Usage of the H3 variants during the S-phase of the cell cycle in Physarum polycephalum.

DNA replication occurring in S-phase is critical for the maintenance of the cell fate from one generation to the next, and requires the duplication of epigenetic information. The integrity of the epigenome is, in part, insured by the recycling of parental histones and de novo deposition of newly synthesized histones. While the histone variants have revealed important functions in epigenetic regulations, the deposition in chromatin during S-phase of newly synthesized histone variants remains unclear. The identification of histone variants of H3 and unique features of Physarum polycephalum provides a powerful system for investigating de novo deposition of newly synthesized histones by tracking the incorporation of exogenous histones within cells. The analyses revealed that the rate of deposition of H3.1 and H3.3 is anticorrelated as S-phase progresses, H3.3 is predominately produced and utilized in early S and dropped throughout S-phase, while H3.1 behaved in the opposite way. Disturbing the expression of H3 variants by siRNAs revealed mutual compensation of histone transcripts. Interestingly, the incorporation of pre-formed constrained histone complexes showed that tetramers of H3/H4 are more efficiently utilized by the cell than dimers. These results support the model whereby the histone variant distribution is established upon replication and new histone deposition.

G × G × E effect on phenotype expression in a non-conventional model organism, the unicellular slime mould Physarum polycephalum.

In metazoans, the expression of key phenotypic traits is sensitive to two- and three-way interactions between variation in mitochondrial DNA, nuclear DNA and the external environment. Whether gene-by-environment interactions affect phenotypes in single-celled eukaryotes is poorly studied, except in a few species of yeast and fungi. We developed a genetic panel of the unicellular slime mould, Physarum polycephalum containing strains differing in mitochondrial and nuclear DNA haplotypes. The panel also included two strains harbouring a selfishly replicating mitochondrial-fusion (mF) plasmid that could affect phenotype expression. We assayed movement and growth rate differences among the strains across two temperature regimes: 24° and 28°C. We found that the slime mould's growth rate, but not movement, is affected by G × G × E interactions. Predictably, mtDNA × nDNA interactions significantly affected both traits. The inter-trait correlation across the strains in each temperature regime was positive. Surprisingly, the mF plasmid had no negative effects on our chosen traits. Our study is the first to demonstrate genetic regulation of phenotype expression in a unicellular slime mould. The genetic effect on phenotypes manifests via epistatic interactions with the thermal environment, thus shedding new light on the role of G × G × E interactions in trait evolution in protists.

The Histone Chaperone Network Is Highly Conserved in Physarum polycephalum.

The nucleosome is composed of histones and DNA. Prior to their deposition on chromatin, histones are shielded by specialized and diverse proteins known as histone chaperones. They escort histones during their entire cellular life and ensure their proper incorporation in chromatin. Physarum polycephalum is a Mycetozoan, a clade located at the crown of the eukaryotic tree. We previously found that histones, which are highly conserved between plants and animals, are also highly conserved in Physarum. However, histone chaperones differ significantly between animal and plant kingdoms, and this thus probed us to further study the conservation of histone chaperones in Physarum and their evolution relative to animal and plants. Most of the known histone chaperones and their functional domains are conserved as well as key residues required for histone and chaperone interactions. Physarum is divergent from yeast, plants and animals, but PpHIRA, PpCABIN1 and PpSPT6 are similar in structure to plant orthologues. PpFACT is closely related to the yeast complex, and the Physarum genome encodes the animal-specific APFL chaperone. Furthermore, we performed RNA sequencing to monitor chaperone expression during the cell cycle and uncovered two distinct patterns during S-phase. In summary, our study demonstrates the conserved role of histone chaperones in handling histones in an early-branching eukaryote.

Identification of a glutathione S-transferase gene of Physarum polycephalum as a biomarker for nanosized TiO2 exposure under dark conditions.

