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.     

Rhythmicity in the protoplasmic streaming of a slime mold,Physarum polycephalum. II. Theoretical treatment 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 back and forth protoplasmic streaming along the strand. When atmospheric pressure at a part of the plasmodium is increased (about 10 cm. H₂O), the electric potential at this part becomes positive (0 to 20 mv.) to another part with a time constant of 2 to 15 minutes. If the atmospheric pressure at a part of the plasmodium is changed (about 10 cm. H₂O) periodically, the electric potential rhythm also changes with the same period as that of the applied pressure change, and the amplitude of the former grows to a new level (i.e., forced oscillation). The electric potential rhythm, in this case, is generally delayed about 90° in phase angle from the external pressure change. The period of the electric potential rhythm which coincided with that of the pressure change is maintained for a while after stopping the application of the pressure change, if the period is not much different from the native flow rhythm. Such a pressure effect is brought about by the forced transport of protoplasm and is reversible as a rule. In the statistical analysis made by Kishimoto (1958) and in the rheological treatment made in the report, the rhythmic deformation of the contractile protein networks is supposed to be the cause of the protoplasmic flow along the strand and of the electric potential rhythm. The role of such submicroscopic networks in the protoplasm in various kinds of protoplasmic movement is emphasized.     

Fully decentralized control of a soft-bodied robot inspired by true slime mold

Animals exhibit astoundingly adaptive and supple locomotion under real world constraints. In order to endow robots with similar capabilities, we must implement many degrees of freedom, equivalent to animals, into the robots’ bodies. For taming many degrees of freedom, the concept of autonomous decentralized control plays a pivotal role. However a systematic way of designing such autonomous decentralized control system is still missing. Aiming at understanding the principles that underlie animals’ locomotion, we have focused on a true slime mold, a primitive living organism, and extracted a design scheme for autonomous decentralized control system. In order to validate this design scheme, this article presents a soft-bodied amoeboid robot inspired by the true slime mold. Significant features of this robot are twofold: (1) the robot has a truly soft and deformable body stemming from real-time tunable springs and protoplasm, the former is used for an outer skin of the body and the latter is to satisfy the law of conservation of mass; and (2) fully decentralized control using coupled oscillators with completely local sensory feedback mechanism is realized by exploiting the long-distance physical interaction between the body parts stemming from the law of conservation of protoplasmic mass. Simulation results show that this robot exhibits highly supple and adaptive locomotion without relying on any hierarchical structure. The results obtained are expected to shed new light on design methodology for autonomous decentralized control system.     

A Simple Method for Making Tetrahedra to Illustrate Stereochemicai Principles

Actin and myosin, the major proteins of the contractile complex actomyosin, have now been demonstrated to be important constituents of many eukaryotic cells. As in muscle, their role is primarily that of a contractile system. This system is thought to underly all aspects of cellular motility: locomotion, shape change, mitosis and meiosis, cell division, cytoplasmic streaming, organelle motion, endo-cytosis (pinocytosis, phagocytosis), and exocytosis.

We describe here a simple experimental system to demonstrate quantitatively aspects of motility and its regulation in the slime mould Physarum polycephalum     

A study of protoplasmic streaming inPhysarum by laser Doppler spectroscopy

Summary Protoplasmic streaming in the slime moldPhysarum polycephalum has been characterized using laser Doppler spectroscopy. Measurement of the spectrum of scattered laser light permits simultaneous determination of the velocities of all particles in the laser beam, with the relative intensity from each particle proportional to its light scattering cross-section. Simple experimental modifications allow the tracking of the oscillations of the streaming velocities. Rhythmic wall contractions can be monitored simultaneously with the flow velocities. Interpretation of the Doppler spectra shows that a small fraction of the particles in the flowing protoplasm are moving with velocities two to four times greater than the characteristic velocities reported by optical microscopy. Transverse velocities in the tubes are nearly as great as the longitudinal velocities. The shape of the Doppler spectrum at the maximum of the oscillation cycle is consistent with a spatial velocity profile which is sharper than parabolic, presumably because of a viscosity gradient from the center to the walls of the plasmodial tubes. The shape of the Doppler spectrum of depolarized scattered light is of approximately the same form. The response of the plasmodium to increased temperature is an increase in the frequency of the velocity oscillations with little change in the magnitude of the velocities. The response of the plasmodium to very high intensities of laser light is to gel at the point of incidence.     

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.     

