Steady compensation of gravity effects in Physarum polycephalum.
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Cold Temperature↗
Biomedical subjects
K E Wohlfarth-Bottermann
Publications and source records attributed to K E Wohlfarth-Bottermann.
Reactivation of cell-free models of endoplasmic drops from Physarum polycephalum after glycerol extraction at low ionic strength.
The effect of calcium ions on the reactivation of cytoplasmic actomyosin contraction in cell-free models of endoplasmic drops from Physarum polycephalum after glycerol extraction at low ionic strength depends on the duration of the extraction procedure: Ca++ prevents contraction in 20-h extracted specimens, whereas after several days of extraction this Ca++-sensitivity is lost. These results indicate an inhibitory effect of Ca++ on cytoplasmic actomyosin contraction.
Actins↗
Reactivation of a cell-free model from Physarum polycephalum: studies on cryosections indicate an inhibitory effect of Ca++ on cytoplasmic actomyosin contraction.
Cell-free models should offer "in situ conditions" to study the physiology of cytoplasmic actomyosin in its natural environment, while, if possible, still associated with its regulatory control proteins and other cytoplasmic components. Detergents and glycerol as the usual media to permeabilize the plasmalemma and to extract a portion of the cytoplasmic components, are accompanied by several disadvantages. We investigated a cell-free model consisting of cryosections of plasmodial strands that were previously enriched with "stress fibrils" and fluorescently labelled with phallotoxins and that contain the non-denatured structures that are to be reactivated in situ. The contraction reaction can be directly observed in the fluorescence microscope. This procedure allows the study of contraction conditions in the natural environment of the fibrils. The aim of these reactivation experiments was to identify the role of calcium ions. According to our results, a reactivation of cryosections is not Ca++ dependent but is partly inhibited at concentrations of 10(-4) to 5 X 10(-2)M Ca++. Complete inhibition occurs at 10 to 20 mM Ca++. Electron microscopical investigations revealed that the fluorescently labelled contracting structures were identical to actomyosin fibrils.
Actins↗
Enrichment of fibrillar cytoplasmic actomyosin in protoplasmic strands of Physarum polycephalum for the production of cell-free models.
The treatment of isolated protoplasmic strands of Physarum polycephalum with 2.5% ethanol in a physiological salt solution under isometric conditions induces the formation of a large amount of mostly longitudinally organized actomyosin fibrils in the endoplasmic channel, a region normally free of actomyosin fibrils. The quantity of fibrillogenesis as well as the concomitant force output during the induced contractures are dependent on the Ca++-content and the temperature of the test solution. The method was developed to optimize the structure of the plasmodial strands before their subsequent transformation into cell-free models by permeabilization and extraction of the strands. Cryosections of plasmodial strands containing cytoplasmic actomyosin fibrils stained with fluorescently labeled phallotoxins offer a further assay for the study of their contraction physiology under cell-free conditions.
Actomyosin↗
Immunocytochemistry of the acellular slime mold Physarum polycephalum. V. Cryosectioning now allows analysis of non-extracted plasmodia of any and all stages.
The spatial distribution of cytoplasmic actin in endoplasmic drops as well as in plasmodial strands can be demonstrated in cryosections by fluorescently labelled phallotoxins and actin antibodies. Our results on cryosections show an identical fibrillar actin distribution as revealed in semithin sections after conventional fixation and embedding. Thus, it is now possible to apply immunocytochemical analysis to any and all plasmodial stages with or without prior fixation and without using extraction procedures. Consequentially the loss of soluble compounds during processing is avoided. The most protective pretreatment of the living specimens before freezing is a 15 min incubation in 1.5 M sucrose containing 50 mM KCl, 10 mM EGTA and 10 mM PIPES buffer, pH 7.0, at 4 degrees C.
Actins↗
Rhythmicity and tension development of spontaneous contracting capillaries in the mesentery of frogs.
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Animals↗
Preservation and phallotoxin-staining of the microfilament system in Amoeba proteus.
