual generations. Considerable variation exists in the evolved multicellular life cycles, with both cell number and propagule size varying among isolates. Survival assays show that evolved multicellular traits provide effective protection against predation. These results support the hypothesis that selection imposed by predators may have played a role in some origins of multicellularity.

As most of eukaryotic diversity lies in single-celled protists, they represent unique opportunities to ask questions about the balance of conservation and innovation in cell biological features. Among free-living protists the ciliates offer ease of culturing, a rich array of experimental approaches, and versatile molecular tools, particularly in Tetrahymena thermophila and Paramecium tetraurelia. These attributes have been exploited by researchers to analyze a wealth of cellular structures in these large and complex cells. This mini-review focuses on 3 aspects of ciliate membrane dynamics, all linked with endolysosomal trafficking. First is nutrition based on phagocytosis and maturation of food vacuoles. Secondly, we discuss regulated exocytosis from vesicles that have features of both dense core secretory granules but also lysosome-related organelles. The third topic is the targeting, breakdown and resorption of parental nuclei in mating partners. For all 3 phenomena, it is clear that elements of the canonical membrane-trafficking system have been retained and in some cases repurposed. In addition, there is evidence that recently evolved, lineage-specific proteins provide determinants in these pathways.

International audience; DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.

Variable antigens are large proteins located on the outer membrane of parasitic but also of free-living protists. Multigene families encoding surface antigens demonstrate an exclusive expression of proteins. The resulting presence of just one protein species on the cell surface is required for surface antigen function; therefore, the molecular mechanism of exclusive expression is of main interest. Regulation of gene expression and mechanisms establishing switching of antigens are hardly understood in any organism. Here we report on the reaction of Paramecium to the artificial knock down of surface antigen 51A expression by bacteria-mediated RNAi. This technique involves the feeding of dsRNA-producing bacteria. We analyzed different fragments of the target gene for dsRNA template regarding their specific knock down efficiency and found great differences. Treatment of Paramecia with RNAi against the 51A antigen demonstrated that although a massive amount of mRNA was present, the protein was not detected on the cell surface. Moreover, a minor abundance of 51D transcripts resulted in an exclusive presence of 51D proteins on the cell surface. This posttranscriptional regulation was confirmed by the transcript ratio (51A/51D) determined by real-time (RT) PCR of single cells. Because we were able to document unexclusive transcription also in wild-type cells our results indicate that this posttranscriptional regulation is a main factor of enabling exclusive gene expression. The comparison of serotype shifts, caused by efficient and inefficient knock down, indicates an involvement of full-length transcripts in regulation of gene expression. Thus, our study gives new insights into the mechanism of exclusive expression on the molecular level: (i) exclusive transcription does not occur, (ii) posttranscriptional regulation is a powerful factor enabling exclusive antigen expression, and (iii) surface antigen mRNA is shown to be involved in this mechanism in a regulating way.

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires’ disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries’ disease, understanding the ecology of L. pneumophila in the environment is essential.

The occurrence of ciliates, especially the Paramecium aurelia complex, has not yet been studied in many parts of the world, or sampling was done only occasionally. Generally, the southern hemisphere still awaits investigation. In North America only the USA was studied in greater detail; the majority of species of the complex were there recorded. In Asia, more frequent sampling was performed only in Japan and Asiatic Russia. Europe was studied more carefully, however, a different number of habitats was studied in particular zones of Europe, the least in the southern zone. New stands of P. tetraurelia , P. sexaurelia, P. octaurelia, and P. novaurelia were revealed as a result of the present investigations carried out in Africa (Mozambique--P. tetraurelia, P. sexaurelia), Asia (Indonesia--P. sexaurelia), borderland of Asia and Europe (Georgia--P. octaurelia), and Europe (Macedonia--P. tetraurelia and Romania--P. novaurelia). Are climatic zones the main factor limiting the occurrence of species of the P. aurelia complex? Analysis of data on the distribution of the P. aurelia species complex in warm "tropical" zones on different continents may suggest such preferences for some species, including P. sexaurelia, P. octaurelia, P. tredecaurelia, P. quadecaurelia. The first two of these species were recorded herein in warm or "tropical" zone.