The estimation of the motive force for protoplasmic streaming inNitella

Summary By use of a theoretical model for a section of a cell ofNitella and assumptions regarding the form of the stress/rate of strain relation for the streaming protoplasm, it has been possible to determine possible velocity profiles for the streaming in normal and disturbedNitella cells. A match of velocities from these theoretical studies to those measured in real systems has led to a re-estimation of the motive force and of the viscosity coefficient as well as to a first estimate of the thickness of the layer over which the force must be distributed.These new results show the motive force field to be restricted to a layer of about 0.1m thickness alongside the sol/gel interface (the outside boundary of the streaming layer), the force per unit area of this interface to be about 0.36 Nm^–2 (3.6 dyne cm^–2) and a possible stress/rate of strain relation to be of the form (stress)=(viscosity coefficient) × (rate of strain)1/3.Although this latter relation is similar to that obtained by Kamiya and Kuroda (1965) for isolated protoplasm, their viscosity coefficient is about twelve times the present estimate (0.027 Nm^–2s 1/3) suggesting that the fluidin situ is much less viscous than their isolated material. The estimate for the motive force is about double that of previous workers. (Kamiya andKuroda 1958,Tazawa 1968).     

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.     

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.     

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.     

Ultrastructure of the plasmodial slime moldPerichaena vermicularis

Summary The development of the peridium ofPerichaena vermicularis has been examined using light and electron microscopy and acid phosphatase localization. A newly formed fruiting body consists of undifferentiated protoplasm which is enveloped by a slime coat. Almost immediately after formation of the plasmodiocarp, the protoplasm differentiates into autolytic and fruiting regions. The autolytic region is located at irregular intervals between the slime coat and the fruiting region and separated from both of them by membranes.Soon after the autolytic region has formed, additional signs of degeneration appear in the autolytic region including unusual appearance of nuclei, increase in autophagic vacuoles, and the presence of clear areas in the ground substance. The plasma membrane, which once completely separated the slime coat from the autolytic region, is no longer continuous. Electron micrographs of the autolytic region from later developmental stages show formation of extensive channels which contain protoplasm in various stages of degradation. Acid phosphatase is present in the channels of the autolytic region. The morphological evidence and the presence of hydrolytic enzyme suggest the region is being digested and re-adsorbed.After the autolytic region has been digested, an even layer of peridial wall material is laid down, and at regular intervals additional wall material is produced. The additional wall material forms the reticulation on the inside of the peridial wall.This work was supported by National Sciences Foundation grants (GB-5883 and GB-8537) to Dr.Ian K.Ross and an NSF Traineeship (GZ 445 and 796) to I.Charvat.This constitutes a portion of a thesis presented to the Regents of the University of California by the first author in partial fulfillment of the requirements for the Ph. D. degree.     

THE EFFECT OF AUXINS UPON PROTOPLASMIC STREAMING

1. Evidence has accumulated that the action of auxins in promoting growth is exerted not upon the cell wall but upon the cell contents; i.e., the protoplasm. Following indications previously obtained, therefore, the effect of auxins on the rate of protoplasm streaming in the Avena coleoptile was studied. 2. Indole-3-acetic acid, the most active auxin available in pure form, was found to increase the rate of streaming in the epidermal cells of the Avena coleoptile at concentrations between 0.5 and 0.002 mg. per liter, the maximum increase being brought about at 0.01 mg. per liter. This concentration is approximately that which, applied in agar to one side of the decapitated coleoptile, would give a curvature of 1°; i.e., it is well within the range of concentrations active in growth promotion. It is, however, much less than that which produces maximum elongation in immersed sections of Avena coleoptiles. 3. This accelerating effect is readily determined quantitatively by comparison with the streaming in control coleoptiles in pure water, which, if thoroughly aerated, maintain a constant rate for over an hour. The accelerating effect takes place immediately and is over within about 30 minutes. 4. Concentrations of indole-3-acetic acid greater than 0.5 mg.per liter inhibit the streaming, the effect being also over in about 30 minutes, and its extent increasing with increasing auxin concentration. This parallels the effect of high auxin concentrations in inhibiting elongation, although the inhibition of streaming is obtained at much lower concentrations than inhibit elongation. 5. The effects of indole-3-acetic acid on streaming are not specific for that substance, but appear to be common to auxins in general. Thus coumaryl-3-acetic acid and allocinnamic acid, both of which bring about cell enlargement, root formation, and bud inhibition, i.e. are typical auxins, also cause an immediate acceleration of the rate of streaming, and as with indole-acetic add the effect is over in about 30 minutes. The concentrations of these two substances which produce the maximum effect are about ten times that of indole-acetic acid, which approximately corresponds with their relative auxin activities. The curves relating concentrations of these substances to their effects on streaming are very similar to that for indole-acetic acid. 6. On the other hand, certain substances which are known to affect streaming in other materials do not produce any effect comparable to that of auxin. Ethylene chlorhydrin, histidine, and urea in all concentrations were without effect on streaming in the Avena coleoptile within the first 30 minutes of treatment. 7. The effects produced by the auxins were not due to pH. 8. The action on streaming here studied is evidently quite different from the re-starting of streaming after its cessation, studied by Fitting in Vallisneria. Correspondingly histidine, which in Fitting''s experiments showed activity down to 10^–7 M, is inactive here. 9. Per contra, the effect of auxin here studied is on normal streaming. It takes place immediately and at concentrations in the same range as those which produce growth. The curve of effect against concentration parallels that for growth although the actual concentration values differ. It is therefore reasonable to suppose that the effect of auxin on streaming is closely connected with one of the first stages of its effect on the growth process.     