The spatial organization of the microfilament system as the main component of the cytoskeleton in Amoeba proteus was preserved by a glutaraldehyde-lysine-fixation and visualized with fluorescent phallotoxins (NBD- phallacidin , R-phalloidin). Results obtained by means of this method coincide exactly with observations gained from immunocytochemical, ultrastructural and molecular cytochemical studies, i.e., the microfilament system is mainly displayed beneath the cell membrane, at the hyalo - granuloplasmic border and around the cell nucleus. The preparation procedure employed is suitable for the rapid demonstration of cytoplasmic microfilaments in cells difficult to preserve by chemical fixation.
Amanitins↗
Reactivation of NBD-phallacidin-labelled actomyosin fibrils in cryosections of Physarum polycephalum: a new cell-free model.
Application of ATP to cryosections of plasmodial strands from Physarum polycephalum leads to an isotonic contraction of the cytoplasmic actomyosin fibrils: when the fibrils are labelled with NBD-phallacidin, their contraction can be observed in the fluorescence microscope. While performing contraction, the fibrils separate into many small units and the formerly continuous fibrils exhibit the appearance of beaded chains. The possibility of visualizing directly the contraction of cytoplasmic actomyosin fibrils in the fluorescence microscope represents a favourable condition for the study of their physiological contraction mechanism, because this new and convenient cell-free model offers in situ contractile structures that are non-denatured and non-extracted.
Actomyosin↗
Energy metabolic regulation of oscillatory contraction activity in Physarum polycephalum.
The influence of inhibitors of respiration (KCN), glycolysis (2-deoxy-D-glucose alone or in combination with monoiodo-acetate) and anaerobic conditions (N2 or 95% N2 + 5% CO2), as well as the effect of application of appropriate substrates (D-glucose, sodium acetate, alpha-ketoglutarate and pyruvate) on contraction behavior (of plasmodia) of Physarum polycephalum was investigated under low intensity red-light illumination. Application of inhibitors of respiration or glycolysis leads to significantly different periods of the force oscillations (2.0 +/- 0.4 min and 4.1 +/- 0.5 min, in the presence of inhibitors of glycolysis and respiration, respectively). In both cases, the force amplitude of oscillations is diminished in comparison to undisturbed energy production. The persistence of oscillations in the presence of inhibitors of glycolysis seems to rule out an exclusively glycolytic origin of the force oscillations. A continuous production of ATP is necessary since a blockade of the energy production causes a cessation of the oscillations. Application of substrates for respiration in the presence of inhibitors of glycolysis leads to an increase in the force amplitude and a slight prolongation of the oscillation period. In contrast, an application of glucose in the presence of a respiration inhibitor has no effect on the force amplitude and period.
Adenine Nucleotides↗
Cytoplasmic actin patterns in Physarum as revealed by NBD-phallacidin staining.
Fluorescently labeled phallacidin, a F-actin specific drug, was used to demonstrate the morphological variety in the cytoskeletal actin pattern of thin-spread plasmodia of the acellular slime mould Physarum polycephalum. The patterns observed in phallacidin-stained specimens consisted of a polygonal network in the anterior region, and of longitudinal as well as helically twisted fibrils in plasmodial strands of the posterior region. These observations are in complete accordance with our recent results obtained on comparable plasmodia by immunofluorescence microscopy using specific antibodies against actin.
Actins↗
An extraction resistant tensile protein in the protoplasmic matrix of Physarum.
A thorough extraction of plasmodia of Physarum polycephalum by sequential treatment with 1% Triton x-100, 0.6 M KI, 4% SDS plus 7 M urea leaves behind an elastic cell ghost, which represents a cytoplasmic matrix protein arranged as a continuous network in all cell regions. The protein is present in the ectoplasm as well as in the endoplasm. The extraction-resistant ghosts reveal filaments 2-3 nm in diameter, perform a conspicuous volume condensation upon the addition of mM-concentrations of di- and trivalent cations and can be partially solubilized in 4.5 M guanidinium chloride plus 25% 2-mercaptoethanol at 70 degrees C. SDS-gel electrophoresis shows a distinct band at 43,000 daltons and a faint high molecular weight component suggesting a similarity to muscle connection.
Contractile Proteins↗
Immunocytochemistry of the acellular slime mould Physarum polycephalum. I. Preparation, morphology, and reliability of results concerning cytoplasmic actomyosin patterns in sandwiched plasmodia.