Caedibacter taeniospiralis is an obligate endosymbiont living in the cytoplasm of Paramecium tetraurelia . C. taeniospiralis causes the so-called killer trait, eliminating intraspecific competitors of its host when released into the medium by the concerted action of the unusual protein structure R-body (refractile body) in addition to an as-yet-unknown toxin.

5-methylcytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have not been discovered in published ciliate genomes, recent studies performed in the stichotrichous ciliate Oxytricha trifallax suggest de novo cytosine methylation during macronuclear development. In this study, we applied bisulfite genome sequencing, DNA mass spectrometry and antibody-based fluorescence detection to investigate the presence of DNA methylation in Paramecium tetraurelia. While the antibody-based methods suggest cytosine methylation, DNA mass spectrometry and bisulfite sequencing reveal that levels are actually below the limit of detection. Our results suggest that Paramecium does not utilize 5-methylcytosine DNA methylation as an integral part of its epigenetic arsenal.

International audience; In the ciliate Paramecium tetraurelia, functional genes are reconstituted during development of the somatic macronucleus through the precise excision of similar to 45 000 single-copy Internal Eliminated Sequences (IESs), thought to be the degenerate remnants of ancient transposon insertions. Like introns, IESs are marked only by a weak consensus at their ends. How such a diverse set of sequences is faithfully recognized and precisely excised remains unclear: specialized small RNAs have been implicated, but in their absence up to similar to 60% of IESs are still correctly excised. To get further insight, we designed a mutagenesis screen based on the hypersensitivity of a specific excision event in the mtA gene, which determines mating types. Unlike most IES-containing genes, the active form of mtA is the unexcised one, allowing the recovery of hypomorphic alleles of essential IES recognition/excision factors. Such is the case of one mutation recovered in the Piwi gene PTIWI09, a key player in small RNA-mediated IES recognition. Another mutation identified a novel protein with a C2H2 zinc finger, mtGa, which is required for excision of a small subset of IESs characterized by enrichment in a 5-bp motif. The unexpected implication of a sequence-specific factor establishes a new paradigm for IES recognition and/or excision.

Synthetic Pyrethroids are considered to be safe over other insecticides; however, data indicate that their use may pose risk to environmental biota, especially, aquatic organisms. Therefore, the aim of the present study was to investigate the effect of cypermethrin, a widely used insecticide and one of the most common contaminants in freshwater aquatic system on the oxidative stress biomarkers of the freshwater ciliate Paramecium tetraurelia. After the treatment of paramecium cells with increasing concentrations of cypermethrin (0.05, 0.5,1, 2�g/l), we followed up the growth kinetics, generation time and the response percentage. Also, we studied the variation in biomarkers of stress such as: Proteins, GSH content, GST and CAT activities. Our results showed a significant decrease in the proliferation of cell, we denote a difference of

The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size.

International audience; Better understanding of uranyl–protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF‐hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X‐ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time‐resolved laser‐induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X‐ray absorption fine structure (EXAFS) data evidence a short U−Oeq distance, in addition to distances compatible with mono‐ and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono‐ and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl–protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein–uranyl complexes.

