Memory inception and preservation in slime moulds: the quest for a common mechanism.

Learning and memory are indisputably key features of animal success. Using information about past experiences is critical for optimal decision-making in a fluctuating environment. Those abilities are usually believed to be limited to organisms with a nervous system, precluding their existence in non-neural organisms. However, recent studies showed that the slime mould Physarum polycephalum, despite being unicellular, displays habituation, a simple form of learning. In this paper, we studied the possible substrate of both short- and long-term habituation in slime moulds. We habituated slime moulds to sodium, a known repellent, using a 6 day training and turned them into a dormant state named sclerotia. Those slime moulds were then revived and tested for habituation. We showed that information acquired during the training was preserved through the dormant stage as slime moulds still showed habituation after a one-month dormancy period. Chemical analyses indicated a continuous uptake of sodium during the process of habituation and showed that sodium was retained throughout the dormant stage. Lastly, we showed that memory inception via constrained absorption of sodium for 2 h elicited habituation. Our results suggest that slime moulds absorbed the repellent and used it as a 'circulating memory'. This article is part of the theme issue 'Liquid brains, solid brains: How distributed cognitive architectures process information'.

Nano-sized TiO2 (nTiO2) induces metabolic perturbations in Physarum polycephalum macroplasmodium to counter oxidative stress under dark conditions.

Nano-sized TiO2 (nTiO2) exerts an oxidative effect on cells upon exposure to solar or UV irradiation and ecotoxicity of the nTiO2 is an urgent concern. Little information is available regarding the effect of TiO2 on cells under dark conditions. Metabolomics is a unique approach to the discovery of biomarkers of nTiO2 cytotoxicity, and leads to the identification of perturbed metabolic pathways and the mechanism underlying nTiO2 toxicity. In the present study, gas chromatography mass spectrometry (GC/MS)-based metabolomics was performed to investigate the effect of nTiO2 on sensitive cells (P. polycephalum macroplasmodium) under dark conditions. According to the multivariate pattern recognition analysis, at least 60 potential metabolic biomarkers related to sugar metabolism, amino acid metabolism, nucleotide metabolism, polyamine biosynthesis, and secondary metabolites pathways were significantly perturbed by nTiO2. Notably, many metabolic biomarkers and pathways were related to anti-oxidant mechanisms in the living organism, suggesting that nTiO2 may induce oxidative stress, even under dark conditions. This speculation was further validated by the biochemical levels of reactive oxygen species (ROS), 8-hydroxy-2-deoxyguanosine (8-OHdG), and total soluble phenols (TSP). We inferred that the oxidative stress might be related to nTiO2-induced imbalance of cellular ROS. To the best of our knowledge, the present study is the first to investigate the nTiO2-induced metabolic perturbations in slime mold, provide a new perspective of the mechanism underlying nTiO2 toxicity under dark conditions, and show that metabolomics can be employed as a rapid, reliable and powerful tool to investigate the interaction among organisms, the environment, and nanomaterials.

Effect of oxidative stress from nanoscale TiO2 particles on a Physarum polycephalum macroplasmodium under dark conditions.

Information regarding the effect of nanoscale titanium dioxide particles (nTiO2) on the environment under dark conditions is scarce, and the effect of nTiO2 on fungi is largely unknown. Due to its huge size and high sensitivity to external stimuli, the slime mold fungi cell, Physarum polycephalum macroplasmodium, was utilized as a novel subject for the toxicity investigations in the present study, and oxidative stress from nTiO2 on the macroplasmodium was assessed under dark conditions. Short exposure (2-3 h) caused an intracellular reactive oxygen species (ROS) imbalance, and an anti-oxidative mechanism was activated from intermediate doses of nTiO2 (5-18 mg/mL). At long exposure times (~3 days), relatively low doses of nTiO2 (≤9 mg/mL) stimulated the growth of macroplasmodium and oxidative stress without DNA damage, whereas higher doses of nTiO2 (≥15 mg/mL) led to growth inhibition, significant DNA oxidative damage, and activation of the DNA single-strand repairing system. Although DNA oxidative damage was decreased to the same level as the control group by the supplementation of the anti-oxidant vitamin C, growth of the macroplasmodium failed to be completely restored. We inferred that nTiO2 induced a complicated toxicity effect on P. polycephalum in addition to DNA oxidative damage. Taken as a whole, the present study implied the probability of using P. polycephalum macroplasmodium for toxicity studies at the single-cell level, indicating that nTiO2 could induce oxidative stress or damage in P. polycephalum even under dark conditions and suggesting that the release of nTiO2 could lead to a growth imbalance of slime molds in the environment.