Small phaneroplasmodia of Physarum polycephalum migrate, under sandwich conditions between two agar sheets and a membrane of cellophane, as thin protoplasmic sheets. This method suitably simulates the situation in the natural habitat of acellular slime moulds; i.e. the narrow clefts of the forest soil. The highly differentiated system of cytoplasmic fibrils displayed under these conditions survives both long-term extraction with glycerol and fixation with methanol, procedures that remove the strong inherent autofluorescence, thus allowing the use of immunocytochemical studies. The complicated fibrillar system of sandwiched plasmodia consists of: (1) a membrane-associated cortical filament layer in the anterior region; (2) a more or less regular polygonal fibrillar network in the intermediate region; and (3) a helically twisted fibrillar system encircling endoplasmic pathways as well as isolated strands in the posterior region. So far, three different cytoskeletal proteins have been identified immunocytochemically as constituents of the fibrillar structures: actin, myosin and AM-protein (fragmin). No positive identification of alpha-actinin, filamin and tropomyosin was obtained using antibodies against vertebrate proteins. Electron microscopy of glycerol-extracted specimens treated with antibodies against actin and myosin revealed that the 6 nm filaments consist of actin, whereas the electron-dense material between single actin filaments appears to be myosin. The AM-protein modulating the polymer status of actin is located in all fibrillar structures.
Actins↗
Contractile and structural reactions to impediments of Ca2+-homeostasis in Physarum polycephalum.
A combined application of 5 mM KCN and 19 microM Ca++-ionophore A-23187 leads to pronounced contractures of plasmodial strands of Physarum polycephalum. The appearance of the contractures is independent of the amount of Ca++ in the external medium. Tensiometric registrations of longitudinal contraction activity (isometric regime) reveal an average tension increase of 50 mp compared with the preceding tension level before the addition of KCN and ionophore. This high force output during the contracture coincides with a pronounced increase in the number of cytoplasmic actomyosin fibrils. Their ultrastructure is seen as a high lateral density of strictly parallel arranged F-actin filaments; the state of cytoplasmic actomyosin during this isometric contracture corresponds to the ultrastructure of isometrically contracted fibrils during the normal contraction-relaxation cycle of this organism. A simultaneous impediment of respiration and Ca++ homeostasis strongly favours a shift of the actin equilibrium to the high polymeric side in the form of fibrils and may thus be used as a preparatory step improving the specimens in the context of other investigations, e.g., for immunocytochemical investigations or for the preparation of cell-free models to be reactivated after extraction procedures.
Actomyosin↗
Pinocytosis and locomotion of amoebae. XIX. Immunocytochemical demonstration of actin and myosin in Amoeba proteus.
The spatial distribution of cytoplasmic actin and myosin in 1. normal locomoting, 2. immobilized, and 3. pinocytosing Amoeba proteus was demonstrated by indirect immunofluorescence microscopy. In orthotactic and polytactic cells fixed during normal locomotion actin is mainly located in a cortical layer delineating the granuloplasm from the peripheral hyaloplasm. In cell areas lacking a hyaloplasmic sheet the actin layer immediately borders the plasma membrane. The amount of actin within the continuous layer seems to increase from the advancing front to the middle cell region and to decrease again toward the uroid. The distribution of myosin is largely congruent to the display of actin, with the exception that the myosin-based fluorescence of the cortical layer gradually increases from the front to the uroid. A considerable amount of actin and myosin is also distributed around the nucleus and the contractile vacuole. In immobilized cells contracted by the external application of 10(-4)M procaine hydrochloride the cortical layer distinctly increases in thickness. In contrast to normal locomoting cells actin and myosin show a uniform distribution within the cell cortex along the entire surface. In pinocytosing cells, up to three cortical layers conspicuously rich in actin are produced during the process of channel formation. One of these layers is located in close proximity to the plasma membrane of the pinocytotic channels and the vacuoles. The immunocytochemical results are discussed with respect to earlier observations on the distribution of actin and myosin in Amoeba proteus as obtained by other methods.
Actins↗
Immunocytochemistry of the acellular slime mold Physarum polycephalum. III. Distribution of myosin and the actin-modulating protein (fragmin) in sandwiched plasmodia.
