Heat resistance of 2 types of protoplasmic movement in Physarum polycephalum plasmodia]

The thermostabilities of the "unordered" and shuttle protoplasmic streamings in myxomycete Physarum polycephalum plasmodia was studied. A comparison of these thermostabilities has revealed that the cessation of the former streaming occurs at temperatures higher than those required for arresting the shuttle streaming. The difference between the two types of protoplasmic streamings is better seen in the rate of repair of protoplasmic streaming halted by a 10 minutes heating at 38-41 degrees C. For example, the unordered streaming is restored 2 minutes after heating plasmodia at 39 degrees for 10 min., while the shuttle streaming is resumed in 24 minutes. It is supposed that the two protoplasmic streamings are independent to an appreciable extent, and that the shuttle streaming, being more complex and coordinated, has appeared in the evolution at later stages than the unordered one. The higher heat sensitivity of the shuttle streaming substantiates a view of the lower stability to injury in regulatory mechanisms if compared to the stability of motile mechanisms.     

Effects of supraoptimal temperatures on the myxomycete Phyrasum polycephalum

Summary The effects of heat shock on the protoplasmic streaming, respiration and leakage of plasmodial constituents absorbing at 260 nm (products of nucleic acid metabolism), 280 nm (products of protein metabolism), and 415 nm (the yellow pigments of the plasmodia) were studied in plasmodia of the myxomycete Physarum polycephalum.Plasmodia grown on a semidefined medium displayed a lower primary thermoresistance of the protoplasmic streaming, and had a lower Q ₁₀ coefficient of the heat injury of this function compared to those grown on rolled oats. They are able to repair thermal injuries during heating. The primary thermoresistance of the protoplasmic streaming is not changed during the mitotic cycle.A 10 min heating at 32°C lowers the rate of protoplasmic streaming and results in a leakage of plasmodial pigments. After a 10 min exposure at 37–38°C the protoplasmic streaming is stopped, the respiration reduced, and products of nucleic acid metabolism are detectable in the heating fluid. Leakage of protein metabolits was observed after 10 min heatshocks at 41°C. A heating of the plasmodia to 47–50°C caused the highest level of leaked substances and the complete cessation of respiration.In contrast to higher plants, the respiration and leakage of the pigments are thermolabile indicators of the condition of Physarum polycephalum plasmodia.     

Acceleration of mitosis induced by mitotic stimulators of Physarum polycephalum

Extracts of the myxomycete Physarum polycephalum exhibit an accelerating effect on nuclear division which fluctuates during the synchronous nuclear division cycle. Extracts from late G2 phase plasmodia can advance mitosis in recipient test plasmodia by up to 30% of the length of the control cycle. The advancing capacity of extracts is heat- and ammonium sulphate-precipitable, non-dialysable and destroyed by pronase, suggesting that the active substance is a protein. The advance of mitosis is in strong correlation with the applied dose of stimulatory material.     

The Effect of ATP (Disodium Salt) upon Rotational Streaming

Through the continuous treatment with various solutions of ATP disodium salt the rotational streaming of the cytoplasma in barley root hairs has been stimulated about 1.2–1.7 times. With the concentrations employed the stimulation of the streaming did not depend on the external ATP supply, but on the initial rate of streaming. It is assumed that the main source of energy supporting the protoplasmic streaming is ATP. Therefore, the results obtained may be interpreted on the basis of variations in ATP content and its degradation products. The differences between initial rates of streaming are supposed to be due to variations of the endogenous ATP level. The ATP taken up probably stimulates the rotational streaming both through the supply of delivered energy and by lowering the cytoplasm viscosity. On the contrary, products of ATP hydrolysis increase the cytoplasm viscosity and induce a lowering or even cessation of the streaming.     