Spectral imaging method for studying Physarum polycephalum growth on polyaniline surface.

The features of spectrophotometric scanner, generally exploited in the artwork field, are here considered in a non-conventional context to characterize the networks created by Physarum polycephalum slime mold during its motion on glass substrates covered with polyaniline: a polymer that varies its color and conductive properties according to the redox state. The used technique allowed the investigation of the effects coming out from the interaction between P. polycephalum and polyaniline. Thus, the contactless method of the analysis of polyaniline conductivity state resulted from the slime mold metabolism was suggested. Indeed, it is here demonstrated that P. polycephalum can modify properties of polyaniline due to its internal activity in contact zones.

Inhibitory effects of extracellular self-DNA: a general biological process?

Self-inhibition of growth has been observed in different organisms, but an underlying common mechanism has not been proposed so far. Recently, extracellular DNA (exDNA) has been reported as species-specific growth inhibitor in plants and proposed as an explanation of negative plant-soil feedback. In this work the effect of exDNA was tested on different species to assess the occurrence of such inhibition in organisms other than plants. Bioassays were performed on six species of different taxonomic groups, including bacteria, fungi, algae, plants, protozoa and insects. Treatments consisted in the addition to the growth substrate of conspecific and heterologous DNA at different concentration levels. Results showed that treatments with conspecific DNA always produced a concentration dependent growth inhibition, which instead was not observed in the case of heterologous DNA. Reported evidence suggests the generality of the observed phenomenon which opens new perspectives in the context of self-inhibition processes. Moreover, the existence of a general species-specific biological effect of exDNA raises interesting questions on its possible involvement in self-recognition mechanisms. Further investigation at molecular level will be required to unravel the specific functioning of the observed inhibitory effects.

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.

Antioxidant potential and oxidative DNA damage preventive activity of unexplored endemic species of Curcuma.

Free radical scavenging activity, ferrous ion chelating capacity, reducing power and genoprotective effect of the aqueous leaf extracts of four unexplored endemic Curcuma spp. (C. vamana, C. neilgherrensis, C. mutabilis, C. haritha) were found to be dose-dependent and were highest in C. vamana. DNA protection property of the extracts was evaluated against H202/UV-induced oxidative damage. DNA-methyl green displacement assay showed that these extracts were free of DNA intercalating compounds. Further, hemolysis assay also showed that the extracts were non-toxic to human erythrocytes. The results highlight C. vamana as a promising source for herbal preparations possessing high antioxidant potential and genoprotective activity.

Assessment of cell cycle phase-specific effects of zerumbone on mitotically synchronous surface cultures of Physarum polycephalum.

Zerumbone, a natural cyclic sesquiterpene, has been the focus of recent research as it has been found to exhibit selective toxicity towards cancer cells compared to normal cells. Studies on the cell cycle phase-specific effects of this interesting compound, however, remain sparse. Hence, concentration and time-dependent effects of zerumbone were evaluated employing a suitable model system, the naturally synchronous surface cultures of Physarum polycephalum. Zerumbone treatment in S, early, and late G2 phases resulted in G2 arrest. Early G2 phase exhibited the highest sensitivity (P < 0.001) to the compound. Protein profiles showed a complete inhibition of cyclin B1 expression following zerumbone treatment. Furthermore, FACS and comet analysis revealed that zerumbone inhibited DNA synthesis (P < 0.001) without being genotoxic at the concentrations tested. Differential display of mRNA showed distinct zerumbone-induced variations in transcript profiles, an analysis of which suggested a likely link between cellular networks involving stress-related gene expression and G2 arrest in P. polycephalum.

[Involvement of cyclic adenosine monophosphate in the control of motile behavior of Physarum polycephalum plasmodium].

Possible involvement of autocrine factors into the control of motile behavior via a receptor-mediated mechanism was investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the auto-oscillatory mode of motility. Cyclic adenosine monophosphate (cAMP) and extracellular cAMP-specific phosphodiesterase, its involvement into the control of plasmodium motile behavior was proved by action of its strong inhibitor, were regarded as putative autocrine factors. It was shown that the plasmodium secreted cAMP. When it was introduced into agar support, 0,1-1 mM cAMP induced a delay of the plasmodium spreading and its transition to migration. When locally applied, cAMP at the same concentrations induced typical for attractant action the increase in oscillation frequency and the decrease of ectoplasm elasticity. The ability to exhibit positive chemotaxis in cAMP gradient and the dependence of its realization were shown to depend on the plasmodium state. Chemotaxis test specimens obtained from the migrating plasmodium, unlike those obtained from growing culture, generate alternative fronts which compete effectively with fronts oriented towards the attractant increment. The results obtained support our supposition stated earlier that advance of the Physarum polycephalum plasmodium leading edge is determined by local extracellular cAMP gradients arising from a time delay between secretion and hydrolysis of the nucleotide.