Das Ziel dieser Arbeit war die Entwicklung eine Gerätes, welches die Überwachung von mehreren hundert Mikrobioreaktoren ermöglicht. Dabei sollte es, neben der Reduzierung von Kosten und Arbeitskraft, die konventionellen Pipettiermethoden in der zeitliche Auflösung übertreffen. Weiterhin soll es über eine Gradientenerzeugung verfügen, um sehr feine Variationen der Zusammensetzung des verwendeten Mediums zu ermöglichen. Dafür wurde ein Analysator für fluoreszierenden Tropfen (Fluorescent Droplet Analyser) entwickelt, mit dem ein segmentierter Fluss von mehreren hundert Tropfen erzeugt werden kann und für viele Tage gemessen werden kann. Für die Messung wurde der Analysator mit einer flexiblen Fluoreszenzoptik ausgestattet um unterschiedliche Fluoreszenzfarbstoffe oder Molekühle detektieren zu können. Mehrere Experimente wurden durchgeführt, welche das Potentials des Gerätes zeigen. Einzelne Zellen des Pantoffeltierchen (Paramecium tetraurelia) konnten in einzelnen Tropfen eingeschlossen werden und mit einem metabolischen Farbstoffe ihre Stoffwechselaktivität gemessen werden. Ebenfalls wurden viele Experimente mit Pseudomonas fluorescens und E.coli YFP durchgeführt. Durch die flexible Fluoreszenzoptik konnte das Wachstum beider Arten in eine Experiment gemessen werden.:1 Introduction 1 1.1 Motivation 1 1.2 Scope of this thesis 2 1.3 State of the art 3 2 Fundamentals 2.1 Millifluidics vs. microfluidics 5 2.1.1 Droplet based microfluidics 6 2.1.2 Surfactant in droplet-based millifluidics 7 2.2 The model of microorganism growth 8 2.3 The fluorescence based detection 9 3 Materials & Methods 3.1 The culture of the microorganisms and preparation 11 3.1.1 The Paramecium tetraurelia 11 3.1.2 Pseudomonas fluorescens 12 3.1.3 Escherichia coli 12 3.1.4 The bacteria culture 13 3.2 The Poisson distribution . 15 3.3 The composition of the emulsion 16 3.4 The fluidic components 17 3.5 Modules of the Fluorescent Droplet Analyser 18 3.5.1 The optocoupled relay card 18 3.5.2 The used fluidic pumps 19 3.5.3 The photonic elements of the FDA 19 3.5.4 The light sources 21 3.5.5 The optical fibres 23 3.5.6 The used detectors 23 3.6 The operating Software 24 4 The Fluorescent Droplet Analyser 4.1 The fluidic network for droplet generation and shuttling 26 4.1.1 Generation of a droplet sequence 28 4.1.2 Measuring a droplet sequence 30 4.2 The electric schematics of the FDA 31 4.3 The light source, guidance, and detection 32 4.3.1 Preparation of the fibres 33 4.3.2 The detection setup 35 4.4 The developed LabView program for operating the FDA 37 4.4.1 The automated measurement process 40 4.4.2 The peak mode 41 4.4.3 The time mode 42 4.4.4 The combined mode 42 4.4.5 The dynamic threshold 42 4.5 The developed VI program for analysing the data of the FDA 44 4.6 Flow characteristics of the FDA 46 4.6.1 Basic flow characteristics 46 4.6.2 Characteristics for shuttling of a droplet sequence 47 4.6.3 Influence of the hydrodynamic resistance 48 5 Monitoring of Paramecium tetraurelia 5.1 Introduction 51 5.2 Generation of a droplet sequence for Paramecium tetraurelia 53 5.3 Metabolic dynamics in droplets 54 5.4 Ecotoxicity assays 58 6 Monitoring of bacteria 6.1 Introduction 63 6.2 Generation of a droplet sequence for the bacteria experiments 64 6.3 Cell number calibration of the FDA 65 6.4 Monitoring of the bacteria growth 68 6.5 Investigation of the of the fluorescence signal 70 6.6 One bacteria cell in a droplet 71 6.7 The determination of the minimum inhibitory concentration 74 6.8 Long-term monitoring 80 6.9 Sectioning of the droplet sequence 80 6.10 Multicolor fluorescence detection 83

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism.

International audience; In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.

Summary In eukaryotes, small RNAs (sRNAs) have key roles in development, gene expression regulation, and genome integrity maintenance. In ciliates, such as Paramecium , sRNAs form the heart of an epigenetic system that has evolved from core eukaryotic gene silencing components to selectively target DNA for deletion. In Paramecium , somatic genome development from the germline genome accurately eliminates the bulk of typically gene-interrupting, noncoding DNA. We have discovered an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later during Paramecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs ("scan" RNAs [scnRNAs]) in targeting DNA for elimination. We show that whole-genome duplications have facilitated successive differentiations of Paramecium Dicer-like proteins, leading to cooperation between Dcl2 and Dcl3 to produce scnRNAs and to the production of iesRNAs by Dcl5. These innovations highlight the ability of sRNA systems to acquire capabilities, including those in genome development and integrity.

