Morphological and molecular characterization of a new succulenticolous Physarum (Myxomycetes, Amoebozoa) with unique polygonal spores linked in chains.

A new plasmodiocarpic and sporocarpic species of myxomycete in the genus Physarum is described and illustrated. This new species appeared on decayed leaves and remains of succulent plants (Agave, Opuntia, Yucca) growing in arid zones. It differs from all other species in the genus in having polyhedral spores linked in chains like a string of beads, a unique feature within all known myxomycetes. Apart from detailed morphological data, partial sequences of both the small-subunit ribosomal RNA and elongation factor 1-alpha genes, generated from four isolates collected in two distant regions, i.e., Mexico and Canary Islands, are also provided in this study. Combined evidence supports the identity of the specimens under study as a new species.

Two-Layer Elastographic 3-D Traction Force Microscopy.

Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum's Poisson's ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson's ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson's ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.

Transient expression of RAD51 in the late G2-phase is required for cell cycle progression in synchronous Physarum cells.

The homologous recombination factor RAD51 is highly conserved. This criterion enabled us to identify a RAD51 ortholog in Physarum polycephalum. We found that the Physarum protein presents a high homology to the human protein and cross-reacted with antibodies directed against the human RAD51. Taking advantage of the natural synchrony of millions of nuclei within a single cell of Physarum, we investigated the fluctuation of the amount of the PpRAD51 throughout the cell cycle. Our results showed that in the late G2-phase, RAD51 was transiently expressed in a large quantity. Furthermore, knocking-down RAD51 in the G2-phase abolished this transient expression before mitosis and affected cell cycle progression. These results support the idea that RAD51 plays a role in the progression of the cell cycle in the late G2-phase.

Accumulation of noncrystalline cellulose in Physarum microplasmodia.

Physarum plasmodium lives as a slimy mass of protoplast in the dark fragments into small multinucleated microplasmodia (mPL) in a liquid medium. When mPL are exposed to several unfavorable environments, they transform into "spherules" with a cell wall. Using a synchronous spherule-induction system for mPL, we examined the effect of 2,6-dichlorobenzonitrile on the synthesis of cellulose in mPL, by observing mPL under a fluorescence microscope, and isolated cellulose from mPL to identify them morphologically under scanning electron microscopy. Moreover, we examined in vivo labeling to determine when cellulose synthesis is activated in step 2. We found that the nourishment medium in step 2 was essential for mPL prior to spherulation and that the conversion starts at 48 h in step 2 of our system. From the experiments using Updegraff reagent for the sedimentation of cellulose in the cell wall fraction from mPL, we propose that cellulose produced in mPL is likely noncrystalline cellulose. We conclude that mPL of multinucleated protoplasts without the cell wall structure synthesize cellulose under constitutive condition and accumulate abundantly noncrystalline cellulose, in preparation for unfavorable environments that may occur in the future in which mPL must initiate the program to form the cell wall of spherules.

A model of network formation by Physarum plasmodium: interplay between cell mobility and morphogenesis.

The plasmodium of Physarum polycephalum has attracted much attention due its intelligent adaptive behavior. In this study, we constructed a model of the organism and attempted to simulate its locomotion and morphogenetic behavior. By modifying our previous model, we were able to get closer to the actual behavior. We also compared the behavior of the model with that of the real organism, demonstrating remarkable similarity between the two.

Physarum machines: encapsulating reaction-diffusion to compute spanning tree.

The Physarum machine is a biological computing device, which employs plasmodium of Physarum polycephalum as an unconventional computing substrate. A reaction-diffusion computer is a chemical computing device that computes by propagating diffusive or excitation wave fronts. Reaction-diffusion computers, despite being computationally universal machines, are unable to construct certain classes of proximity graphs without the assistance of an external computing device. I demonstrate that the problem can be solved if the reaction-diffusion system is enclosed in a membrane with few 'growth points', sites guiding the pattern propagation. Experimental approximation of spanning trees by P. polycephalum slime mold demonstrates the feasibility of the approach. Findings provided advance theory of reaction-diffusion computation by enriching it with ideas of slime mold computation.