Routing Physarum with repellents.

Plasmodium of Physarum polycephalum is a single cell with many nuclei. Plasmodium is an easy-to-experiment-with biological substrate, a multi-functional bio-material used to implement novel and future computing architectures. The plasmodium exhibits typical features of excitable chemical systems and capable for distributed sensing, parallel information processing and decentralized actuation. Plasmodium of P. polycephalum is proved to be a universal storage modification machine. Actively growing zones of the plasmodium are considered to be elementary processors of the growing computing machine, as well as messages traveling in the spatially extended non-linear medium. Controlling propagation of the messages and computing processes is a prerequisite for a successful implementation of working prototypes of plasmodium machines. In laboratory experiments and computer simulation we show that active growing zones of plasmodium can be precisely routed using repelling diffusion gradients generated by crystals of sodium chloride. We demonstrate how to achieve controllable reflection, splitting/multiplication and merging of plasmodium's active zones.

[The role of phosphoinositide-3-kinase in controlling the shape and directional movement in Physarum polycephalum plasmodium].

The influence of wortmannin and LY294002, specific inhibitors of phosphoinosite-3-kinase, on the shape, motile behavior, and chemotaxis toward glucose has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motion. Both inhibitors were shown to cause a reduction of the plasmodium frontal edge and a decrease in the efficiency of mass transfer during migration. They also suppress chemotaxis toward glucose and eliminate characteristic changes in autooscillatory behavior normally observed in response to the treatment of the whole plasmodium with glucose. The manifestation of these effects depends on the inhibitor concentration, the duration of treatment, and the size of plasmodium. The involvement of phosphoinosite-3-kinase in creating the frontal edge and in controlling the chemotaxis of Physarum plasmodium suggests that the interrelation of polar shape and directional movement of amoeboid cells with the distribution of phosphoinositides in the plasma membrane has the universal nature.

[Chemotaxis as a method for testing of the biological effects of silver nanoparticles].

A method for assessing the abiotic efficiency of water-dispersed nano-sized silver particles is suggested. Nanoparticles were obtained by the method of biochemical synthesis in reversed micelles made from anionic surfactant bis-(2-ethylhexyl) sodium sulfosuccinate (AOT). A water dispersion of nanoparticles was prepared from the micellar solution in organic solvent by means of a special procedure. The abiotic efficiency assay is based on the capacity of cells to respond by the negative chemotaxis to chemical agents with harmful metabolic action. In plasmodium of myxomicete Physarum polycephalum, the biocide and repellent effects of silver nanoparticles, Ag+ ions, and AOT were tested in order to compare the abiotic efficiency of these substances in water solution and after introduction into agar substrate. The increase in the oscillation period, the reduction in the area of spreading, and the avoidance reaction in the spatial test, taken together, revealed a much higher repellent efficiency of silver nanoparticles as compared with that of Ag+ ions and AOT. The NSP concentrations lethal for Physarum were similar to those found earlier for bacteria and viruses. The chemotaxis-based tests applied in this study allow one to quantitatively assess cell reactions and monitor their time course. Besides, they have a much higher resolving capacity than the tests based on lethal effects of abiotic substances.

Function of c-Fos-like and c-Jun-like proteins on trichostatin A-induced G2/M arrest in Physarum polycephalum.

The homologs of transcription factors c-Fos and c-Jun have been detected in slime mold Physarum polycephalum during progression of the synchronous cell cycle. Here we demonstrated that c-Fos-like and c-Jun-like proteins participated in G2/M transition by the regulation of the level of Cyclin B1 protein in P. polycephalum. The study of antibody neutralization revealed that interruption of the functions of c-Fos-like and c-Jun-like proteins resulted in G2/M transition arrest, implicating their functional roles in cell cycle control. When G2/M transition was blocked by histone deacetylase inhibitor trichostatin A, changes in c-Fos- and c-Jun-like protein levels, and hyperacetylation of c-Jun-like protein, were observed. The data suggest that in P. polycephalum, c-Fos- and c-Jun-like proteins may be the key factors in the regulation of histone acetylation-related G2/M transition, involving the coordinated expression and hyperacetylation of these proteins.

