Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

An adaptive and robust biological network based on the vacant-particle transportation model

A living system reveals local computing by referring to a whole system beyond the exploration-exploitation dilemma. The slime mold, Physarum polycephalum, uses protoplasmic flow to change its own outer shape, which yields the boundary condition and forms an adaptive and robust network. This observation suggests that the whole Physarum can be represented as a local protoplasmic flow system. Here, we show that a system composed of particles, which move and are modified based upon the particle transformation that contains the relationship between the parts and the whole, can emulate the network formed by Physarum. This system balances the exploration-exploitation trade-off and shows a scale-free sub-domain. By decreasing the number of particles, our model, VP-S, can emulate the Physarum adaptive network as it is attracted to a food stimulus. By increasing the number of particles, our model, VP-D, can emulate the pattern of a growing Physarum. The patterns produced by our model were compared with those of the Physarum pattern quantitatively, which showed that both patterns balance exploration with exploitation. This model should have a wide applicability to study biological collective phenomena in general.     

RNA editing of the col mRNA throughout the life cycle of Physarum polycephalum

Editing of RNA via the insertion, deletion or substitution of genetic information affects gene expression in a variety of systems. Previous characterization of the Physarum polycephalum cytochrome c oxidase subunit I (col) mRNA revealed that both nucleotide insertions and base substitutions occur during the maturation of this mitochondrial message. Both types of editing are known to be developmentally regulated in other systems, including mammals and trypanosomatids. Here we show that the col mRNA present in Physarum mitochondria is edited via specific nucleotide insertions and C to U conversions at every stage of the life cycle. Primer extension sequencing of the RNA indicates that this editing is both accurate and efficient. Using a sensitive RT-PCR assay to monitor the extent of editing at individual sites of C insertion, we estimate that greater than 98% of the steady-state amount of col mRNA is edited throughout the Physarum developmental cycle.     

Asymmetry in the self-sustained oscillation ofPhysarum plasmodial strands

Summary The characteristics of the self-sustained oscillation in the plasmodial strand ofPhysarum polycephalum have been investigated in one steady and two transient conditions. An isolatedPhysarum strand changes its length periodically when it is suspended. In the behaviour of the self-sustained oscillation under the conditions, we provide the first demonstration that the changes in the periods of the oscillation can be ascribed to effects on the shortening phase, while the lengthening periods are almost unaffected. This result means that the asymmetric self-sustained oscillation of thePhysarum strand is composed of an active contracting process, presumably due to actin filaments and myosin-like molecules in the strand, and a passive lengthening process which is merely an extension of the strand under a load.     

Towards Physarum Robots: Computing and Manipulating on Water Surface

Plasmodium of Physarum polycephalum is an ideal biological substrate for implementing concurrent anu parauel computation, including combinatorial geometry and optimization on graphs. The scoping experiments on Physarum computing in conditions of minimal friction, on the water surface were performed. The laboratory and computer experimental results show that plasmodium of Physarum is capable of computing a basic spanning tree and manipulating of light-weight objects. We speculate that our results pave the pathways towards the design and implementation of amorphous biological robots.     

Dynamic complexity inPhysarum polycephalum shuttle streaming

Summary The nature of the oscillator controlling shuttle streaming inPhysarum polycephalum is not well understood. To examine the possibility of complex behavior in shuttle streaming, the time between reversal of streaming direction was measured over several hours in an intact plasmodium to produce a time series. Time series data were then used to analyze shuttle streaming dynamics. Complexity in shuttle streaming is revealed by an inverse frequency (1/f) power spectrum where the amplitude of reversals is plotted against their frequency. The complex dynamics of shuttle streaming is also shown by a trajectory in phase space typical of a strange attractor. Finally, shuttle streaming time series data have a dominant Lyapunov exponent of approximately zero. Dynamic systems with a Lyapunov exponent of zero exist in a state at the edge of chaos. Systems at the edge exhibit self-organized criticality, which produces complex behavior in many physical and biological systems. We propose that complex dynamics inPhysarum shuttle streaming is an example of self-organized criticality in the cytoplasm. The complex behavior ofPhysarum is an emergent phenomenon that probably results from the interaction of actin filaments, myosin, ATP, and other components involved in cell motility.     