In this study, a glutathione S-transferase gene (gst) from sensitive Physarum polycephalum was selected for its ability to detect nanosized TiO2 (nTiO2 ) exposure under dark conditions. The concentration of nTiO2 (25, 40 and 60 nm) for subsequent assays was first determined (5-18 mg ml-1 ) and total GST enzyme activity of P. polycephalum was confirmed to be increased 6-44 fold in groups treated with nTiO2 . Second, an RNA-seq study was performed to identify candidate gst genes before isolation of an optimum gst gene of P. polycephalum (Ppgst), which encoded 223 amino acids. Third, the transcriptional level of the Ppgst gene was further confirmed to be positively correlated with nTiO2 exposure within the concentration range of (5-15 mg ml-1 ) by qPCR. In conclusion, these results indicated that the transcriptional level of Ppgst can reflect nTiO2 exposure, suggesting that it may be employed as a new biomarker for nTiO2 pollution under dark conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identifies a new gst gene for indicating nanosized TiO2 under dark conditions and provides a new option for detection of nanosized TiO2 pollution under dark conditions.

Replication forks reverse at high frequency upon replication stress in Physarum polycephalum.

The addition of hydroxyurea after the onset of S phase allows replication to start and permits the successive detecting of replication-dependent joint DNA molecules and chicken foot structures in the synchronous nuclei of Physarum polycephalum. We find evidence for a very high frequency of reversed replication forks upon replication stress. The formation of these reversed forks is dependent on the presence of joint DNA molecules, the impediment of the replication fork progression by hydroxyurea, and likely on the propensity of some replication origins to reinitiate replication to counteract the action of this compound. As hydroxyurea treatment enables us to successively detect the appearance of joint DNA molecules and then of reversed replication forks, we propose that chicken foot structures are formed both from the regression of hydroxyurea-frozen joint DNA molecules and from hydroxyurea-stalled replication forks. These experiments underscore the transient nature of replication fork regression, which becomes detectable due to the hydroxyurea-induced slowing down of replication fork progression.

Phenotypic variability in unicellular organisms: from calcium signalling to social behaviour.

Historically, research has focused on the mean and often neglected the variance. However, variability in nature is observable at all scales: among cells within an individual, among individuals within a population and among populations within a species. A fundamental quest in biology now is to find the mechanisms that underlie variability. Here, we investigated behavioural variability in a unique unicellular organism, Physarum polycephalum. We combined experiments and models to show that variability in cell signalling contributes to major differences in behaviour underpinning some aspects of social interactions. First, following thousands of cells under various contexts, we identified distinct behavioural phenotypes: 'slow-regular-social', 'fast-regular-social' and 'fast-irregular-asocial'. Second, coupling chemical analysis and behavioural assays we found that calcium signalling is responsible for these behavioural phenotypes. Finally, we show that differences in signalling and behaviour led to alternative social strategies. Our results have considerable implications for our understanding of the emergence of variability in living organisms.

Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants.

Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

Physarum polycephalum mutants in the photocontrol of sporulation display altered patterns in the correlated expression of developmentally regulated genes.

Physarum polycephalum is a lower eukaryote belonging to the amoebozoa group of organisms that forms macroscopic, multinucleate plasmodial cells during its developmental cycle. Plasmodia can exit proliferative growth and differentiate by forming fruiting bodies containing mononucleate, haploid spores. This process, called sporulation, is controlled by starvation and visible light. To genetically dissect the regulatory control of the commitment to sporulation, we have isolated plasmodial mutants that are altered in the photocontrol of sporulation in a phenotypic screen of N-ethyl-N-nitrosourea (ENU) mutagenized cells. Several non-sporulating mutants were analyzed by measuring the light-induced change in the expression pattern of a set of 35 genes using GeXP multiplex reverse transcription-polymerase chain reaction with RNA isolated from individual plasmodial cells. Mutants showed altered patterns of differentially regulated genes in response to light stimulation. Some genes clearly displayed pairwise correlation in terms of their expression level as measured in individual plasmodial cells. The pattern of pairwise correlation differed in various mutants, suggesting that different upstream regulators were disabled in the different mutants. We propose that patterns of pairwise correlation in gene expression might be useful to infer the underlying gene regulatory network.

A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages.

Complete characterization of the edited transcriptome of the mitochondrion of Physarum polycephalum using deep sequencing of RNA.

