Rhythmicity in the protoplasmic streaming of a slime mold,Physarum polycephalum. I. A statistical analysis of the electric potential rhythm

The electric potential difference (1 to 15 mv.) between two loci of the slime mold connected with a strand of protoplasm changes rhythmically with the same period (60 to 180 seconds) as that of the back and forth protoplasmic streaming along the strand. Generally some phase difference is observed between them. Periods of the electric potential rhythm show a Gaussian distribution. Amplitudes give a somewhat different distribution curve. Wave forms are not always simple harmonic ones, but are distorted more or less. However, auto-correlation analysis proves that there is a dominant rhythm of a nearly constant period which coincides with the mean period of the Gaussian distribution curve. Calculations made on an assumption that the electric potential rhythm is the result of many elementary rhythms (i.e., same periodicity, arbitrary phase angles) distributed throughout the plasmodium, give a satisfactory coincidence with the observed distribution for the amplitude. The predominance of a rhythm of a nearly constant periodicity suggests the existence of well organized interactions among components of a contractile protein network, the rhythmic deformation of which is supposed to be responsible for the protoplasmic streaming and for the electric potential rhythm.     

Studies on the velocity distribution of the protoplasmic streaming in the myxomycete plasmodium

Summary It was shown that the velocity distribution of the intracapillary streaming of protoplasm in a plasmodium ofPhysarum polycephalum is the same no matter whether the flow is spontaneous or whether it is induced artificially by external local air pressure applied to the plasmodium. Thus we conclude that the protoplasmic flow in the plasmodium is caused by local difference in endoplasm pressure. The view that the seat of the motive force responsible for the flow is located in the streaming protoplasm itself is untenable for this type of streaming.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : I. EFFECTS OF GUAIACOL ON VALONIA

In normal cells of Valonia the order of the apparent mobilities of the ions in the non-aqueous protoplasmic surface is K > Cl > Na. After treatment with 0.01 M guaiacol (which does not injure the cell) the order becomes Na > Cl > K. As it does not seem probable that such a reversal could occur with simple ions we may assume provisionally that in the protoplasmic surface we have to do with charged complexes of the type (KX _I)⁺, (KX _II)⁺, where X _I and X _II are elements or radicals, or with chemical compounds formed in the protoplasm. When 0.01 M guaiacol is added to sea water or to 0.6 M NaCl (both at pH 6.4, where the concentration of the guaiacol ion is negligible) the P.D. of the cell changes (after a short latent period) from about 10 mv. negative to about 28 mv. positive and then slowly returns approximately to its original value (Fig. 1, p. 14). This appears to depend chiefly on changes in the apparent mobilities of organic ions in the protoplasm. The protoplasmic surface is capable of so much change that it does not seem probable that it is a monomolecular layer. It does not behave like a collodion nor a protein film since the apparent mobility of Na⁺ can increase while that of K⁺ is decreasing under the influence of guaiacol.     

淡黄绒泡菌和全白绒泡菌原生质流向的观察

显型原质团是绒泡菌目黏菌的营养生长阶段,其最明显的现象是往返原生质流,但一直并不清楚原生质流反向流动的原因。观察研究了淡黄绒泡菌和全白绒泡菌原质团中的原生质流,结果表明：由于菌脉中堵塞或是在原质团前缘尚未分化通道引起反向原生质流,从而引起原质团多方向生长使原质团前缘呈现扇面状。原生质流总的方向是扇面端,并完成原质团运动。     

Electron Microscope Observations on Spore Formation in the True Slime Mold Didymium nigripes

SYNOPSIS. Developing and mature sporangia of the true slime mold Didymium nigripes were studied with the electron microscope to follow the course of spore formation. The sporangium forms from the plasmodium as a protoplasmic bleb which differentiates into a stalk and an apical sphere containing a mass of protoplasm. Nuclei within this protoplasmic mass undergo synchronous division (presumably meiosis). The division spindle forms within the nuclear membrane which is retained intact throughout the division; centrioles have not been observed at the spindle poles. At the same time the nuclei are dividing, the protoplasm cleaves to give ultimately uninucleate spheres—the incipient spores. Capillitial threads come to lie in the furrows created by the cleaving protoplasm. A wall consisting of an inner thick component and an outer thin component forms about each sphere. Cyto-chemical tests suggest that the inner wall of the spore is cellulose-containing and that the outer component might contain chitin.     