Physarum myosin light chain one: A potential regulatory factor in cytoplasmic streaming

Summary Physarum myosin is composed of a heavy chain of about 225,000 daltons and two small polypeptides of 17,700 and 16,100 daltons, called light chain one (LC 1) and two (LC 2). Light chain one is shown to belong to the general class of regulating light chains by two independent criteria. After denaturation, purification and renaturation of thePhysarum light chains only LC 1 will combine with scallop myofibrils in which one myosin regulatory light chain has been removed. This LC 1 can restore inhibition of the ATPase activity of the myofibrils at 10^–8 M Ca⁺⁺ just as well as light chains from rabbit skeletal myosin. Secondly, this LC 1 is the only component of the myosin that is significantly phosphorylated by an endogenous kinase present in crude actomyosin. An active phosphatase is also present. Preliminary results could not detect calcium sensitivity for either kinase or phosphatase, nevertheless the importance of phosphorylation in affecting activity of biological systems suggests that LC 1 may serve some regulating function for plasmodial actomyosin.     

The intranuclear microtubule organizing centre in the plasmodium of the myxomycete Physarum polycephalum

Physarum possesses two different microtubule cytoskeletons. In amoebae, cytoplasmic and mitotic microtubules are nucleated by a typical centrosome. In contrast, it has been reported that plasmodia have an intranuclear spindle organizing centre (SPOC) devoid of centrioles. We present genetic evidence suggesting that the SPOC located in the centrosome is very similar to the intranuclear plasmodial SPOC. The immunostaining properties of a new monoclonal antibody against Physarum centrosome has been used to compare these different MTOCs. Moreover, a dense plasmodial microtubule network was present in interphase plasmodia and absent in plasmodia undergoing mitosis. MTOCs responsible for the nucleation of the cytoplasmic microtubule network and intranuclear SPOCs were located in two different compartments of the plasmodium.     

Thermotaxis and protoplasmic oscillations inPhysarum plasmodia analysed in a novel device generating stable linear temperature gradients

Summary The application of sublethal temperature gradients offers a simple, non-invasive means for in vivo studies of thermotaxis and other temperature-dependent processes in various organisms. Development, for instance, can be dramatically desynchronized, and the resulting development gradients allow to analyze physiological inter-dependencies between locally separated subsystems. For this purpose a simple device has been developed, by which a stable linear gradient of 8 °C/cm is established on an inert metal sheet with the aid of Peltier elements. The effects of linear temperature gradients on fusion, growth, and migration of plasmodia of the slime moldPhysarum polycephalum was filmed by 16 mm film time-lapse technique, and their local contraction—relaxation cycles analysed by multistrip kymography, which represents a graphic documentation of the spatio-temporal pattern of protoplasmic movements that occur along well-defined regions within the giant cell.Physarum plasmodia preferentially fuse, and grow, in the range of 24–26 °C. Different parts of a single macroplasmodium can simultaneously show positive and negative thermotaxis. The contraction—relaxation cycles generating the protoplasmic shuttle streaming within the network of veins essentially depend on local temperatures and are instantaneously desynchronized by the temperature gradient. Thus they cannot be controlled by a central pacemaker or an overall electric signal. However, there is a strong tendency to locally synchronize the various oscillation frequencies present within the giant cell if temperature differences do not exceed 2 °C.     

ATP oscillation inPhysarum plasmodium

Summary Using bioluminescence of luciferin-luciferase, we showed that ATP leaked out rhythmically from a strand segment ofPhysarum plasmodium made permeable with caffeine-arsenate. With simultaneous measurement of isometric tension rhythm of the strand, it was revealed that the period and phase of oscillation in ATP leakage correspond well with those of tension production. Further, microinjection of luciferin-luciferase into the plasmodial strand indicated that the intracellular luminescence of luciferin-luciferase also oscillates with the same period and in the same phase as the tension rhythm.The free ATP concentration in a homogenate ofPhysarum plasmodium was of the order of 10 M, but if the homogenate was heated in boiling water, the intensity of luminescence suddenly increased 10–100 fold. ATP available for mechanical workin vivo is thus supposed to be at a much lower level than the total average, which was found in the range of 0.2–0.7 mM.     