RNAs transcribed from the mitochondrial genome of Physarum polycephalum are heavily edited. The most prevalent editing event is the insertion of single Cs, with Us and dinucleotides also added at specific sites. The existence of insertional editing makes gene identification difficult and localization of editing sites has relied upon characterization of individual cDNAs. We have now determined the complete mitochondrial transcriptome of Physarum using Illumina deep sequencing of purified mitochondrial RNA. We report the first instances of A and G insertions and sites of partial and extragenic editing in Physarum mitochondrial RNAs, as well as an additional 772 C, U and dinucleotide insertions. The notable lack of antisense RNAs in our non-size selected, directional library argues strongly against an RNA-guided editing mechanism. Also of interest are our findings that sites of C to U changes are unedited at a significantly higher frequency than insertional editing sites and that substitutional editing of neighboring sites appears to be coupled. Finally, in addition to the characterization of RNAs from 17 predicted genes, our data identified nine new mitochondrial genes, four of which encode proteins that do not resemble other proteins in the database. Curiously, one of the latter mRNAs contains no editing sites.

Genetic Diversity in the mtDNA of Physarum polycephalum.

The mtDNA of the myxomycete Physarum polycephalum can contain as many as 81 genes. These genes can be grouped in three different categories. The first category includes 46 genes that are classically found on the mtDNA of many organisms. However, 43 of these genes are cryptogenes that require a unique type of RNA editing (MICOTREM). A second category of gene is putative protein-coding genes represented by 26 significant open reading frames. However, these genes do not appear to be transcribed during the growth of the plasmodium and are currently unassigned since they do not have any apparent similarity to other classical mitochondrial protein-coding genes. The third category of gene is found in the mtDNA of some strains of P. polycephalum. These genes derive from a linear mitochondrial plasmid with nine significant, but unassigned, open reading frames which can integrate into the mitochondrial DNA by recombination. Here, we review the mechanism and evolution of the RNA editing necessary for cryptogene expression, discuss possible origins for the 26 unassigned open reading frames based on tentative identification of their protein product, and discuss the implications to mtDNA structure and replication of the integration of the linear mitochondrial plasmid.

RNA editing of 10 Didymium iridis mitochondrial genes and comparison with the homologous genes in Physarum polycephalum.

Regions of the Didymium iridis mitochondrial genome were identified with similarity to typical mitochondrial genes; however, these regions contained numerous stop codons. We used RT-PCR and DNA sequencing to determine whether, through RNA editing, these regions were transcribed into mRNAs that could encode functional proteins. Ten putative gene regions were examined: atp1, atp6, atp8, atp9, cox1, cox2, cytb, nad4L, nad6, and nad7. The cDNA sequences of each gene could encode a functional mitochondrial protein that was highly conserved compared with homologous genes. The type of editing events and editing sequence features were very similar to those observed in the homologous genes of Physarum polycephalum, though the actual editing locations showed a variable degree of conservation. Edited sites were compared with encoded sites in D. iridis and P. polycephalum for all 10 genes. Edited sequence for a portion of the cox1 gene was available for six myxomycetes, which, when compared, showed a high degree of conservation at the protein level. Different types of editing events showed varying degrees of site conservation with C-to-U base changes being the least conserved. Several aspects of single C insertion editing events led to the preferential creation of hydrophobic amino acid codons that may help to minimize adverse effects on the resulting protein structure.

Two forms of RNA editing are required for tRNA maturation in Physarum mitochondria.

The mitochondrial genome of Physarum polycephalum encodes five tRNAs, four of which are edited by nucleotide insertion. Two of these tRNAs, tRNA(met1) and tRNA(met2), contain predicted mismatches at the beginning (proximal end) of the acceptor stem. In addition, the putative 5' end of tRNA(met2) overlaps the 3' end of a small, abundant, noncoding RNA, which we term ppoRNA. These anomalies led us to hypothesize that these two Physarum mitochondrial tRNAs undergo additional editing events. Here, we show that tRNA(met1) and tRNA(met2) each has a nonencoded G at its 5' end. In contrast to the other nucleotides that are added to Physarum mitochondrial RNAs, these extra G residues are likely added post-transcriptionally based on (1) the absence of added G in precursor transcripts containing inserted C and AA residues, (2) the presence of potential intermediates characteristic of 5' replacement editing, and (3) preferential incorporation of GTP into tRNA molecules under conditions that do not support transcription. This is the first report of both post-transcriptional nucleotide insertions and the addition of single Gs in P. polycephalum mitochondrial transcripts. We postulate that tRNA(met1) and tRNA(met2) are acted upon by an activity similar to that present in the mitochondria of certain other amoebozoons and chytrid fungi, suggesting that enzymes that repair the 5' end of tRNAs may be widespread.