Ionomycin and 2,5'-di(tertbutyl)-1,4,-benzohydroquinone elicit Ca2+-induced Ca2+ release from intracellular pools in Physarum polycephalum.

Calcium level in organelles of the slime mold Physarum polycephalum was monitored by chlortetracycline, a low-affinity calcium indicator. It was found that 2,5'-di(tertbutyl)-1,4,-benzohydroquinone (BHQ) at a concentration of 100 microM, but not the highly specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (1-10 microM), elicited calcium release from the CTC-stained intracellular calcium pool. Ionomycin also caused a calcium release (23.7+/-5.1%), which was less than that induced by BHQ (30.1+/-6.0%). Procaine (10 mM), a blocker of ryanodine receptor, completely abolished the responses to BHQ and ionomycin. Another blocker, ryanodine (100 microM), only slightly diminished the responses to ionomycin and BHQ. Apparently, BHQ and ionomycin acting as a Ca2+-ATPase inhibitor and an ionophore, respectively, elicit an increase in [Ca2+]i, which in turn triggers a calcium-induced calcium release (CICR) via the ryanodine receptor. Caffeine, an activator of ryanodine receptor, at a concentration of 25-50 mM produced a Ca2+-release (5.6-16.0%), which was not similar in magnitude to CICR. The response to 25 mM caffeine was only moderately inhibited by 25 mM procaine, and almost completely abolished by 50 mM procaine and 100 microM ryanodine.

Beta-poly(L-malate) production by non-growing microplasmodia of Physarum polycephalum. Effects of metabolic intermediates and inhibitors.

The production of beta-poly(L-malate) (PMLA) by non-growing microplasmodia of Physarum polycephalum was investigated. Growth was minimal in culture medium devoid of nitrogen source, but PMLA production occurred at a substantial rate. The addition of metabolic intermediates, malate, fumarate, succinate, and oxaloacetate, and the omission of hematin showed considerable growth inhibition in the presence of the nitrogen source, while PMLA production per unit biomass increased significantly. The results indicated that PMLA production was dissociated from biomass production under these conditions. The stimulating effect of carbonate on PMLA production was independent on growth. Cultivation in the absence of the nitrogen source and hematin or in the presence of the metabolites may be a useful technique for efficient PMLA production at a minimum of biomass production.

[Effects of cytochalasin B upon mitosis of Physarum polycephalum].

Cytochalasin B, known as a functional inhibitor of actin, was microinjected into naturally synchronous plasmodia of Physarum polycephalum, and the mitotic behaviours of both CB-treated specimens and the control were examined with light and electron microscopy. Mitosis in the CB-treated specimens began about 20 to 60 minutes later than that of the control. It was delayed 35 minutes in the specimens treated with CB in the S phase of the cell cycle, and the delayed time was 20 minutes and 45 minutes, respectively. In the specimens treated with CB in early and middle G2 phase, the longest delay was 60 minutes found in the specimens treated in late G2 phase, indicating that mitosis was affected in Physarum polycephalum when the function of actin was inhibited by CB treatment. The CB-treated specimens and the control showed similarities in the process of mitosis and dynamic changes of nuclear structures, suggesting that the main effect of CB treatment upon mitosis may be to delay the triggering of the mitosis.

Is beta-poly(L-malate) synthesis catalysed by a combination of beta-L-malyl-AMP-ligase and beta-poly(L-malate) polymerase?

beta-Poly(L-malate) is supposed to function in the storage and transport of histones, DNA polymerases and other nuclear proteins in the giant syncytical cells (plasmodia) of myxomycetes. Here we report on the biosynthesis of [14C]beta-poly(L-malate) from injected L-[14C]malate in the plasmodium of Physarum polycephalum. The effects of KCN, arsenate, adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, guanosine 5'-(beta, gamma-methylene)triphosphate, desulfo coenzyme A and phenylarsinoxid on beta-poly(L-malate) synthesis were studied after their coinjection with L-[14C]malate. The synthesis was not affected by KCN or desulfo coenzyme A, but was blocked by arsenate and adenosine 5'-(alpha,beta-methylene)triphosphate. The plasmodium lysate catalysed an L-malate-dependent ATP-[32P]pyrophosphate exchange, but was devoid of beta-poly(L-malate) synthetic activity under all experimental conditions tested. The results suggested an extramitochondrial synthesis of beta-poly(L-malate), involving the polymerization of beta-L-malyl-AMP. It is assumed that the lack of synthesis in the lysate is caused by the inactivation of beta-poly(L-malate) polymerase involving a cell injury kinase pathway. Because injected guanosine 5'-(beta, gamma-methylene)triphosphate blocks the synthesis, the injury signal is likely to be GTP dependent.