The acellular slime mold Physarum forms very thin plasmodia when sandwiched between two agar sheets. After extraction with glycerol-containing buffers, suitable objects for immunofluorescence microscopy are obtained, and an analysis of the cytoskeletal and contractile system of Physarum becomes possible. Plasmodia were stained with antibodies against myosin and fragmin, a protein factor involved in actin filament length regulation. The microanatomy and topography of cellular structures containing these proteins were investigated at the light and electron microscopic levels. The patterns obtained with the two antibodies are closely related to those obtained with actin antibody [25]. In both cases the complex system of cytoplasmic fibrils is stained selectively. The fibrils form a more or less regular network in the advancing front zone with the fibrils being interconnected by focal nodes. In the posterior region of the plasmodium, where endoplasmic pathways and protoplasmic veins are differentiated, larger fibrils are detected, running obliquely or longitudinally to the veins. With both antibodies the fluorescent pattern of the fibrils is continuous without indications of periodic interruptions or striations, which would be expected in the case of sarcomere-like subunits. With anti-myosin unstained patches are frequently seen at or close to the nodes of the fibrillar network in the anterior region. The small lobopodia, which are rich in actin, are apparently not stained by the myosin antibody, a result similar to the situation in "ruffling edges¿ of cultured vertebrate cells. Electron microscopic investigations of antibody-labeled fibrils in embedded and sectioned plasmodia allow the identification of antibody molecules at specific sites along the fibrils with a different distribution pattern for each of the two antibodies.
Animals↗
Consequences of impeding in mitochondrial function in Physarum polycephalum. I. Reversible effects of anoxia, KCN, and influences of the Ca2+ ionophore A-23187.
5 mM KCN as well as anoxia have corresponding effects on Physarum plasmodia: Both induce a disintegration of the plasmalemma invaginations, an increase in cytoplasmic vacuoles, formation of a thick cortical actomyosin layer and an increase of cytoplasmic actomyosin fibrils. Both KCN treatment and anoxia cause a temporary increase in the level of the force oscillations and a reversible prolongation of the periods of the contraction-relaxation cycle of cytoplasmic actomyosin. The normal pattern of oscillation can be restored by the addition of 10 mM alpha-ketoglutarate +5 mM AMP to the solution containing 5 mM KCN. A combination of the Ca2+ ionophore A-23187 and KCN induces a strong contracture, whereas a combination of KCN, ionophore, alpha-ketoglutarate and AMP prevents this effect. The state of contracture is characterised by a pronounced increase in the Young's modulus and an increased fibrillogenesis of cytoplasmic actomyosin. The isometrically contracted state during the high force output represents a strict parallel arrangement of F-actin. The capability of alpha-ketoglutarate in combination with AMP to restore the normal pattern of oscillation as well as to inhibit contractures is interpreted as a stimulation of the alternate pathway of respiration. It is suggested that this stimulation leads to a restoration of cellular Ca2+-homeostasis originally disturbed by the impediment of cell respiration.
Adenosine Monophosphate↗
Consequences of impeding in mitochondrial function in Physarum polycephalum. II. Influence on "de novo" generation of contractile activities and responses to glucose and blue light.
The "de novo" generation of longitudinal contractile activity in endoplasmic veins is inhibited by 5 mM KCN, whereas 10 mM alpha-ketoglutarate combined with 5 mM AMP abolishes this inhibiting effect in spite of a continued presence of KCN. An analysis of the Young's modulus and studies on the morphogenesis of endoplasmic veins reveal morphological effects of an impediment of cell respiration: (1) an increased fibrillogenesis and changes in the spatial distribution of cytoplasmic actomyosin fibrils, (2) an impediment of the "de novo" generation of the plasmalemma invaginations, and (3) the appearance of a thick cortical layer of ground-plasm. These effects of KCN do not appear in the presence of alpha-ketoglutarate and AMP, and disappear by their subsequent application. Impediment of cell respiration by 5 mM KCN inhibits the tensiometrically registrable responses to glucose and blue light. Both reactions are restored in the presence of KCN by an additional application of 10 mM alpha-ketoglutarate combined with 5 mM AMP. The importance of mitochondrial function with respect to morphogenetic events and to the perception and transduction of external signals as well as to locomotory reactions of Physarum plasmodia is discussed.
Adenosine Monophosphate↗
Lateral apertures as passage-ways between ectoplasm and endoplasm in plasmodial strands of Physarum.
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Cytoplasmic Streaming↗