Semi-in vitro detection of Mg2+-dependent DNase that specifically digest mitochondrial nucleoids in the zygote of Physarum polycephalum.

The maternal/uniparental inheritance of mitochondria is controlled by the selective elimination of paternal/uniparental mitochondria and digestion of their mitochondrial DNA (mtDNA). In isogamy, the selective digestion of mtDNA in uniparental mitochondria is initiated after mating and is completed prior to the elimination of mitochondria, but the molecular mechanism of the digestion of uniparental mtDNA remains unknown. In this study, we developed a semi-in vitro assay for DNase, wherein the digestion of mitochondrial nucleoids (mt-nucleoids) was microscopically observed using isolated mitochondria from Physarum polycephalum and the DNase involved in uniparental inheritance was characterized. When myxamoebae of AI35 and DP246 are crossed, mtDNA and mt-nucleoid from only the DP246 parent are digested. The digestion of mt-nucleoids was observed in zygotes 3 h after plating for mating. During the digestion of mt-nucleoids, mitochondrial membrane integrity was maintained. In the semi-in vitro assay, the digestion of mt-nucleoids was only observed in the presence of Mg2+ at pH 7.5-9.0. Moreover, such Mg2+-dependent DNase activity was specifically detected in mitochondria isolated from zygotes 3 h after plating for mating. Therefore, Mg2+-dependent DNase is potentially involved in uniparental inheritance. Our findings provide insights into the DNase involved in uniparental inheritance and its regulatory mechanism.

RNase-dependent discontinuities associated with the crossovers of spontaneously formed joint DNA molecules in Physarum polycephalum.

Transient four stranded joint DNA molecules bridging sister chromatids constitute an intriguing feature of replicating genomes. Here, we studied their structure and frequency of formation in Physarum polycephalum. By "3D gels", we evidenced that they are not made of four continuous DNA strands. Discontinuities, which do not interfere with the unique propensity of the joint DNA molecules to branch migrate in vitro, are linked to the crossover, enhanced by RNaseA, and affect at most half of the DNA strands. We propose a structural model of joint DNA molecules containing ribonucleotides inserted within one strand, a gapped strand, and two continuous DNA strands. We further show that spontaneous joint DNA molecules are short-lived and are as abundant as replication forks. Our results emphasize the highly frequent formation of joint DNA molecules involving newly replicated DNA in an untreated cell and uncover a transitory mechanism connecting the sister chromatids during S phase.

Distinct roles for sequences upstream of and downstream from Physarum editing sites.

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within approximately 9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.

Abundant 5S rRNA-like transcripts encoded by the mitochondrial genome in amoebozoa.

5S rRNAs are ubiquitous components of prokaryotic, chloroplast, and eukaryotic cytosolic ribosomes but are apparently absent from mitochondrial ribosomes (mitoribosomes) of many eukaryotic groups including animals and fungi. Nevertheless, a clearly identifiable, mitochondrion-encoded 5S rRNA is present in Acanthamoeba castellanii, a member of Amoebozoa. During a search for additional mitochondrial 5S rRNAs, we detected small abundant RNAs in other members of Amoebozoa, namely, in the lobose amoeba Hartmannella vermiformis and in the myxomycete slime mold Physarum polycephalum. These RNAs are encoded by mitochondrial DNA (mtDNA), cosediment with mitoribosomes in glycerol gradients, and can be folded into a secondary structure similar to that of bona fide 5S rRNAs. Further, in the mtDNA of another slime mold, Didymium nigripes, we identified a region that in sequence, potential secondary structure, and genomic location is similar to the corresponding region encoding the Physarum small RNA. A mtDNA-encoded small RNA previously identified in Dictyostelium discoideum is here shown to share several characteristics with known 5S rRNAs. Again, we detected genes encoding potential homologs of this RNA in the mtDNA of three other species of the genus Dictyostelium as well as in a related genus, Polysphondylium. Taken together, our results indicate a widespread occurrence of small, abundant, mtDNA-encoded RNAs with 5S rRNA-like structures that are associated with the mitoribosome in various amoebozoan taxa. Our working hypothesis is that these novel small abundant RNAs represent radically divergent mitochondrial 5S rRNA homologs. We posit that currently unrecognized 5S-like RNAs may exist in other mitochondrial systems in which a conventional 5S rRNA cannot be identified.