In vivo topoisomerase II cleavage sites in the ribosomal DNA of Physarum polycephalum.

We have analyzed the topoisomerase II cleavage sites in the extrachromosomal ribosomal DNA of the lower eukaryote Physarum polycephalum using the topoisomerase II-specific inhibitor, 6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid. Most of the in vivo topoisomerase II cleavage sites were found either in the transcribed region of ribosomal DNA or in the palindromic region surrounded by the replication origins. Two classes of sites were identified: those which correlate with DNase I hypersensitive sites and corresponding to an open chromatin configuration (transcribed region) and internucleosomal cleavage sites (in the region of replication origins). Topoisomerase II drug-induced cleavage in the ribosomal DNA was considerably reduced upon Physarum differentiation to a dormant stage of life, the spherules. In contrast, the amount of drug-dependent cleavage was found to increase during the metaphase of mitosis, when rDNA transcription is shut off. These findings suggest a role for topoisomerase II in the ribosomal DNA minichromosomes segregation, in addition to its role in transcription. Finally, the similarity between in vivo sites and those observed following drug treatment of isolated nuclei indicates that no profound change occurs in rDNA chromatin conformation during nuclei isolation. By contrast, in vitro cleavage sites with purified topoisomerase II weakly correlate to in vivo, indicating a prominent role for chromatin structure in determining the interaction sites of topoisomerase II with DNA in vivo.

Microtubules are required in amoeba chemotaxis for preferential stabilization of appropriate pseudopods.

Amoebae of Physarum polycephalum exhibit chemotactic responses to glucose and to cAMP. The chemotaxing amoebae exhibit alternating locomotive movements: relatively linear locomotion and movements that change the direction of the locomotion. Such locomotive activity is tightly coupled with the changes in the number and the positions of the pseudopods; cells have one pseudopod at the leading edge during their linear locomotion, while they have multiple pseudopods when they are changing the direction of locomotion. Treatment of cells with microtubule-disrupting reagents inhibited the chemotaxis of the cells. To characterize the role of the microtubule system in chemotaxis, we quantitatively analyzed the relationship between the positions of multiple pseudopods of the amoebae and the relative stability of the pseudopods during reorientation. No significant differences were observed in the pseudopod dynamics between the untreated and the treated amoebae. In both cases, one pseudopod at the leading edge continued to expand during linear locomotion. It then split into two to three pseudopods in the reorientation phase, and the positions of the multiple pseudopods were random. Among multiple pseudopods, however, the pseudopods closer to the microneedle tip were selectively stabilized more often than those distant from the tip in the presence of the microtubule system. By contrast, such preferential stabilization of the appropriate pseudopods was completely abolished by microtubule inhibitors. The microtubule-dependent selection of appropriately located pseudopods enables amoebae to turn correctly at the reorientation step.

Information propagation by spatio-temporal pattern change of Ca2+ concentration throughout Physarum polycephalum with repulsive stimulation.

The development of a spatio-temporal pattern of Ca2+ concentration (Ca2+ pattern) in the plasmodium of Physarum polycephalum during repulsive response was studied using fura-2. In the migrating cell, the gradient of the Ca2+ concentration (Ca2+ gradient) immediately showed a decrease in local concentration in the area (S-site) stimulated by 50 mM KCl. The concentration rose and then decreased in a site neighboring the S-site. This transient increase of Ca2+ concentration, the duration of which was approx. 10 minutes, was propagated to the site most distant from the S-site. There, the Ca2+ concentration gradually rose and remained at a high level. Twenty-five minutes after stimulation, a new Ca2+ gradient was established throughout the plasmodium. The migratory direction of the cell as a whole then changed. In this process, although the period of Ca2+ oscillation changed at the S-site, this change was only local to the site. During the information processing of the local repulsive stimulus, the transient Ca2+ increase propagated the local information about the stimulus to the non-stimulation sites (NS-sites), leading to the generation of a new pattern and the start of coordinated migration of the plasmodium.