RESUMEN
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.
Asunto(s)
Physarum polycephalum , Physarum polycephalum/genética , ADN Mitocondrial/genética , Secuencia de Bases , Mitocondrias/genética , Variación Genética/genéticaRESUMEN
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.
Asunto(s)
Physarum polycephalum , Physarum polycephalum/genética , ADN Mitocondrial/genética , Mitocondrias/genética , Plásmidos , FenotipoRESUMEN
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.
Asunto(s)
Histonas , Physarum polycephalum , Animales , Histonas/metabolismo , Physarum polycephalum/genética , Physarum polycephalum/metabolismo , Chaperonas de Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMEN
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.
Asunto(s)
ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Desoxirribonucleasas/metabolismo , Desoxirribonucleasas/fisiología , Magnesio/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Physarum polycephalum/genética , Physarum polycephalum/metabolismo , Cigoto , Concentración de Iones de Hidrógeno , Mitocondrias/enzimología , Membranas Mitocondriales/metabolismo , Physarum polycephalum/fisiologíaRESUMEN
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.
Asunto(s)
Histonas , Physarum polycephalum , Ciclo Celular/genética , Cromatina/genética , Replicación del ADN/genética , Histonas/genética , Histonas/metabolismo , Nucleosomas , Physarum polycephalum/genética , Physarum polycephalum/metabolismoRESUMEN
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 (â¼0.5% 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.
Asunto(s)
Intrones , Physarum polycephalum , Empalmosomas , Physarum polycephalum/genética , Empalme del ARN , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
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.
Asunto(s)
Nanopartículas , Physarum polycephalum , Animales , Humanos , Metaboloma , Nanopartículas/toxicidad , Physarum polycephalum/genética , Titanio/toxicidadRESUMEN
Single-celled organisms show a fascinating faculty for integrating spatial information and adapting their behaviour accordingly. As such they are of potential interest for elucidating fundamental mechanisms of developmental biology. In this mini-review we highlight current research on two organisms, the true slime mould Physarum polycephalum and the ciliates Paramecium and Tetrahymena. For each of these, we present a case study how applying physical principles to explain behaviour can lead to the understanding of general principles possibly relevant to developmental biology.
Asunto(s)
Paramecium/crecimiento & desarrollo , Physarum polycephalum/crecimiento & desarrollo , Fenómenos Físicos , Tetrahymena/crecimiento & desarrollo , Conducta/fisiología , Biología Evolutiva/tendencias , Paramecium/genética , Physarum polycephalum/genética , Tetrahymena/genéticaRESUMEN
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.
Asunto(s)
Contaminantes Ambientales/análisis , Glutatión Transferasa/metabolismo , Nanopartículas del Metal/análisis , Physarum polycephalum/metabolismo , Titanio/análisis , Secuencia de Aminoácidos/genética , Biomarcadores , Glutatión Transferasa/genética , Physarum polycephalum/genéticaRESUMEN
Activation of a phytochrome photoreceptor triggers a program of Physarum polycephalum plasmodial cell differentiation through which a mitotic multinucleate protoplasmic mass synchronously develops into haploid spores formed by meiosis and rearrangement of cellular components. We have performed a transcriptome-wide RNAseq study of cellular reprogramming and developmental switching. RNAseq analysis revealed extensive remodeling of intracellular signaling and regulation in switching the expression of sets of genes encoding transcription factors, kinases, phosphatases, signal transduction proteins, RNA-binding proteins, ubiquitin ligases, regulators of the mitotic and meiotic cell cycle etc. in conjunction with the regulation of genes encoding metabolic enzymes and cytoskeletal proteins. About 15% of the differentially expressed genes shared similarity with members of the evolutionary conserved set of core developmental genes of social amoebae. Differential expression of genes encoding regulators that act at the transcriptional, translational, and post-translational level indicates the establishment of a new state of cellular function and reveals evolutionary deeply conserved molecular changes involved in cellular reprogramming and differentiation in a prototypical eukaryote.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Redes Reguladoras de Genes/fisiología , Genes Protozoarios/fisiología , Physarum polycephalum/crecimiento & desarrollo , Proteínas Protozoarias/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/efectos de la radiación , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Redes Reguladoras de Genes/efectos de la radiación , Luz , Células Fotorreceptoras de Invertebrados/fisiología , Células Fotorreceptoras de Invertebrados/efectos de la radiación , Physarum polycephalum/genética , Physarum polycephalum/efectos de la radiación , Fitocromo/genética , Fitocromo/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal/genética , Transcriptoma/fisiología , Transcriptoma/efectos de la radiaciónRESUMEN
Histone modifications have been widely correlated with genetic activities. However, how these posttranslational modifications affect the dynamics and the structure of chromatin is poorly understood. Here, we describe the incorporation of the exogenous histone proteins into the slime mold Physarum polycephalum, which has been revealed to be a valuable tool for examining different facets of the function histones in chromatin dynamics like replication-coupled chromatin assembly, histone exchange, and nucleosome turnover.
Asunto(s)
Histonas/metabolismo , Physarum polycephalum/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Código de Histonas/genética , Código de Histonas/fisiología , Nucleosomas/metabolismo , Physarum polycephalum/genética , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.
