Transcriptome reprogramming during developmental switching in Physarum polycephalum involves extensive remodeling of intracellular signaling networks.

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.

Decision-making ability of Physarum polycephalum enhanced by its coordinated spatiotemporal oscillatory dynamics.

An amoeboid unicellular organism, a plasmodium of the true slime mold Physarum polycephalum, exhibits complex spatiotemporal oscillatory dynamics and sophisticated information processing capabilities while deforming its amorphous body. We previously devised an 'amoeba-based computer (ABC),' that implemented optical feedback control to lead this amoeboid organism to search for a solution to the traveling salesman problem (TSP). In the ABC, the shortest TSP route (the optimal solution) is represented by the shape of the organism in which the body area (nutrient absorption) is maximized while the risk of being exposed to aversive light stimuli is minimized. The shortness of the TSP route found by ABC, therefore, serves as a quantitative measure of the optimality of the decision made by the organism. However, it remains unclear how the decision-making ability of the organism originates from the oscillatory dynamics of the organism. We investigated the number of coexisting traveling waves in the spatiotemporal patterns of the oscillatory dynamics of the organism. We show that a shorter TSP route can be found when the organism exhibits a lower number of traveling waves. The results imply that the oscillatory dynamics are highly coordinated throughout the global body. Based on the results, we discuss the fact that the decision-making ability of the organism can be enhanced not by uncorrelated random fluctuations, but by its highly coordinated oscillatory dynamics.

Futile attempts to differentiate provide molecular evidence for individual differences within a population of cells during cellular reprogramming.

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.

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.

Beyond input-output computings: error-driven emergence with parallel non-distributed slime mold computer.

The emergence derived from errors is the key importance for both novel computing and novel usage of the computer. In this paper, we propose an implementable experimental plan for the biological computing so as to elicit the emergent property of complex systems. An individual plasmodium of the true slime mold Physarum polycephalum acts in the slime mold computer. Modifying the Elementary Cellular Automaton as it entails the global synchronization problem upon the parallel computing provides the NP-complete problem solved by the slime mold computer. The possibility to solve the problem by giving neither all possible results nor explicit prescription of solution-seeking is discussed. In slime mold computing, the distributivity in the local computing logic can change dynamically, and its parallel non-distributed computing cannot be reduced into the spatial addition of multiple serial computings. The computing system based on exhaustive absence of the super-system may produce, something more than filling the vacancy.

The sequence of regulatory events in the sporulation control network of Physarum polycephalum analysed by time-resolved somatic complementation of mutants.

The developmental decision for sporulation of Physarum polycephalum plasmodia is under sensory control by environmental factors like visible light or heat shock and endogenous signals like glucose starvation. Several hours after perceiving an inductive stimulus, plasmodia become committed to sporulation; thereby, they lose their unlimited replicative potential and execute a developmental program that involves differentiation into various cell types required to form a mature fruiting body. Plasmodia are multinuclear single cells which spontaneously fuse upon physical contact. Fusion of mutant plasmodia and cytoplasmic mixing allows complementation studies to be performed at the functional level. Mutant cells altered in their ability to sporulate in response to phytochrome activation by far-red light were cured by fusion with wild-type or other mutant plasmodia. Phytochrome activation in one plasmodium and subsequent fusion with a non-induced plasmodium revealed that complementation of the two mutations depended on (i) which of two genetically distinct plasmodial cells was stimulated; and (ii) on the delay time elapsed between stimulation and cytoplasmic mixing. Such experiments allow us to determine the kinetics and the causal sequence of the regulatory events tagged by mutation.

Light irradiation induces fragmentation of the plasmodium, a novel photomorphogenesis in the true slime mold Physarum polycephalum: action spectra and evidence for involvement of the phytochrome.