Locomotive mechanism of Physarum plasmodia based on spatiotemporal analysis of protoplasmic streaming

We investigate how an amoeba mechanically moves its own center of gravity using the model organism Physarum plasmodium. Time-dependent velocity fields of protoplasmic streaming over the whole plasmodia were measured with a particle image velocimetry program developed for this work. Combining these data with measurements of the simultaneous movements of the plasmodia revealed a simple physical mechanism of locomotion. The shuttle streaming of the protoplasm was not truly symmetric due to the peristalsis-like movements of the plasmodium. This asymmetry meant that the transport capacity of the stream was not equal in both directions, and a net forward displacement of the center of gravity resulted. The generality of this as a mechanism for amoeboid locomotion is discussed.     

Extraction of an actin-like protein from the plasmodium of a myxomycete and its interaction with myosin A from rabbit striated muscle

An actin-like protein was obtained from the plasmodia of a myxomycete, Physarum polycephalum. It forms a complex with muscle myosin A which behaves similarly to the actomyosin from rabbit striated muscle. On the addition of ATP the complex of this protein with myosin A shows a viscosity drop at high concentrations of KCl (～0.5 M). At low concentrations of KCl (～0.05 M) this complex superprecipitates from solutions containing 1 mM MgCl₂ and shows Mg-activated ATPase activity. That is, the actin-like protein converts the ATPase of myosin A to the actomyosin type.     

A simple method for the isolation of actin from myxomycete plasmodia.

A new, simple method for the isolation of actin from myxomycete plasmodia has been developed. Plasmodium myosin B was incubated at 55 degrees C for 15 min in the presence of ATP or was treated with 90% acetone. By this treatment myosin was denatured completely. Actin was then extracted with a dilute ATP and cysteine solution from the heat- or acetone-treated myosin B. The method is simple and almost pure actin was obtained in high yield. The purified G-actin polymerized to F-actin on addition of 0.1 M KCl or 2 mM MgCl2. The viscosity of the purified F-actin was 8-10 dl/g. The F-actin activated muscle myosin ATPase, and actomyosin synthesized from the F-actin and muscle myosin showed superprecipitation on addition of ATP.     

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.     

Immunocytochemistry of the acellular slime mold Physarum polycephalum

Abstract. The polygonal arrangement of actomyosin fibrils in different stages of the acellular slime mold Physarum polycephalum is correlated with morphogenetic processes at the cell surface. Light and electron microscopic investigations on both endoplasmic drops and thin-spread small plasmodia demonstrate that the differentiation of a polygonal pattern depends on a transient deficiency of plasma membrane invaginations.
Glycerol-extracted specimens show condensation and drastic spatial changes in the organization of the polygonal net after addition of ATP, thus indicating contractile properties of this system. Observations with the polarizing microscope reveal rhythmic changes in fibrillar birefringence intensity corresponding to the protoplasmic streaming activity, i.e., birefringence increases during contraction and decreases during relaxation. Cell fusion experiments, local irradiation with blue light (450 nm), and chemical treatment by impeding the mitochondria1 function with DNP (2,4-di-nitrophenol) demonstrate morphological as well as physiological interdependences of the actomyosin system, the motive force generation, and the expression of a locomotor polarity in plasmodia of Physarum polycephalum.     

Protoplasmic streaming during the cell cycle of Physarum polycephalum

It has been reported that protoplasmic streaming stops during the synchronous mitosis exhibited by growing plasmodia of P. polycephalum. Our data reveal that at no time during the mitotic cycle did streaming stop. However, during a 3–5 min period at anaphase the percent of each oscillation period accounted for by an outward flow was precisely equal in duration to the corresponding inward flow. At all other periods the duration of outward flow exceeded that of inward flow. Plasmodial migration or locomotion was briefly arrested at telophase, although shuttle streaming persisted.     