Studies on contraction rhythm of the plasmodial strand IV. Site of active oscillation in an advancing plasmodium

Summary From the anterior region of an advancingPhysarum plasmodium, a slender rectangular piece of cytoplasmic gel was removed and its contractile properties were compared with those of a strand segment isolated from the network region of the same plasmodium. The anterior piece, in spite of injury caused by the surgical operation, began to contract and relax periodically without an appreciable lag stage after it had been excised. The segment of strand (vein) obtained from the network behind the advancing front showed no significant rhythm immediately after it had been isolated. Only after a lag period lasting for 10–20 minutes (stage I.Yoshimoto andKamiya 1978 a) did the strand gradually begin to oscillate under both isometric and isotonic conditions. Such behaviour of the isolated strand is interpreted as showing that the active site of the rhythmic contraction and relaxation in the advancing plasmodium is restricted to its anterior zone and the rear network structure plays no leading part in bringing about rhythmicity when it isin situ. Rhythmicity of a strand segment excised from the plasmodial network occurs only after it has been isolated as an independent system. Whether or not the strand is under tension is not important for this functional reorganization.The present work was supported in part by grants-in-aid from the Mitsubishi Foundation and the Japanese Ministry of Education, Science, and Culture.     

Protoplasmic streaming of an internodal cell of Nitella flexilis; its correlation with electric stimulus

The sudden cessation or sudden decrease in velocity of the protoplasmic streaming of Nitella flexilis is observed whenever an action potential is elicited. The action potential can be generated by an electric stimulus after its refractory period, whether the flow is at a complete standstill or on the way to recovery. The membrane potential is generally decreased more or less when the rate of flow is decreased on application of salts or other agents. There is, however, no parallelism between these two. The membrane potential decreases proportionally with applied voltage of subthreshold intensity, while the rate of flow does not change appreciably. Only on application of a superthreshold voltage does the flow stop suddenly. In one case the rate of flow decreased to half without appreciable decrease in membrane potential. In another case it continued flowing at about one-half rate, although the membrane potential was almost zero. The Q₁₀ of the rate of flow is about 2, while it is 1.1 to 1.5 for the membrane potential. The sudden cessation of the protoplasmic streaming is supposed to be caused by the temporary formation of certain interlinkages among contractile protein networks in the endoplasm during excitation at the cathodal half of Nitella.     

THE VARIATION OF ELECTRICAL RESISTANCE WITH APPLIED POTENTIAL : III. IMPALED VALONIA VENTRICOSA

Electrical resistance and polarization were measured during the passage of direct current across a single layer of protoplasm in the cells of Valonia ventricosa impaled upon capillaries. These were correlated with five stages of the P.D. existing naturally across the protoplasm, as follows: 1. A stage of shock after impalement, when the P.D. drops from 5 mv. to zero and then slowly recovers. There is very little effective resistance in the protoplasm, and polarization is slight. 2. The stage of recovery and normal P.D., with values from 8 to 25 mv. (inside positive). The average is 15 mv. At first there is little or no polarization when small potentials are applied in either direction across the protoplasm, nor when very large currents pass outward (from sap to sea water). But when the positive current passes inward there is a sudden response at a critical applied potential ranging from 0.5 to 2.0 volts. The resistance then apparently rises as much as 10,000 ohms in some cases, and the rise occurs more quickly in succeeding applications after the first. When the potential is removed there is a back E.M.F. displayed. Later there is also an effect of such inward currents which persists into the first succeeding outward flow, causing a brief polarization at the first application of the reverse potential. Still later this polarization occurs at every exposure, and at increasingly lower values of applied potentials. Finally there is a "constant" state reached in which the polarization occurs with currents of either direction, and the apparent resistance is nearly uniform over a considerable range of applied potential. 3. A state of increased P.D.; to 100 mv. (inside positive) in artificial sap; and to 35 or 40 mv. in dilute sea water or 0.6 M MgSO₄. The polarization response and apparent resistance are at first about as in sea water, but later decrease. 4. A reversed P.D., to 50 mv. (outside positive) produced by a variety of causes, especially by dilute sea water, and also by large flows of current in either direction. This stage is temporary and the cells promptly recover from it. While it persists the polarization appears to be much greater to outward currents than to inward. This can largely be ascribed to the reduction of the reversed P.D. 5. Disappearance of P.D. caused by death, and various toxic agents. The resistance and polarization of the protoplasm are negligible. The back E.M.F. of polarization is shown to account largely for the apparent resistance of the protoplasm. Its calculation from the observed resistance rises gives values up to 150 mv. in the early stages of recovery, and later values of 50 to 75 mv. in the "constant" state. These are compared with the back E.M.F. similarly calculated from the apparent resistance of intact cells. The electrical capacitance of the protoplasm is shown by the time curves to be of the order of 1 microfarad per cm.² of surface.     