First report of sporangia of a myxomycete (Physarum pusillum) on the body of a living animal, the lizard Corytophanes cristatus.

Myxomycetes are protists whose life cycle depends on aerially dispersed spores that germinate into motile myxamoebae, which then pair and fuse to form a larger, motile plasmodium. The plasmodium seeks out a suitable fruiting site (usually atop vegetative material or detritus) and transforms into fruiting bodies that release the spores. In this paper we report the first known instance of a myxomycete, in this case Physarum pusillum, sporulating on the body of a living animal, the cryptic lizard Corytophanes cristatus, which was collected in eastern Honduras in 2003.

Smart behavior of true slime mold in a labyrinth.

Even for humans it is not easy to solve a maze. But the plasmodium of true slime mold, an amoeba-like unicellular organism, has shown an amazing ability to do so. This implies that an algorithm and a high computing capacity are included in the unicellular organism. In this report, we discuss information processing in the microorganism to focus on the issue as to whether the maze-solving behavior is akin to primitive intelligence.

Transcription and RNA editing in a soluble in vitro system from Physarum mitochondria.

The dissection of RNA editing mechanisms in PHYSARUM: mitochondria has been hindered by the absence of a soluble in vitro system. Based on our studies in isolated mitochondria, insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs is closely linked to transcription. Here we have fractionated mitochondrial lysates, enriching for run-on RNA synthesis, and find that editing activity co-fractionates with pre-formed transcription elongation complexes. The establishment of this soluble transcription-editing system allows access to the components of the editing machinery and permits manipulation of transcription and editing substrates. Thus, the availability of this system provides, for the first time, a means of investigating roles for cis-acting elements, trans-acting factors and nucleotide requirements for the insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs. This methodology should also be broadly applicable to the study of RNA processing and editing mechanisms in a wide range of mitochondrial systems.

Interaction between cell shape and contraction pattern in the Physarum plasmodium.

The relationship between cell shape and rhythmic contractile activity in the large amoeboid organism Physarum polycephalum was studied. The organism develops intricate networks of veins in which protoplasmic sol moved to and fro very regularly. When migrating on plain agar, the plasmodium extends like a sheet and develops dendritic veins toward the rear. After a particular stimulation, the vein organization changes into veinless or vein-network structures. In both structures, the mixing rate of the protoplasm, which is related to communication among contraction oscillators, decreased compared with that of the dendritic one. Accompanying these changes in vein structure, the spatio-temporal pattern of the rhythmic contraction changed into a small-structured pattern from a synchronized one. In the above process, cell shape affects the contraction pattern, but, conversely, the contraction pattern effects the cell shape. To demonstrate this, a phase difference in the rhythmic contraction was induced artificially by entraining the intrinsic rhythm to external temperature oscillations. New veins then formed along the direction parallel to the phase difference of the rhythm. Consequently, the vein organization of the cell interacts with the contractile activity to form a feedback loop in a mechanism of contraction pattern formation.

Acceleration-sensitivity threshold of Physarum.

Free-living cells show distinct gravisensitivities and often use the gravity ('g') vector for their spatial orientation. The rhythmic contractions of the ameboid Myxomycete (acellular slime mold) Physarum polycephalum are a sensitive parameter which can be modified by external stimuli. Space experiments and ground-based 0 x g simulation studies established that the contraction period transiently decreases after a transition from 1 x g to 0 x g with a back-regulating process starting after 30 min. For determination of the threshold of acceleration sensitivity, a slow-rotating centrifuge microscope (NIZEMI--Niedergeschwindigkeits-Zentrifugenmikroskop) was used, providing in space accelerations from 0 x g to 1.5 x g. A stepwise acceleration increase revealed that the lowest acceleration level capable of inducing a response was 0.1 x g. The response to the acceleration increase was an increase in contraction period, in contrast to a stimulus deprivation, which led to a period decrease. The time schedule of the acceleration responses and back-regulating process seems to be fixed, suggesting that every acceleration being above the threshold can induce a complete response-regulation process. The low acceleration-sensitivity threshold favors rather large and dense cell organelles as candidates for the gravity receptor in Physarum.

