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.

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.

Gravisensitivity of the acellular slime mold Physarum polycephalum demonstrated on the fast-rotating clinostat.

The acellular slime mold Physarum polycephalum was used to investigate a postulated general gravisensitivity of cells. Physarum was subjected i) to a rotation on the fast-rotating clinostat, which enables the simulation of weightlessness (0 g), and ii) to single horizontal turns of 180 degrees. On the fast-rotating clinostat the response consists of a frequency increase in radial contractile activity, an oscillation of the mean values (frequency regulation phenomena) and an increase in standard deviation.

Confirmation of gravisensitivity in the slime mold Physarum polycephalum under near weightlessness.

We have investigated Physarum polycephalum, a unicellular organism with no special gravity receptors, on its ability to react to gravity. The first experiments were 0 g-simulation experiments on the fast-rotating clinostat conducted with plasmodial strands of this acellular slime mold. In these earth-bound experiments the observed parameters were periodicity of the contractions and dilatations of the strand's ectoplasm as well as the periodicity and velocity of the striking cytoplasmic (endoplasmic) shuttle streaming.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The blue-light reaction in plasmodia of Physarum polycephalum is coupled to respiration.

The influence of inhibitors of energy metabolism (2-deoxy-D-glucose, monoiodoacetate, KCN) as well as various substrates for respiration (sodium acetate, glycine, glutamine, α-ketoglutarate, pyruvate) were investigated with respect to the effect of blue light (450 nm) on contractile behaviour of plasmodial strands of Physarum polycephalum. When the energy metabolism is not experimentally modified, blue light induces a prolongation of the period of the contraction-relaxation cycle. This effect appears within 2-3 min and seems to represent the primary reaction of this organism to blue light.

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.

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.

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.

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.

Endoplasmic veins from plasmodia of Physarum polycephalum: a new strand model defined age, structure, and behavior.

Pure endoplasm from plasmodia of Physarum polycephalum taken up into glass pipettes and subsequently extruded into water forms a long protoplasmic cylinder uniform in diameter, which is designated as an "endoplasmic vein". This new experimentally obtained model has proven advantageous for tensiometric investigation of contraction activity. During the process of aging, this model undergoes processes of 1.