A morphogen for the sporulation of Physarum polycephalum detected by cell fusion experiments

The light stimulus, which under conditions of starvation induces the development of sporangia in the slime mold Physarum polycephalum, can be transferred from the light-exposed part to the unexposed part of a plasmodium by means of plasma circulation. A small quantity of protoplasm from a sporulating donor plasmodium, which had passed through the premorphogenetic phase, was transferred by a short period fusion with a briefly starved, light-induction-incompetent acceptor plasmodium. This led to sporulation and even to a reduction of the premorphogenetic phase from 9 down to 3 h in the acceptor plasmodium. After fusion with a sporulating plasmodium, a highly starved plasmodium from a non-sporogenic culture line or a growing plasmodium from a normal line prevents further morphogenesis of sporangia in the sporulating partner.     

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.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : II. THE EFFECTS OF POTASSIUM ON TWO SPECIES WITH DIFFERENT SAPS

The potential difference across the protoplasm of impaled cells of two American species of Halicystis is compared. The mean value for H. Osterhoutii is 68.4 mv.; that for H. ovalis is 79.7 mv., the sea water being positive to the sap in both. The higher potential of H. ovalis is apparently due to the higher concentration of KCl (0.3 M) in its vacuolar sap. When the KCl content of H. Osterhoutii sap (normally 0.01 M or less) is experimentally raised to 0.3 M, the potential rises to values about equal to those in H. ovalis. The external application of solutions high in potassium temporarily lowers the potential of both, probably by the high mobility of K⁺ ions. But a large potential is soon regained, representing the characteristic potential of the protoplasm. This is about 20 mv. lower than in sea water. The accumulation of KCl in the sap of H. ovalis is apparently not due to the higher mobility of K⁺ ion in its protoplasm, since the electrical effects of potassium are practically identical in H. Osterhoutii, where KCl is not accumulated.     

The role of water in protoplasmic permeability and in antagonism

The behavior of the cell depends to a large extent on the permeability of the outer non-aqueous surface layer of the protoplasm. This layer is immiscible with water but may be quite permeable to it. It seems possible that a reversible increase or decrease in permeability may be due to a corresponding increase or decrease in the water content of the non-aqueous surface layer. Irreversible increase in permeability need not be due primarily to increase in the water content of the surface layer but may be caused chiefly by changes in the protoplasm on which the surface layer rests. It may include desiccation, precipitation, and other alterations. An artificial cell is described in which the outer protoplasmic surface layer is represented by a layer of guaiacol on one side of which is a solution of KOH + KCl representing the external medium and on the other side is a solution of CO₂ representing the protoplasm. The K⁺ unites with guaiacol and diffuses across to the artificial protoplasm where its concentration becomes higher than in the external solution. The guaiacol molecule thus acts as a carrier molecule which transports K⁺ from the external medium across the protoplasmic surface. The outer part of the protoplasm may contain relatively few potassium ions so that the outwardly directed potential at the outer protoplasmic surface may be small but the inner part of the protoplasm may contain more potassium ions. This may happen when potassium enters in combination with carrier molecules which do not completely dissociate until they reach the vacuole. Injury and recovery from injury may be studied by measuring the movements of water into and out of the cell. Metabolism by producing CO₂ and other acids may lower the pH and cause local shrinkage of the protoplasm which may lead to protoplasmic motion. Antagonism between Na⁺ and Ca⁺⁺ appears to be due to the fact that in solutions of NaCl the surface layer takes up an excessive amount of water and this may be prevented by the addition of suitable amounts of CaCl₂. In Nitella the outer non-aqueous surface layer may be rendered irreversibly permeable by sharply bending the cell without permanent damage to the inner non-aqueous surface layer surrounding the vacuole. The formation of contractile vacuoles may be imitated in non-living systems. An extract of the sperm of the marine worm Nereis which contains a highly surface-active substance can cause the egg to divide. It seems possible that this substance may affect the surface layer of the egg and cause it to take up water. A surface-active substance has been found in all the seminal fluids examined including those of trout, rooster, bull, and man. Duponol which is highly surface-active causes the protoplasm of Spirogyra to take up water and finally dissolve but it can be restored to the gel state by treatment with Lugol solution (KI + I). The transition from gel to sol and back again can be repeated many times in succession. The behavior of water in the surface layer of the protoplasm presents important problems which deserve careful examination.     