A new photomorphogenesis was found in the plasmodium of the true slime mold Physarum polycephalum: the plasmodium broke temporarily into equal-sized spherical pieces, each containing about eight nuclei, about 5 h after irradiation with light. Action spectroscopic study showed that UVA, blue and far-red lights were effective, while red light inhibited the far-red-induced fragmentation. Difference absorption spectra of both the living plasmodium and the plasmodial homogenate after alternate irradiation with far-red and red light gave two extremes at 750 and 680 nm, which agreed with those for the induction and inhibition of the fragmentation, respectively. A kinetic model similar to that of phytochrome action explained quantitatively the fluence rate-response curves of the fragmentation. Our results indicate that one of the photoreceptors for the plasmodial fragmentation is a phytochrome.

Action spectrum for sporulation and photoavoidance in the plasmodium of Physarum polycephalum, as modified differentially by temperature and starvation.

The plasmodium of the myxomycete Physarum polycephalum sporulates in bright natural environments, suggesting a relationship between photobehavior and sporulation. Thus, the action spectra for two light-dependent phenomena as well as the effects of other environmental conditions have been studied. Sporulation like photo-avoidance responded to UVC (near 270 nm) and near IR (near 750 nm) in addition to the well-documented UVA (near 350 nm) and blue (near 460 nm) regions. Sporulation and photoavoidance had similar sensitivities in the shorter wavelengths, while the former was about 100 times more sensitive in near IR. The plasmodium moved away from light in a wide spectral range. Starvation and high temperature at 31 degrees C (25 degrees C in standard conditions) reduced photoavoidance to UVA and to blue light, respectively. A high fluence rate of UVC suppressed the rhythmic contraction of the plasmodium, and the action spectrum peaked at 270 nm. These results indicate that the Physarum plasmodium may stay at brighter places not by positive phototaxis but by weakening the negative phototaxis to sunlight or by other possible taxes such as hydrotaxis. There may be at least four different photo-systems in the plasmodium.

Functional mapping of the branched signal transduction pathway that controls sporulation in Physarum polycephalum.

Sporulation of starving plasmodia of Physarum polycephalum was found to be induced by far-red light, blue light or heat shock, each of which is perceived by a different input receptor system. The branched signal transduction pathway was mapped and the time-dependent formation of some of its components analyzed.

A photoreceptor with characteristics of phytochrome triggers sporulation in the true slime mould Physarum polycephalum.

Phytochrome is a ubiquitous photoreceptor in plants that controls a variety of responses to light, including gene expression, differential cell growth and intracellular movement of organelles. All phytochromes analysed so far are reversibly interconverted by light between an inactive and an active conformation, each of which has a different and characteristic absorbance spectrum. Based on photophysiological measurements we provide evidence, that a photoreceptor with these unique properties of phytochrome triggers sporulation in the true slime mould Physarum polycephalum.

Time-resolved detection of three intracellular signals controlling photomorphogenesis in Physarum polycephalum.

Incompetent plasmodia of Physarum polycephalum exposed to a light pulse sporulated after reaching the competent stage. Fusion of irradiated plasmodia with dark-incubated plasmodia and analysis of sporulation indicated the presence of a morphogenetic signal. It is concluded that a logic AND gate integrates the photoreceptor signal and the competence signal and controls the formation of the morphogenetic signal.

Reduction of UV-induced mitotic delay by caffeine in BUdR-substituted plasmodia of Physarum polycephalum.

Chromosomal DNA of the synchronously mitotic plasmodia of P. polycephalum was substituted with 5-bromo-2'-deoxyuridine, by growing the plasmodia during S phase, on a medium containing this nucleoside analog. A strong synergism was observed between bromodeoxyuridine and UV-irradiation, in late G2-irradiated plasmodia in that, the mitotic delay obtained in them was much more than a simple sum of the delays induced by these two agents individually. It was also observed that the mitotic delay in this system is reduced significantly by different concentrations of caffeine applied immediately after irradiation and there was a stage specificity in this effect. The reduction in mitotic delay was maximum (80%) in those plasmodia irradiated 20-30 min before control metaphase, when mitogenic factors also reach their maximum activity in this system. It is proposed that the mitotic delay reducing effect of caffeine is due to its ability to promote the activity of the mitogenic factors, largely independent of the system which is responsible for monitoring the state of the chromosomal DNA.