Patterns of oscillation during mitosis in plasmodia ofPhysarum polycephalum

Summary The rhythmic contraction pattern in plasmodia ofPhysarum polycephalum was studied to determine whether characteristic changes occur during the synchronized nuclear division. An electrical method that measures the contraction rhythm in situ during several cell cycles was used. Biopsies of the plasmodia were taken at 17 min intervals for precise determination of the cell cycle stages and were correlated with the simultaneously measured contraction rhythm. All measurements were performed in a temperature controlled environment (27 °C) at 100% relative humidity with the plasmodia (less than 24 h old) growing on a semi-defined agar medium. A total of 14 different plasmodia have been examined, and on one occasion the plasmodium was followed through 3 subsequent mitoses. The mitotic stages were identified with aceto-orcein coloring techniques and by fluorescence methods. Except for a few cases where a mitotic asynchrony of 2–3 min was observed, the mitotic events occurred simultaneously in the nuclei within a single plasmodium. Both the occurrence of the first mitosis after inoculation and the intermitotic times were highly variable. Our study indicates that the contraction rhythm in plasmodia ofPhysarum is unperturbed during the synchronized nuclear division. However, in 5 of the 17 examined mitoses an amplitude decay was observed. We discuss possible explanations for the obtained results with emphasis on the applied techniques, interpretation of the oscillation patterns, and possible restrictions in the cell itself.     

Studies on microplasmodia ofPhysarum polycephalum

Summary The new technique of fluorescent analog cytochemistry was used to investigate the cell surface morphology (RITC-WGA staining), the organization of the microfilament system (Rh-phalloidin staining) and the spatial distribution of mitochondria (Rh-123 staining) in the various growth stages of axenically cultured living and fixed microplasmodia ofPhysarum polycephalum. The differentiation degree of the cell surface is generally size- and age-dependent: the invagination system develops by degrees from small spherical stages (50–100 m) without invaginations to large vein-like or dumbbell-shaped specimens (300–1,000 (m long) with extensive invagination systems. The microfilaments are always organized in a cortical system along the entire cell surface and sometimes in a fibrillar system as well, extending throughout the cytoplasmic matrix. Results on living microplasmodia demonstrate that the cortical microfilament system is mainly involved in motive force generation for changes of cell surface morphology and protoplasmic streaming activity, whereas the fibrillar system rather serves a stabilizing and adhering function. Moreover, the functional differences of the two microfilament systems are indicated by the position of a large population of stationary mitochondria in close vicinity to the cell surface, thus pointing to a reasonable arrangement of the energy-supplying and energy-transforming system.     

Periodic events and cell cycle regulation in the plasmodia ofPhysarum polycephalum

The multinucleated plasmodia ofPhysarum polycephalum, a myxomycete, have been extensively used in cell cycle studies. The natural synchrony of mitosis and DNA synthesis, easy culture methods, the ready fusions obtainable between plasmodia, and the amenability to phase specific studies, employing physical and chemical perturbers, are some of the attractive features of this organism. Because of the absence of a Gl phase in the plasmodia, there is a crowding of cell cycle specific marker events at the G2/M boundary, which reflect features of both the G2/M and the Gl/S boundaries of a typical eukaryotic cell. Prominent among these are the synthesis and overall activity of thymidine kinase, the co-triggering of tubulin and histone genes, translation of their mRNA, the organization and duplication of the microtubular organizing centres of the mitotic spindle and the triggering of cdc 2 kinase activity. These above events have not only served as good markers to monitor the progress of the plasmodial cell cycle, but have also been fairly thoroughly analysed by means of specific perturbers such as DNA synthesis inhibitors, antimicrotubular drugs, UV-irradiation, heat-shock etc. Along with fusion studies, these perturbation studies have been helpful in the formulation of various models on regulation of mitosis. These above aspects as well as prospects for future studies employing this organism are discussed This paper is dedicated to the memory of the late Prof. S C K Nair, formerly University Professor of Physics.     

Relationship between endoplasmic and ectoplasmic oscillations during chemotaxis ofPhysarum polycephalum

Summary The plasmodium ofPhysarum polycephalum usually migrates coordinately as one whole body even in a complicated environment. By measuring oscillation phenomena in endoplasm and ectoplasm separately during chemotactic process, we studied the mechanism of information processing to achieve such a coordination. (1) The interaction between endoplasmic oscillators was long-range, competitive according to the length of period, and fast (18 cm/min). Ectoplasmic one was short-range. (2) After a partial stimulation of attractant to the organism, the period at the stimulated portion decreased first, and a global phase gradient appeared in endoplasm. Then ectoplasm at the non-stimulated portion was entrained to the endoplasmic pattern, and the migration direction at each part changed in accordance with the phase gradient as a whole body. (3) When the endoplasmic interaction was interrupted, the above coordinated response was not observed. These facts suggest that two-layer coupled oscillator system composed of endoplasm and ectoplasm play important roles for such an information integration.     