DISSIMILARITY OF INNER AND OUTER PROTOPLASMIC SURFACES IN VALONIA. II

In measurements of P.D. across the protoplasm in single cells, the presence of parallel circuits along the cell wall may cause serious difficulty. This is particularly the case with marine algae, such as Valonia, where the cell wall is imbibed with a highly conducting solution (sea water), and hence has low electrical resistance. In potential measurements on such material, it is undesirable to use methods in which the surface of the cell is brought in contact with more than one solution at a time. The effect of a second solution wetting a part of the cell surface is discussed, and demonstrated by experiment. From further measurements with improved technique, we find that the value previously reported for the P.D. of the chain Valonia sap | Valonia protoplasm | Valonia sap is too low, and also that the P.D. undergoes characteristic changes during experiments lasting several hours. The maximum P.D. observed is usually between 25 and 35 mv., but occasionally higher values (up to 82 mv.) are found. The appearance of the cells several days after the experiment, and the P.D.''s which they give with sea water, indicate that no permanent injury has been received as a result of exposure to artificial sap. If such cells are used in a second measurement with artificial sap, however, the form of the P.D.-time curve indicates that the cells have undergone an alteration which persists for a long time. On the basis of the theory of protoplasmic layers, an attempt has been made to explain the observed changes in P.D. with time, assuming that these changes are due to penetration of KCl into the main body of the protoplasm.     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : I. EFFECTS OF FORMALDEHYDE ON NITELLA

When protoplasm dies it becomes completely and irreversibly permeable and this may be used as a criterion of death. On this basis we may say that when 0.2 M formaldehyde plus 0.001 M NaCl is applied to Nitella death arrives sooner at the inner protoplasmic surface than at the outer. If, however, we apply 0.17 M formaldehyde plus 0.01 M KCl death arrives sooner at the outer protoplasmic surface. The difference appears to be due largely to the conditions at the two surfaces. With 0.2 M formaldehyde plus 0.001 M NaCl the inner surface is subject to a greater electrical pressure than the outer and is in contact with a higher concentration of KCl. In the other case these conditions are more nearly equal so that the layer first reached by the reagent is the first to become permeable. The outer protoplasmic surface has the ability to distinguish electrically between K⁺ and Na⁺ (potassium effect). Under the influence of formaldehyde this ability is lost. This is chiefly due to a falling off in the partition coefficient of KCl in the outer protoplasmic surface. At about the same time the inner protoplasmic surface becomes completely permeable. But the outer protoplasmic surface retains its ability to distinguish electrically between different concentrations of the same salt, showing that it has not become completely permeable. After the potential has disappeared the turgidity (hydrostatic pressure inside the cell) persists for some time, probably because the outer protoplasmic surface has not become completely permeable.     

Mathematical Model for Rhythmic Protoplasmic Movement in the True Slime Mold

The plasmodium of the true slime mold Physarum polycephalum is a large amoeboid organism that displays “smart” behavior such as chemotaxis and the ability to solve mazes and geometrical puzzles. These amoeboid behaviors are based on the dynamics of the viscoelastic protoplasm and its biochemical rhythms. By incorporating both these aspects, we constructed a mathematical model for the dynamics of the organism as a first step towards understanding the relation between protoplasmic movement and its unusual abilities. We tested the validity of the model by comparing it with physiological observation. Our model reproduces fundamental characteristics of the spatio-temporal pattern of the rhythmic movement: (1) the antiphase oscillation between frontal tip and rear when the front is freely extending; (2) the asynchronous oscillation pattern when the front is not freely extending; and (3) the formation of protoplasmic mounds over a longer time scale. Both our model and physiological observation suggest that cell stiffness plays a primary role in plasmodial behaviors, in contrast to the conventional theory of coupled oscillator systems.     

Chemotaxis in Physarum polycephalum : Effects of chemicals on isometric tension of the plasmodial strand in relation to chemotactic movement

The effects of chemicals were examined on the isometric tension of the plasmodial strand of the true slime mold Physarum polycephalum, and chemotactic motive forces were compared with the contractile properties of the strand. The results were:

1. 1. Isometric tension changed rhythmically within a period of 3–4 min and a few mg in amplitude. Application of attractants (glucose, galactose, maltose, Ca(H₂PO₄)₂, and K(H₂PO₄) led to a decrease in the amplitude of the tension. Contrary to this, repellents (sucrose, inorganic salts) increased the amplitude of the tension. The base line of the tension did not change appreciably unless the concentration of chemicals applied was not too high as compared with respective thresholds.

2. 2. Changes in the isometric tension, F, induced by application of chemicals were analysed quantitatively in terms of integral of isometric tension with respect to time during a period as defined by S=F dt. The values of S changed gradually with increase of concentration of chemicals above their respective thresholds.

3. 3. The threshold concentrations of chemicals determined by measurements of the isometric tension agreed with those obtained from chemotactic motive force and from membrane potential changes.