Repair of functional and ultrastructural alterations after thermal injury of Physarum polycephalum

Repair of thermal injury of Physarum polycephalum Schw. plasmodia has been studied by light and electron microscopy. As a result of heating the plasmodia for 10 min at 42°C both the unordered and shuttle protoplasmic streaming were arrested; the outer plasmodial membrane showed alterations at sites of contact with water; the onset of the next mitosis was considerably delayed. The plasmodial ultrastructure was markedly disturbed, including disappearance of the granular component of the nucleoili and a compact, almost fibrillar structure of the latter. The mitochondria became distorted and their intracristal spaces enlarged while the outer and inner membranes appeared in some places to be separated. Glycogen particles disappeared from the cytoplasm. Recovery of both types of protoplasmic streaming of the motility of the plasmodium, of the resistance of its membrane to contact with water, and of the ability of the organism to go through the cell cycle went all hand in hand with the normalization of the structure of nucleoli, mitochondria and cytoplasm. All of the functional and structural characteristics are normalized within ca. 9 h following heating.     

The isolation of myxomyosin,an ATP-sensitive protein from the plasmodium of a myxomycete

1. A procedure has been developed for the preparation of an active concentrate from the myxomycete, Physarum polycephalum. This concentrate responds with a lowered viscosity to the addition of small amounts of ATP. The preparation recovers in viscosity, and the process may be repeated. 2. In the most active concentrates, 75 per cent of the non-dialyzable material moves as a single boundary both in the descending limb in electrophoresis and in the ultracentrifuge. It contains about 10 per cent ribonucleic acid, which is at least in part reversibly bound to the protein. 3. The active material has been designated myxomyosin because of its origin and its similarity to actomyosin in ATP response.     

Nutrient mobilization by plasmodium of myxomycete Physarum rigidum in deadwood

To assess the nutrient mobilization ability of myxomycete plasmodia in deadwood, a microcosm experiment was conducted. Alive or dead plasmodia of a common lignicolous myxomycete, Physarum rigidum, were inoculated on field-collected crushed wood powder of white-rot or brown-rot pine wood and incubated for 24 d. The activity of living plasmodia led to increased concentrations of Ca²⁺, K⁺, Mg²⁺, Na⁺, and Cl⁻, but lower PO₄^3– concentrations in the wood powder. For NO₃⁻ concentration, the effect of living plasmodia was negative in white-rotted wood but positive in brown-rotted wood. These results suggest that the plasmodium of P. rigidum has the ability to mobilize nutrients in deadwood.     

The pathway of photosensory transduction in Physarum polycephalum

Irradiation of the plasmodia of Physarum with blue and white light results in a transient change of their oscillatory contraction frequency. This reaction to light decreases with increasing distance from the illuminated area (Block and Wohlfarth-Bottermann, 1981).The first local appearance of light response in non-illuminated parts of the plasmodia was used to analyse the sensory pathway of the light stimulus modulating the contractile apparatus. Different experimental assays revealed that the direction of photosensory transduction is determined by the momentary direction of protoplasmic shuttle streaming. The endoplasmic flow carries the signal responsible for photosensory transduction and light reaction to the force generating ectoplasmic tube.     

Ca2+ effect on protoplasmic streaming in Nitella internodal cell

Ca²⁺ ion effect on protoplasmic streaming in an internodal cell of Nitella has been investigated for various temperatures. We have found that the protoplasmic streaming at low temperature is remarkably affected by the Ca²⁺ ions in the internodal cell but larger concentrations of the Ca²⁺ ions are needed to suppress the streaming velocity at higher temperatures. These streaming behaviors of the protoplasm, furthermore, have been elucidated on the basis of the reaction equations which take into account ATP hydrolysis due to actin-myosin molecules and inactivity of the molecules due to the Ca²⁺ ions.     