Information processing for the organization of chemotactic behavior ofPhysarum polycephalum studied by micro-thermography

Summary The spatial and temporal pattern of oscillating temperatures on the cell surface of a plasmodial strand ofPhysarum polycephalum was measured with a sensitive thermal image camera. The longitudinal tension of the strand was studied simultaneously. In the absence of chemical stimulation, the phases of the temperature oscillation observed at various portions of the strand were entrained with almost coincidental phase. The temperature and tension oscillation were synchronized, although the phase difference between them was occasionally changed. With local chemical stimulation, the phase of the temperature oscillation advanced in the portion to which the plasmodium would be induced to migrate. The phases between temperature and tension oscillations then became constant. The mechanism by which the plasmodium processes local information of chemical stimulus to global information for the migration is discussed.     

Composition,secretion kinetics and radioactive labelling of the extracellular slime polysaccharide secreted during spherulation ofPhysarum polycephalum

The myxomycetePhysarum polycephalum synthesizes copious amounts of slime when differentiating into the hard walled resting stage. The chemical composition of slime obtained after introduction of spherulation in a nutrient and a non-nutrient salt-medium has been analysed and compared. The composition of slime is almost identical after the two different induction methods. This slime could be labelled with radioactived-[U-¹⁴C]glucose,³²PO₄ ^3–,³⁵SO₄ ^2– and⁷⁵SeO₄ ^2–. The kinetics of slime secretion after both induction methods has been followed using different criteria. The sulfate analog⁷⁵SeO₄ ^2– seems to be incorporated into the slime, partially replacing sulfate groups of the sulfated polysaccharide. Furthermore,d-[U-¹⁴C]glucose was used to labe the spherule walls. Determination of an alkali resistent polysaccharide component serves as a new method to follow wall formation.     

Studies on microplasmodia ofPhysarum polycephalum: II. Fine structure and function of the mucous layer

Summary Microplasmodia of the acellular slime moldPhysarum polycephalum have developed an extensive extracellular slime layer which amounts to 75% of the volume of the total biomass. The slime layer covers the entire plasma membrane including the cell surface invagination system and represents a humid compartment protecting the organism against damaging influence from the surrounding environment.Histochemical treatment with cationic substances revealed that the slime is mostly composed of acid mucopolysaccharides: intensive staining was obtained by colloidal or dialyzed iron, Ruthenium Red, acriflavine and lanthanum hydroxide.At the macromolecular level the slime layer is composed of thin filaments showing a parallel course to the plasma membrane. The slime filaments are very sensitive to fixation and dehydration procedures and tend to form thicker aggregates.From feeding experiments using different fine-particulated markers (Thorotrast, Aerosil) it can be concluded that the slime layer exhibits an important function with regard to the controlled transport and uptake of substances. In particular, the indentations of the cell membrane invagination system running perpendicular from the microplasmodial surface to the cell interior represent preferred pathways for the selective diffusion of macromolecules.     

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     

Cytoskeletal changes visualized by fluorescence microscopy during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum

Summary Changes in the intracellular distribution of microtubules and microfilaments during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum were examined by fluorescence microscopy using anti-tubulin antibody and NBD-phallacidin, respectively. Amoebae contained an extensive microtubular cytoskeleton, which was converted to a flagellar cone structure during transformation to flagellates in liquid medium. When flagellates reverted back to amoebae, this conical structure disintegrated prior to flagella resorption. Amoebae showed some microfilament-enriched domains along the periphery, from which numerous filamentous extrusions, probably pseudopods and filopods, emanated. Flagellates contained a ridge, a sheet-like structure, along their dorsal axis, especially in the earlier stages of flagellation. Another microfilament-enriched thick filamentous structure ran along the dorsal axis, starting from the anterior tip of the cell. This structure apparently coincided spatially with one of the bundles of microtubules. During the reversion to amoebae, other localized microfilaments were transiently observed at the posterior end. A model of cytoskeletal changes in the transformations between these two cell types was proposed.     