Relationship between intracellular period modulation and external environment change in Physarum plasmodium.

The relationship between intracellular period modulation and external environment change was investigated from the viewpoint of internal information coding in Physarum plasmodium. For the external conditions, concentration changes of attractant (galactose) and repellent (KCl) were used, and the internal responses were measured as the thickness oscillation of the plasmodium. (i) Period of the intracellular oscillation decreased when the concentration of attractant was increased and when the concentration of repellent was decreased. (ii) The period increased when the attractant was decreased and when the repellent was increased. (iii) The larger concentration change induced the larger period modulation. (iv) These responses were observed when the change of concentration was greater than a threshold value. From these results, it was clarified that the relative change in environmental condition is encoded on the relative period modulation in intracellular oscillation. This means that the period change does not directly represent the environment itself but represents the change of its condition. Thus, it is further suggested that the plasmodium estimates the environmental condition based on the relationship between the previous external condition and the present one.

Purification and partial characterization of transglutaminase from Physarum polycephalum.

An intracellular form of calcium ion-dependent transglutaminase (R-glutaminylpeptide:amine gamma-glutaminyltransferase, EC 2.3.2.13) was purified 818-fold to apparent homogeneity from acetone powder preparations of spherules of the acellular slime mold Physarum polycephalum. The enzyme was purified by combined methods of precipitation with 15% (wt/vol) polyethylene glycol, DEAE-cellulose chromatography, and isoelectric focusing in a pH 5 to 7 gradient. The isoelectric point of the enzyme was 6.1. The molecular mass of the denatured enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 39.6 kDa. A molecular weight of 77,000 was found by gel filtration of the native enzyme on a Superose 12 fast protein liquid chromatography column, indicating that the native functional protein is a dimer. The purified transglutaminase catalyzed the incorporation of [14C]putrescine into protein substrates including casein, N,N'-dimethylcasein, actin purified from P. polycephalum, and actin purified from bovine muscle. Actin was the preferred substrate for the enzyme, both as a purified protein and in crude extracts prepared from P. polycephalum. With N,N'-dimethylcasein as the amine acceptor substrate, [14C]putrescine, [14C]spermidine, and [14C]spermine were all effective amine donor substrates with Km values of 49, 21.4, and 31.7 microM, respectively. All three of these polyamines demonstrated strong substrate inhibition of the enzyme activity between 100 and 200 microM. Upon starvation induced by depletion of a carbon source for growth, the specific activity of this enzyme increased sixfold during the differentiation of P. polycephalum microplasmodia to spherules. This suggests a role for transglutaminase in the construction of spherules, which have the capacity to survive starvation and dessication.

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum.

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.

Synergistic effect of heat-shock and UV-irradiation on mitosis and protein synthesis in G2-phase plasmodia of Physarum polycephalum Schw--reduction in UV-induced mitotic delay in a preheat-shocked system.

The synergistic effect of UV irradiation and heat-shock during the last 3 hr of G2 phase of the cell cycle in the plasmodia of P. polycephalum, in terms of mitotic delay and inhibition of protein synthesis, has been evaluated. The mitotic delay due to both perturbers coordinately increased closer to mitosis. Maximum mitotic delay was obtained in plasmodia heat-shocked after UV irradiation, indicating the presence in this system of either a heat-labile mitogenic substance which is comparatively less susceptible to UV or a substance which is made more susceptible to hyperthermia by UV. A protective role for heat-shock applied before irradiation has been observed in that, radiation-induced mitotic delay is significantly reduced in this combination. There was severe inhibition of translation in all the perturbed classes. Organelle level effects which are independent of major protein synthetic activities or different levels of heat-shock protein production could be the reason for the lack of correlation between percentage inhibition of general protein synthesis and the extent of mitotic delay with respect to the two double-perturbed systems.

A purified cellular extract accelerates the cell cycle in Physarum polycephalum.