Steady and transient behaviors of protoplasmic streaming in Nitella internodal cell

Steady and transient behaviors of protoplasmic streaming in Nitella internodal cell have been investigated for various temperatures from 30°C to near 0°C. It has been found that steady velocity of the streaming linearly decreases with increasing inverse temperature but its proportionality coefficient changes at ~ 10°C. Velocity distribution, which reflects temporal fluctuations of the protoplasmic streaming, is nonGaussian and its half width becomes larger at higher temperatures. On the other hand, recovery of the protoplasmic streaming, which is observed after stopping the streaming with a current stimulus to the internodal cell, has been found to show more clear sigmoidal time courses at higher temperatures.     

Oscillations in cell shape and size during locomotion and in contractile activities of Physarum polycephalum, Dictyostelium discoideum, Amoeba proteus and macrophages

Changes in cell shape and size were measured during locomotion, together with the motive force of the protoplasmic streaming, in various amoeboid cells in different stages of their life cycle, and under various environmental conditions. The variations in these measurements with time were examined by Fourier spectral analysis. Notwithstanding a change in cell type in the life cycle of P. polycephalum, myxamoebae and tiny plasmodia showed a similar time pattern of locomotion, exhibiting oscillations having a mixture of several periods. A regular oscillation with protoplasmic streaming appeared in the plasmodium only above a critical cell size. D. discoideum amoebae oscillated with two periods of a few minutes in preaggregation stage, but with a period of 10 min in aggregation stage, the latter being induced by cAMP. Macrophages and A. proteus also oscillated with periods of a few minutes. Periods of all these oscillations were prolonged severalfold by respiratory inhibition with NaCN, but were unaffected by glycolytic inhibition with 2-deoxyglucose. Cell fragments of A. proteus containing fewer granules oscillated more slowly and with a larger amplitude than those containing more granules. Among the granules, the nucleus was excluded as a possible modifier of the oscillation. The oscillation in Physarum plasmodium was reversibly suppressed by combining respiratory and ATPase inhibitions in mitochondria with NaCN and oligomycin, intracellular ATP concentration being kept at an appropriate level. The present results show that amoeboid motility, as well as cell shape, is oscillatory and that mitochondria are involved in time keeping.     

Extraction of an actomyosin-like pootein from amoebae of Dictyostelium discoideum

An actomyosin-like protein has been extracted from amoebae of Dictyostelium discoideum, V-12. The purified protein exhibited a reversible change in viscosity upon addition of ATP, indicating an ATP sensitivity of 75–85% and a specific viscosity of 0.1. At low ionic strength in the presence of Mg⁺⁺ and ATP the amoeba protein displayed the phenomenon of superprecipitation. The protein extract was found to be an adenosinetriphosphatase (ATP'ase) hydrolyzing ATP to ADP and inorganic phosphate. Both Mg⁺⁺ and Ca⁺⁺ at low ionic strength accelerated the ATP ase activity whereas at high ionic strength only Ca⁺⁺ stimulated ATP hydrolysis. The ATP'ase activity was inhibited by ethylene-diamine-tetraacetic-acid, Mersayl and p-chloromercuribenzoate. The physico-chemical and enzymatic properties of the extracted amoeba protein are qualitatively comparable to those of muscle actomyosin, and very similar in quantitative properties to smooth muscle actomyosin and the actomyosin-like proteins of blood platelets, leucocytes and slime mold plasmodia. The significance of the presence of this actomyosin-like protein in Dictyostelium amoebae is discussed in relation to amoeboid form and movement.     

Production of DL-DOPA from acellular slime-mould Stemonitis herbatica

3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter precursor and hence has wide therapeutic applications. Our earlier studies have shown that the melanin in sporangia of the myxomycete species Stemonitis herbatica is DOPA melanin, and that?it?is?possible to extract the precursor DL-DOPA from differentiating plasmodia. The production of DL-DOPA from this species might be commercially viable.     

Active streaming in actomyosin solutions

Solutions of actomyosin, extracted from plasmodia of Physarum polycephalum or from rabbit's striated muscle, were introduced into microcapillaries in the presence of ATP. Vigorous and sometimes oscillatory streaming was observed which resembled in many respects cytoplasmatic streaming.