Studies on microplasmodia ofPhysarum polycephalum: Endocytotic activity,morphology of the vacuolar apparatus and defecation mechanism

Summary The investigation of endocytotic processes in axenically cultured microplasmodia ofPhysarum polycephalum is considerably complicated by the development of an extensive cell membrane invagination system. Cross-sections through single channels of this system are difficult to distinguish from vacuoles formed endocytotically. Therefore the whole system was labelled by staining the extracellular slime with ruthenium red or lanthanum hydroxide. In this way endosomes produced during the incubation period could be clearly identified. Aerosil andThorotrast are suitable markers for food vacuoles because they can easily be detected with the electron microscope. The application of these substances revealed that submerged cultured microplasmodia are able to form endosomes which contain material of extracellular origin. However, the endocytotic uptake of food material is of much less intensity than in normal macroplasmodia. Microplasmodia seem to cover most of their requirements for metabolic substances by active trans-membrane transport.The intracellular digestive system of microplasmodia corresponds to the vacuolar apparatus of other cells. Preexisting lysosomes originating by autophagic processes play a central role in this system: They coalesce with endosomes or secondary lysosomes thus forming digestion vacuoles. Indigestible food components are extruded together withCa-containing granules into the cell surface invagination system by defecation. The physiological significance of theCa-granules is unknown.     

Controlling the geometry and the coupling strength of the oscillator system in plasmodium ofPhysarum polycephalum by microfabricated structure

Summary The plasmodium of the true slime moldPhysarum polycephalum, which shows various oscillatory phenomena, can be regarded as a collective of nonlinear oscillators. Partial bodies in the plasmodium, which are assumed to be nonlinear oscillators, are mutually connected by microscale tubes named plasmodial strand. The interactions among the oscillators can be strongly affected by the geometry and the dimension of the tube network. Investigation of the collective behavior under the condition that the configuration of the network can be manipulated gives significant information on the characteristics of the plasmodium from the viewpoint of nonlinear dynamics. In this study, we have developed a new method to control the geometry and the tube dimension of the plasmodium with a microfabricated structure. It is shown that the geometry of the plasmodium can be manipulated with a microstructure which is fabricated of ultrathick photoresist resin by photolithographic processes. In order to confirm that not only the geometry but also the dimension of the tubes can be controlled with the microstructure, we observed the cross section of the patterned plasmodium with a three-dimensional internal-structure microscope. By observing the oscillatory behavior of the partial bodies of the patterned plasmodium, it was confirmed that the coupling strength between two oscillators, which corresponds to the dimension of the plasmodial strand, can be controlled by the microstructure. It is concluded that the present method is suitable for further studies of the network of Physarum plasmodium as a collective nonlinear oscillator system.     

Behavior of mitochondria and their nuclei during amoebae cell proliferation inPhysarum polycephalum

Summary We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture ofPhysarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells ofP. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system - BrdU 5-bromodeoxyuridine - FITC fluorescein isothiocyanate     

Functional analysis of actin fibrils in Physarum polycephalum

Summary Fluorochromed heavy meromyosin (TRITC-HMM) was microinjected as a molecular probe into small sandwich-plasmodia of Physarum polycephalum with the aim to demonstrate the spatial morphology and to analyze the dynamic activity of the fibrillar actin system in the living state. The plasmodia display different fibrillar organizations with a polygonal arrangement in the front region (FR) and a parallel or helical arrangement along protoplasmic veins in the intermediate (IR) and uroid region (UR). Quantitative evaluations by measuring the total length, lifetime, dynamic activity, long-term stability and optical density of fibrils reveal distinct differences between the three plasmodial regions: The total length (FR = 27.1 ± 18.5 m, IR = 24.8 ± 12.9 m, UR= 12.3 ± 4.7 m), the lifetime (FR = 12.2 ± 3.4 min, IR=10.5 ± 3.7 min, UR = 6.0 ± 3.4 min), and the dynamic activity as measured in length changes per min (FR = 17.9 ± 11.3 m, IR = 13.1 ± 3.9 m, UR = 8.3 ± 3.9 m) distinctly decrease from the front to the uroid region. On the other hand, the greatest stability as determined by lifetime changes in length (FR = -2.4 ± 16.2 m, IR = 0.3 ± 10.1 m, UR = -6.6 ± 8.9 m) and the highest optical density as expressed in grey-values (FR = 57.0 ± 14.1 gv, IR = 115.6 ± 26.1 gv, UR 62.5 ± 8.1 gv) were found for actomyosin fibrils of the intermediate region. The morphological and physiological data of the present paper are discussed with respect to the biological significance of the fibrillar microfilament system in Physarum polycephalum.     

Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: Cycle blockade by Phalloidin injection

1) The injection of a mushroom drug, Phalloidin (750 microgram -1 mg/ml), into the endoplasmic channel of Physarum veins induces an irreversible blockade of the intrinsic contraction-relaxation automaticity of the ectoplasmic tube wall, as measured by tensiometrical methods. 2) The morphological responses to Phalloidin injection include an increase and condensation of cytoplasmic actomyosin sheets bordering the plasmalemma invaginations within the ectoplasmic tube and a more pronounced dense layer of "groundplasm" in the cell cortex. This is in accordance with experiments with other cells as well as with Physarum. 3) The addition of marker particles to the injection solution revealed that the injected substances can be brought into direct contact with the contractile substrate, before newly formed membranes separate off the injection fluid. 4) Since Phalloidin irreversibly transforms oligomeric actin into a filamentous "Phalloidin-actin complex" and because this transformation does not hinder the actin in activating myosin ATPase, it is concluded that the contraction-relaxation cycle of cytoplasmic actomyosin in Physarum involves actin transformations. If these transformations are hindered, e.g. by Phalloidin, one stage and thereby the whole cycle is sustained which results in a blockade of the intrinsic contraction automaticity. 5) The functional importance of actin transformations in the congraction physiology of cytoplasmic actomyosins and cell motility phenomena is discussed.     

RNA Polymerase B levels during the cell cycle ofPhysarum polycephalum

Summary Tritiated -amanitin has been used as a specific and sensitive probe to estimate the number of RNA polymerase B molecules in isolated nuclei, chromatin and nucleoids, obtained from macroplasmodia ofPhysarum polycephalum. During mitosis (metaphase±10 min) there is at least 10-fold less RNA polymerase B than at all phases of the cell cycle, even if DNA replication has been blocked in vivo. It is concluded that many of the RNA polymerase B molecules leave the chromatin during decondensation of the chromosomes in telophase of the synchronous nuclear division ofPhysarum.     

The life cycle of mitochondria in the true slime mould,Physarum polycephalum

The cytoplasmic aspects of mitochondrial biogenesis have been the focus of much recent attention and a review is presented here of studies on the life cycle of mitochom dria inPhysarum polycephalum. Such studies have focused predominantly on behavior of the mitochondrial genome throughout the mitochondrial life cycle and have been designed to reveal details about (1) the role of the DNA-membrane complex in the segregation of the mitoehondrial genome; (2) the regulation of mitochondrial activity associated with changes in ptoidy of the mitoehondrial nucleus; (3) the hierarchical pattern of transmission of the mitochondrial genome as it relates to the mating-type locus (matA) during the sexual development; and (4) the fusion of mitoehondria that is promoted by a mitochondrial plasmid. The results of such studies contribute significantly to efforts towards a better understanding not only of the mitochondrial life cycle inP. potycephalum but also of the biogenesis of mitoehondria and plastids in many other organisms. Recipient of the Botanical Society Award for Young Scientists, 1988.     

Fragmentation of the plasmodium into equally sized pieces by low temperatures in the true slime moldPhysarum polycephalum: A new morphogenesis

Protoplasma - We found a new type of morphogenesis in the plasmodia of the true slime moldPhysarum polycephalum: The plasmodium broke temporarily into pieces with uniform size at low temperatures....     

Development of spatio-temporal pattern of Ca2+ on the chemotactic behaviour ofPhysarum plasmodium

Summary The development of a spatio-temporal pattern of Ca²⁺ concentration by a plasmodium ofPhysarum polycephalum during chemotaxis was studied using fura-2. Whenever the cell displayed coordinated migration in one direction as a whole body, a spatiotemporal pattern was established with a characteristic feature along the longitudinal axis. Calcium concentration oscillated with a period of a few minutes within the cell; the mean concentration at the front was higher than that at the rear. When the cell was given an attractant only at the rear end, the mean concentration rose at the site of application with an immediate increase in the frequency of oscillation. First, the change of the frequency is propagated toward the other end and then the mean level of the Ca²⁺ concentration at the non-stimulated site decreases. As a result, the Ca²⁺ gradient is reversed along the cell, which then begins to migrate in a coordinated manner in the reverse direction. This study showed that the spatiotemporal pattern of Ca²⁺ concentration is closely related to information processing for coordinated migration in chemotaxis. The role of the pattern in that process is discussed.