Asunto(s)
Evolución Molecular , Genoma de Protozoos , Physarum polycephalum/genética , Proteínas Protozoarias/genética , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sitios Genéticos , Proteínas Protozoarias/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , TranscriptomaRESUMEN
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.
Asunto(s)
Señalización del Calcio , Variación Genética , Fenotipo , Physarum polycephalum/fisiología , Modelos Biológicos , Physarum polycephalum/genética , Conducta SocialRESUMEN
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Malatos/administración & dosificación , Terapia Molecular Dirigida/métodos , Nanopartículas/administración & dosificación , Polímeros/administración & dosificación , Animales , Neoplasias de la Mama/enzimología , Femenino , Silenciador del Gen , Humanos , Malatos/síntesis química , Malatos/química , Malatos/metabolismo , Ratones , Ratones Desnudos , Nanopartículas/química , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Physarum polycephalum/genética , Physarum polycephalum/metabolismo , Polímeros/síntesis química , Polímeros/química , Polímeros/metabolismo , Medicina de Precisión/métodos , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Physarum polycephalum/genética , Fase S/genética , Estrés Fisiológico/genética , ADN Cruciforme , ADN Protozoario/metabolismo , Recombinación Homóloga , Hidroxiurea/farmacología , Physarum polycephalum/efectos de los fármacos , Physarum polycephalum/metabolismo , Fase S/efectos de los fármacosRESUMEN
In this paper, we report the dose-dependent antioxidant activity and DNA protective effects of zingerone. At 500 µg/mL, the DPPH radical scavenging activity of zingerone and ascorbic acid as a standard was found to be 86.7 and 94.2 % respectively. At the same concentration, zingerone also showed significant reducing power (absorbance 0.471) compared to that of ascorbic acid (absorbance 0.394). The in vitro toxicity of stannous chloride (SnCl2) was evaluated using genomic and plasmid DNA. SnCl2-induced degradation of genomic DNA was found to occur at a concentration of 0.8 mM onwards with complete degradation at 1.02 mM and above. In the case of plasmid DNA, conversion of supercoiled DNA into the open circular form indicative of DNA nicking activity was observed at a concentration of 0.2 mM onwards; complete conversion was observed at a concentration of 1.02 mM and above. Zingerone was found to confer protection against SnCl2-induced oxidative damage to genomic and plasmid DNA at concentrations of 500 and 750 µg/mL onwards, respectively. This protective effect was further confirmed in the presence of UV/H2O2-a known reactive oxygen species (ROS) generating system-wherein protection by zingerone against ROS-mediated DNA damage was observed at a concentration of 250 µg/mL onwards in a dose-dependent manner. This study clearly indicated the in vitro DNA protective property of zingerone against SnCl2-induced, ROS-mediated DNA damage.
Asunto(s)
Guayacol/análogos & derivados , Peróxido de Hidrógeno/toxicidad , Compuestos de Estaño/toxicidad , Antioxidantes/metabolismo , Antioxidantes/farmacología , Daño del ADN/efectos de los fármacos , Guayacol/farmacología , Physarum polycephalum/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Embryophyta/genética , Eucariontes , Proteínas de Plantas/metabolismo , Edición de ARN , Proteínas de Unión al ARN/metabolismo , Acanthamoeba castellanii/genética , Acanthamoeba castellanii/metabolismo , Embryophyta/metabolismo , Naegleria/genética , Nitella/genética , Nitella/metabolismo , Orgánulos/genética , Orgánulos/metabolismo , Filogenia , Physarum polycephalum/genética , Physarum polycephalum/metabolismo , Proteínas de Plantas/genética , Células Procariotas/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genéticaRESUMEN
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.
Asunto(s)
Perfilación de la Expresión Génica , Células Gigantes/metabolismo , Mutación , Physarum polycephalum/genética , Citoplasma/genética , Corriente Citoplasmática/genética , Expresión Génica/efectos de la radiación , Luz , Physarum polycephalum/citología , Physarum polycephalum/fisiología , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esporas Protozoarias/genética , Esporas Protozoarias/fisiologíaRESUMEN
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.
Asunto(s)
Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Mutación , Physarum polycephalum/genética , Redes Reguladoras de Genes/efectos de la radiación , Genes Protozoarios/genética , Physarum polycephalum/fisiología , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esporas Protozoarias/genética , Esporas Protozoarias/efectos de la radiaciónRESUMEN
The heterogeneity of cell populations and the influence of stochastic noise might be important issues for the molecular analysis of cellular reprogramming at the system level. Here, we show that in Physarum polycephalum, the expression patterns of marker genes correlate with the fate decision of individual multinucleate plasmodial cells that had been exposed to a differentiation-inducing photostimulus. For several hours after stimulation, the expression kinetics of PI-3-kinase, piwi, and pumilio orthologs and other marker genes were qualitatively similar in all stimulated cells but quantitatively different in those cells that subsequently maintained their proliferative potential and failed to differentiate accordingly. The results suggest that the population of nuclei in an individual plasmodium behaves synchronously in terms of gene regulation to an extent that the plasmodium provides a source for macroscopic amounts of homogeneous single-cell material for analysing the dynamic processes of cellular reprogramming. Based on the experimental findings, we predict that circuits with switch-like behaviour that control the cell fate decision of a multinucleate plasmodium operate through continuous changes in the concentration of cellular regulators because the nuclear population suspended in a large cytoplasmic volume damps stochastic noise.