The contractile vacuole complex (CVC) of freshwater protists serves the extrusion of water and ions, including Ca(2+). No vesicle trafficking based on SNAREs has been detected so far in any CVC. SNAREs (soluble NSF [N-ethylmaleimide sensitive factor] attachment protein receptors) are required for membrane-to-membrane interaction, i.e. docking and fusion also in Paramecium. We have identified three v-/R- and three t/Q-SNAREs selectively in the CVC. Posttranscriptional silencing of Syb2, Syb6 or Syx2 slows down the pumping cycle; silencing of the latter two also causes vacuole swelling. Increase in extracellular Ca(2+) after Syb2, Syb6 or Syx2 silencing causes further swelling of the contractile vacuole and deceleration of its pulsation. Silencing of Syx14 or Syx15 entails lethality in the Ca(2+) stress test. Thus, the effects of silencing strictly depend on the type of the silenced SNARE and on the concentration of Ca(2+) in the medium. This shows the importance of organelle-resident SNARE functions (which may encompass the vesicular delivery of other organelle-resident proteins) for Ca(2+) tolerance. A similar principle may be applicable also to the CVC in widely different unicellular organisms. In addition, in Paramecium, silencing particularly of Syx6 causes aberrant positioning of the CVC during de novo biogenesis before cytokinesis.

We report the detailed evaluation of crucial parameters for chromatin immunoprecipitation (ChIP) of macronuclear DNA in the unicellular eukaryote Paramecium tetraurelia. Optimized parameters include crosslinking conditions, chromatin sonication and antibody titration thus providing a detailed protocol for successful ChIP in P. tetraurelia. As this ciliate is bacterivorous and RNAi by feeding represents a powerful tool for analysis of gene function, we moreover determined the effects of ingested nucleic acids by food bacteria. Feasibility of our protocol is demonstrated by characterisation of chromatin remodelling at promoters of cytosolic HSP70 isoforms during transcriptional activation under heat shock conditions by analyzing RNA abundance, nucleosome occupancy and levels of H3 lysine 9 acetylation.

As most of eukaryotic diversity lies in single-celled protists, they represent unique opportunities to ask questions about the balance of conservation and innovation in cell biological features. Among free-living protists the ciliates offer ease of culturing, a rich array of experimental approaches, and versatile molecular tools, particularly in Tetrahymena thermophila and Paramecium tetraurelia. These attributes have been exploited by researchers to analyze a wealth of cellular structures in these large and complex cells. This mini-review focuses on three aspects of ciliate membrane dynamics, all linked with endolysosomal trafficking. First is nutrition based on phagocytosis and maturation of food vacuoles. Secondly, we discuss regulated exocytosis from vesicles that have features of both dense core secretory granules but also lysosome-related organelles. The third topic is the targeting, breakdown and resorption of parental nuclei in mating partners. For all three phenomena, it is clear that elements of the canonical membrane-trafficking system have been retained and in some cases repurposed. In addition, there is evidence that recently evolved, lineage-specific proteins provide determinants in these pathways. published

Summary In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences. Video Abstract, Graphical Abstract, Highlights • In Paramecium, pieces of deleted DNA are transcribed to form regulatory RNAs • Ultra-short DNA segments are concatenated and circularized, allowing transcription • This concatenation is carried out by Ligase IV, which also repairs DNA ends • Concatenation is random, which leads to diversity in the resulting sRNA population, “Junk” DNA can be ligated into circles and transcribed to generate regulatory RNAs.

Background The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. Results We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. Conclusions We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3′ and 5′ UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis regulatory motifs). The P. tetraurelia improved transcriptome resource, gene annotations for P. tetraurelia, P. biaurelia, P. sexaurelia and P. caudatum, and Paramecium-trained EuGene configuration are available through ParameciumDB (http://paramecium.i2bc.paris-saclay.fr). TrUC software is freely distributed under a GNU GPL v3 licence (https://github.com/oarnaiz/TrUC). Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3887-z) contains supplementary material, which is available to authorized users.