Physarum polycephalum macroplasmodium exhibits countermeasures against TiO2 nanoparticle toxicity: A physiological, biochemical, transcriptional, and metabolic perspective.

Concerns about the environmental and human health implications of TiO2 nanoparticles (nTiO2) are growing with their increased use in consumer and industrial products. Investigations of the underlying molecular mechanisms of nTiO2 tolerance in organisms will assist in countering nTiO2 toxicity. In this study, the countermeasures exhibited by the slime mold Physarum polycephalum macroplasmodium against nTiO2 toxicity were investigated from a physiological, transcriptional, and metabolic perspective. The results suggested that the countermeasures against nTiO2 exposure include gene-associated metabolic rearrangements in cellular pathways involved in amino acid, carbohydrate, and nucleic acid metabolism. Gene-associated nonmetabolic rearrangements involve processes such as DNA repair, DNA replication, and the cell cycle, and occur mainly when macroplasmodia are exposed to inhibitory doses of nTiO2. Interestingly, the growth of macroplasmodia and mammal cells was significantly restored by supplementation with a combination of responsive metabolites identified by metabolome analysis. Taken together, we report a novel model organism for the study of nTiO2 tolerance and provide insights into countermeasures taken by macroplasmodia in response to nTiO2 toxicity. Furthermore, we also present an approach to mitigate the effects of nTiO2 toxicity in cells by metabolic intervention.

Expansion and transformation of the minor spliceosomal system in the slime mold Physarum polycephalum.

Spliceosomal introns interrupt nuclear genes and are removed from RNA transcripts ("spliced") by machinery called spliceosomes. Although the vast majority of spliceosomal introns are removed by the so-called major (or "U2") spliceosome, diverse eukaryotes also contain a rare second form, the minor ("U12") spliceosome, and associated ("U12-type") introns.1-3 In all characterized species, U12-type introns are distinguished by several features, including being rare in the genome (∼0.5% of all introns),4-6 containing extended evolutionarily conserved splicing motifs,4,5,7,8 being generally ancient,9,10 and being inefficiently spliced.11-13 Here, we report a remarkable exception in the slime mold Physarum polycephalum. The P. polycephalum genome contains >20,000 U12-type introns-25 times more than any other species-enriched in a diversity of non-canonical splice boundaries as well as transformed splicing signals that appear to have co-evolved with the spliceosome due to massive gain of efficiently spliced U12-type introns. These results reveal an unappreciated dynamism of minor spliceosomal introns and spliceosomal introns in general.

Transcriptomic changes arising during light-induced sporulation in Physarum polycephalum.

BACKGROUND: Physarum polycephalum is a free-living amoebozoan protist displaying a complex life cycle, including alternation between single- and multinucleate stages through sporulation, a simple form of cell differentiation. Sporulation in Physarum can be experimentally induced by several external factors, and Physarum displays many biochemical features typical for metazoan cells, including metazoan-type signaling pathways, which makes this organism a model to study cell cycle, cell differentiation and cellular reprogramming. RESULTS: In order to identify the genes associated to the light-induced sporulation in Physarum, especially those related to signal transduction, we isolated RNA before and after photoinduction from sporulation- competent cells, and used these RNAs to synthesize cDNAs, which were then analyzed using the 454 sequencing technology. We obtained 16,669 cDNAs that were annotated at every computational level. 13,169 transcripts included hit count data, from which 2,772 displayed significant differential expression (upregulated: 1,623; downregulated: 1,149). Transcripts with valid annotations and significant differential expression were later integrated into putative networks using interaction information from orthologs. CONCLUSIONS: Gene ontology analysis suggested that most significantly downregulated genes are linked to DNA repair, cell division, inhibition of cell migration, and calcium release, while highly upregulated genes were involved in cell death, cell polarization, maintenance of integrity, and differentiation. In addition, cell death- associated transcripts were overrepresented between the upregulated transcripts. These changes are associated to a network of actin-binding proteins encoded by genes that are differentially regulated before and after light induction.