Plasmodia of the myxomycete Physarum polycephalum (strain Cl) were collected at different times during the cell cycle and extracts were prepared from homogenates using a buffer optimized for microinjection into plasmodial veins. These extracts were injected into plasmodia during the first 3 h of the cell cycle. The time of the following mitosis was monitored and compared with that of the buffer-injected controls. Extracts of plasmodia homogenized 45 min before late telophase accelerated the onset of mitosis in the injected plasmodium up to 70 min, i.e., an advance of 10-14% compared to the 8- to 10-h cell cycle duration of the controls. The accelerating activity vanished completely after heating, freezing, or protease digestion, thus indicating the peptide nature of the active agent. Purification of the active compound by means of gel filtration revealed a molecular mass of about 2500 Da. The active portion of the extract was further fractionated by HPLC and the activity determined in a single peak.

Regulation of tubulin synthesis during the cell cycle in the synchronous plasmodia of Physarum polycephalum.

Regulation of alpha- and beta-tubulin isotype synthesis during the cell cycle has been studied in the myxomycete Physarum polycephalum, by subjecting synchronous plasmodia to temperature shifts and pharmacological perturbations. Temperature shifts interfered with the regulation of tubulin synthesis. Inhibition of DNA synthesis prevents tubulin degradation after completion of the cell cycle (Ducommun and Wright, Eur. J. Cell Biol., 50:48-55, 1989) but did not perturb the initiation of tubulin synthesis. The constant increase of tubulin synthesis in the presence of tubulin-sequestering drugs and the decrease of tubulin synthesis during a treatment with aphidicolin in late G2 phase suggest the existence of an autoregulatory mechanism of tubulin synthesis. Moreover, the microtubule poison methyl benzimidazole carbamate dissociated synthesis of the alpha 1-tubulin isotype from the generally strictly coordinated synthesis of all tubulin isotypes during the transient interruption of mitosis. These observations show that a microtubular poison can perturb regulation of the synthesis of specific isotubulins.

Polypeptides from the myxomycete Physarum polycephalum interacting in vitro with microtubules.

Microtubule-interacting proteins have been studied in the lower eukaryote Physarum polycephalum. We show for the first time 1) the presence in Physarum amoebal crude extracts of at least six polypeptides that bind specifically to amoebal microtubules, 2) the binding between these proteins and mammalian microtubules, 3) the heat stability of two of these polypeptides (125 and 235 kDa), 4) the functional properties of a fraction containing a heat-soluble 125 kDa polypeptide, and 5) the phosphorylation of the 125 kDa polypeptide during two distinct periods of the cell cycle in Physarum synchronous plasmodia, first at late S/early G2 phase and second at late G2/prophase.

Variation of tubulin half-life during the cell cycle in the synchronous plasmodia of Physarum polycephalum.

The half-life of tubulin has been studied during the cell cycle of the myxomycete Physarum, using a specific quantitative immunological method. In asynchronous microplasmodial and amoebal cultures the apparent half-life of tubulin was greater than 15 h. In contrast, in the naturally synchronous plasmodia, in which tubulin exhibits a cyclic synthesis, we have shown for the first time that tubulin half-life was not constant through the cell cycle. There was no tubulin degradation before mitosis, while tubulin half-life was reduced to about 1 h during the postmitotic period. Such a variation of stability through the cell cycle has not been observed in the case of thymidine kinase, another protein showing a cyclic synthesis in Physarum plasmodia. The decrease of tubulin half-life that occurred during the S-phase immediately following mitosis (no G1-phase in Physarum) was independent of the increase of growth temperature between 22 and 32 degrees C, in contrast with the half-life of thymidine kinase which decreased in the same conditions. Tubulin did not completely disappear after mitosis. A 20% residual amount of tubulin persisted from late S-phase to early G2-phase. We have shown that tubulin degradation was not modified by actinomycin D or cycloheximide but was prevented when DNA synthesis was inhibited by fluorodeoxyuridine and hydroxyurea. In contrast, inhibition of S-phase did not modify the half-life of thymidine kinase. These results indicate that: 1) during the cell cycle, the pool of tubulin is regulated not only at the transcriptional and translational levels but also by a cell cycle-dependent degradative process.(ABSTRACT TRUNCATED AT 250 WORDS)