A charged contaminant prevents Physarum chromatin from aggregating

Unlike typical chromatins, the soluble Physarum chromatin can not be precipitated by salt. Using the reconstituted system based on the H1/H5-depleted chicken erythrocyte chromatin, we demonstrate that this feature is not due to inability of Physarum histone H1 to aggregate chromatin. We show that the abnormal behavior of Physarum chromatin can be explained by its association with charged contaminant.     

Studies on microplasmodia of Physarum polycephalum

Summary Fragments excised from front regions of thinspread Physarum plasmodia were used to examine a possible correlation between the periodical dynamic activity of such specimens and the spatial organization of actin fibrils. Under isotonic conditions, symmetrical contractions and relaxations of the entire fragment alternate with a period of 1–4 min, whereas under isometric conditions local contractions and relaxations occur simultaneously in different regions of the same specimen. Rapid fixation and phalloidin-staining at distinct stages of the contraction-relaxation cycle demonstrates the permanent existence of cytoplasmic actin fibrils under both isometric and isotonic conditions. During the transition from relaxation to contraction the fibrils shorten in length from 25.5 m to 21.0 m and increase in density from 1.2 fibrils/1000 m² to 2.3 fibrils/1000 m². The present results demonstrate that actin fibrils in Physarum plasmodia are not completely decomposed and reformed every contraction-relaxation cycle.Series Studies on microplasmodia of Physarum polycephalum VIII     

The self-organizing exploratory pattern of the argentine ant

Workers of the Argentine ant, Iridomyrmex humilis,start to explore a chemically unmarked territory randomly. As the exploratory front advances, other explorers are recruited and a trail extends from it to the nest. Whereas recruitment trails are generally constructed between two points, these exploratory trails have no fixed destination, and strongly resemble the foraging patterns of army ants. A minimal model shows how the exploratory pattern may be generated by the individual workers' simple trail-laying and -following behavior, illustrating how complex collective structures in insect colonies may be based on self-organization.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Arrangement of highly-repeated sequences

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.     

Multiobjective H2/H∞ synthetic gene network design based on promoter libraries

Some current promoter libraries have been developed for synthetic gene networks. But an efficient method to engineer a synthetic gene network with some desired behaviors by selecting adequate promoters from these promoter libraries has not been presented. Thus developing a systematic method to efficiently employ promoter libraries to improve the engineering of synthetic gene networks with desired behaviors is appealing for synthetic biologists.In this study, a synthetic gene network with intrinsic parameter fluctuations and environmental disturbances in vivo is modeled by a nonlinear stochastic system. In order to engineer a synthetic gene network with a desired behavior despite intrinsic parameter fluctuations and environmental disturbances in vivo, a multiobjective H₂/H_∞ reference tracking (H₂ optimal tracking and H_∞ noise filtering) design is introduced. The H₂ optimal tracking can make the tracking errors between the behaviors of a synthetic gene network and the desired behaviors as small as possible from the minimum mean square error point of view, and the H_∞ noise filtering can attenuate all possible noises, from the worst-case noise effect point of view, to achieve a desired noise filtering ability. If the multiobjective H₂/H_∞ reference tracking design is satisfied, the synthetic gene network can robustly and optimally track the desired behaviors, simultaneously.First, based on the dynamic gene regulation, the existing promoter libraries are redefined by their promoter activities so that they can be efficiently selected in the design procedure. Then a systematic method is developed to select an adequate promoter set from the redefined promoter libraries to synthesize a gene network satisfying these two design objectives. But the multiobjective H₂/H_∞ reference tracking design problem needs to solve a difficult Hamilton–Jacobi Inequality (HJI)-constrained optimization problem. Therefore, the fuzzy approximation method is employed to simplify the HJI-constrained optimization problem to an equivalent linear matrix inequality (LMI)-constrained optimization problem, which can be easily solved by selecting an adequate promoter set from the redefined promoter libraries using the LMI toolbox in Matlab.Based on the confirmation of in silico design examples, we can select an adequate promoter set from the redefined promoter libraries to achieve the multiobjective H₂/H_∞ reference tracking design. The proposed method can reduce the number of trial-and-error experiments in selecting an adequate promoter set for a synthetic gene network with desired behaviors. With the rapid increase of promoter libraries, this systematic method will accelerate progress of synthetic biology design.     