4. 4. The plasmodium of Physarum polycephalum moved away vigorously from high osmolarity by producing a large transient increase of motive force of the protoplasmic movement. Similarly, the isometric tension increased transiently with a high peak when the concentration of sugars and glycerol exceeded 0.2 M. The maximum tension was linearly proportional to the diameter of the strand.

These results indicate that contraction or relaxation of the plasma gel is the primary cause of the negative and positive chemotaxis in the slime molds.     

NATURE OF THE ACTION CURRENT IN NITELLA : III. SOME ADDITIONAL FEATURES

Several forms of the action curve are described which might be accounted for on the ground that the outer protoplasmic surface shows no rapid electrical change. This may be due to the fact that the longitudinal flow of the outgoing current of action is in the protoplasm instead of in the cellulose wall. Hence the action curve has a short period with a single peak which does not reach zero. On this basis we can estimate the P.D. across the inner and outer protoplasmic surfaces separately. These P.D.''s can vary independently. In many cases there are successive action currents with incomplete recovery (with an increase or decrease or no change of magnitude). Some of the records resemble those obtained with nerve (including bursts of action currents and after-positivity).     

ATP oscillation inPhysarum plasmodium

Summary Using bioluminescence of luciferin-luciferase, we showed that ATP leaked out rhythmically from a strand segment ofPhysarum plasmodium made permeable with caffeine-arsenate. With simultaneous measurement of isometric tension rhythm of the strand, it was revealed that the period and phase of oscillation in ATP leakage correspond well with those of tension production. Further, microinjection of luciferin-luciferase into the plasmodial strand indicated that the intracellular luminescence of luciferin-luciferase also oscillates with the same period and in the same phase as the tension rhythm.The free ATP concentration in a homogenate ofPhysarum plasmodium was of the order of 10 M, but if the homogenate was heated in boiling water, the intensity of luminescence suddenly increased 10–100 fold. ATP available for mechanical workin vivo is thus supposed to be at a much lower level than the total average, which was found in the range of 0.2–0.7 mM.     

Oscillations of apoplasmic K+ and H+ activities in Desmodium motorium (Houtt.) Merril. pulvini in relation to the membrane potential of motor cells and leaflet movements

The lateral leaflets of Desmodium motorium exhibit rhythmic upward and downward movements with a period in the minute range. Apoplasmic K⁺ and H⁺ activities were monitored in situ in the abaxial part of the pulvini with ion-selective microelectrodes. An extracellular electric potential was recorded simultaneously. The apoplasmic H⁺ activity of all pulvini exhibiting a regular rhythm of the extracellular electric potential oscillated with the same period between about 10 and 20 mM. The apoplasmic K⁺ activity was high when the membrane potential of the motor cells was depolarized (about 36 mV) and the cells were shrunken. In contrast, the apoplasmic K⁺ activity was low in the swollen state of the motor cells, when the membrane potential was hyperpolarized (about -136 mV). The volatile anesthetic enflurane suppressed reversibly the movement of the leaflets. The same treatment also arrested spontaneous oscillations in the apoplasmic K⁺ activity in the pulvinus. The apoplasmic K⁺ activity oscillated roughly in phase with the K⁺ activity between pH 6.6 and 6.0. Application of white light disturbed the rhythm and increased the extracellular pH. Our results indicate that the physiological mechanism that drives the lateral leaflet movements of Desmodium motorium is closely related to the osmotic motors mediating the leaf movements of Mimosa, Samanea and Phaseolus.Abbreviations E_m membrane potential - E_ex extracellular electric potential - H_ex extracellular H⁺ activity - K_ex extracellular K⁺ activity - R_ex extracellular electrical resistance B. Antkowiak was supported by the Stiftung Volkswagenwerk.     

A theory of self-organization of an excitable membrane formed on the protoplasmic droplet isolated from Nitella

The process of self-organization of an excitable membrane of protoplasmic droplet of Nitella is studied theoretically by taking the interaction of the local electric current caused by the spatially non-uniform distribution of active domains into account. The theoretical model employed is that the surface membrane forms a mosaic structure composed of lipids and protein molecules, and that each element of the mosaic structure (domain) on the membrane has two distinct conformations corresponding to excited and resting states. The molecules constituting the membrane are derived from the inside of the protoplasm by diffusion. The excitability of the surface membrane appears suddenly after a morphogenetic structure of the membrane is formed with time on the surface of droplet. Time courses of the variation in membrane potential and in membrane resistance are calculated, and the results are compared with experimental data obtained with the protoplasmic droplet of Nitella.