Characterization of ard B and ard C actin gene loci of Physarum polycephalum

Summary Physarum polycephalum (strain M₃CVIII) contains four unlinked actin gene loci, each with two alleles (ard A1, ard A2, ard B1, ard B2, ard C1, ard C2, ard D1 and ard D2). The 4.8 kbp HindIII component of the ard C2 locus was isolated as a recombinant phage-, after HindIII fragments of Physarum DNA ranging from 4.3 kbp to 5.5 kbp were cloned into phage- NM1149. The fraction of Physarum DNA cloned contained the ard C locus, and no other actin locus. Small inserts were favoured to reduce the probability of cloning a complete repetitive element, because such elements have been found to adversely affect the stability of recombinants.The coding sequences of the actin gene (approximately 1.1 kbp) spanned more than 3 kbp indicating the presence of introns. A 1.6 kbp HindIII/EcoRI fragment of the ard C locus, which contained some coding sequences, hybridized extensively with HindIII fragments of genomic DNA indicating the presence of repetitive sequences. A 2.3 kbp HindIII/EcoRI fragment containing most of the coding sequences of the C2 allele of the ard C locus hybridized with the C1, allele and both alleles of the ard B locus, but not with the ard A locus or ard D locus. This distinction was used to establish for the ard B and ard C loci the relationship between the EcoRI and HindIII fragments that define an ard locus. The ability to distinguish between ard loci may facilitate studies of the expression of particular actin loci.     

Dispersion relation in oscillatory reaction-diffusion systems with self-consistent flow in true slime mold

In the large amoeboid organism Physarum, biochemical oscillators are spatially distributed throughout the organism and their collective motion exhibits phase waves, which carry physiological signals. The basic nature of this wave behaviour is not well-understood because, to date, an important effect has been neglected, namely, the shuttle streaming of protoplasm which accompanies the biochemical rhythms. Here we study the effects of self-consistent flow on the wave behaviour of oscillatory reaction-diffusion models proposed for the Physarum plasmodium, by means of numerical simulation for the dispersion relation and weakly nonlinear analysis for derivation of the phase equation. We conclude that the flow term is able to increase the speed of phase waves (similar to elongation of wave length). We compare the theoretical consequences with real waves observed in the organism and also point out the physiological roles of these effects on control mechanisms of intracellular communication.     

Spatio-temporal organization of intracellular ATP content and oscillation patterns in response to blue light byPhysarum polycephalum

Summary Phototactic responses in a giant amoeboid cell ofPhysarum plasmodium were studied both by analyzing intracellular ATP content and by applying image processing for recording oscillatory changes in thickness with use of a microcomputer. The ATP content fluctuated and deviated from the initial level upon exposure to light with varying wavelength from 400 to 600 nm. The maximum decrease in the integrated value dt with T=9 minutes occurred at the wavelength 450 nm. The ATP in a migrating plasmodium distributed twice as high in the front as in the rear. This polar pattern became unstable, and a new wavy pattern appeared by stimulating a local frontal part with blue light. In a concentrically extending plasmodium, peripheral and inside regions oscillated in opposite phase to each other. When part of this organism was exposed to light, stimulated and unslimulated regions began to oscillate in opposite phase, and phase waves propagated inward the stimulated region. And the protoplasm there became thinner, the strongest avoidance reaction occurring to 450 nm light as in ATP response. Phototactic behavior inPhysarum is discussed in terms of bifurcation in spatio-temporal organization appearing in a self-organizing system.     

A queueing model for predicting message latency in uni-directional <Emphasis Type="Italic">k</Emphasis>-ary <Emphasis Type="Italic">n</Emphasis>-cubes with deterministic routing and non-uniform traffic

The interconnection network is one of the key architectural components in any parallel computer. The distribution of the traffic injected into the network is among the factors that greatly influences network performance. The uniform traffic pattern has been adopted in many existing network performance evaluation studies due to the tractability of the resulting analytical modelling approach. However, many real applications exhibit non-uniform traffic patterns such as hot-spot traffic. K-ary n-cubes have been the mostly widely used in the implementation of practical parallel systems. Extensive research studies have been conducted on the performance modelling and evaluation of these networks. Nonetheless, most of these studies have been confined to uniform traffic distributions and have been based on software simulation. The present paper proposes a new stochastic model to predict message latency in k-ary n-cubes with deterministic routing in the presence of hot-spot traffic. The model has been validated through simulation experiments and has shown a close agreement with simulation results.