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.     

Chemotaxis toward carbohydrates and amino acids in Physarum polycephalum

A method is described for assaying chemotaxis in the acellular slime mold Physarum polycephalum. It consists of measuring the amount of plasmodium that moves on a strip of nitrocellulose membrane filter Millipore in response to a gradient of an attractant. Time course of chemotactic response of the slime mold is described. Different factors that affect chemotaxis in the slime mold such as: culture care and stage of growth of microplasmodia, substratum used for cell movement, nature of the gradient, effect of salts, pH and temperature are described. From concentration-response curves for different attractants several parameters of the chemotactic effect, such as threshold concentration, half maximal concentration, and maximal effective concentration can be determined. As a group, sugars are more effective chemotactic agents than amino acids. Glucose and galactose, which support the growth of the slime mold, are shown to have high positive chemotactic effect. 3-O-Methyl- -glucose and 2-deoxy- -glucose are two sugars that do not support growth but are very effective attractants. Conversely, fructose which supports slime mold growth is at best a weak attractant. The results support the view that the chemotactic effects of different sugars are not dependent on their growth-supporting value.     

Cyclic AMP and calcium exchange in a cellular slime mold

Cyclic AMP is known to be an effective chemotactic agent for amebae of the cellular slime mold D. discoideum. A large amount of information from experiments on metazoa suggested that one cellular effect of cyclic AMP might be to alter the permeability of the cell membrane to Calcium or Sodium ions. On the basis of this information experiments were designed to test the effect of cyclic AMP on outflow of labeled Calcium or Sodium ions from amebae of D. discoideum. It was found that addition of cyclic AMP at 10^?4M resulted in a large increase of Ca⁴⁵ outflow from cells at the pre-aggregative or aggregative stage of development. No effect was found on Na²² outflow. It is suggested that this effect on Calcium permeability of the membrane is related to the chemotactic influence of ATP by some action on the contractile mechanism for ameboid movement. The phenomenon may be distinct from the enzyme inductive activity of cyclic AMP known for bacteria, and perhaps occurring in the cellular slime molds as well.     

Gravity-dependent polarity of cytoplasmic streaming inNitellopsis

Summary The internodal cells of the characean algaNitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downwardstreaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR<1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K⁺ channels. The K⁺ channel blocker TEA Cl^– inhibits the NR effect.External Ca²⁺ is required for normal graviresponsivness. The [Ca²⁺] of the medium determines the polarity of cytoplasmic streaming. Less than 1 M Ca²⁺ resulted in a PR<1 while greater than 1 M Ca²⁺ resulted in the normal gravity response. The voltage-dependent Ca²⁺ -channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl₃ resulted in a PR<1, indicating the presence of two types of Ca²⁺ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR ofNitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.     

The penetration of radioactive sodium intoValonia andHalicystis

Conclusions In comparing the uptake of Na^* byValonia and that byHalicystis, it was found that the greater proportion was taken up by the protoplasm in the former case, and by the sap in the latter case.In former experiments only the sap was tested for penetration and found to contain negligible concentrations of Na inValonia and comparatively larger concentrations inHalicystis. It is therefore of interest to show that Na^* does penetrateValonia but is taken up by the protoplasm considerable concentrations; but that it does not pass into the sap under normal conditions, thereby showing that the semi-permeable membrane between the sap and the protoplasm is the region of non-penetration. InHalicystis this is not the case since Na^* was found in both the sap and the protoplasm of this cell.Further work on the difference between these two membranes would be of interest in elucidating the movement of K and Na ions through membranes.This paper was assembled by Matilda M. Brooks.     

A NATURALLY OCCURRING DEVELOPMENTAL SYNERGISM BETWEEN THE CELLULAR SLIME MOLD,DICTYOSTELIUM MUCOROIDES AND THE FUNGUS,MUCOR HIEMALIS

We here report the second record of a developmentally aberrant strain of a cellular slime mold from natural populations and demonstrate that this Dictyostelium mucoroides variant is capable of undergoing normal morphogenesis in the presence of the phycomycete fungus, Mucor hiemalis. The synergism is induced by an extracellular product(s) which is diffusable through thin agar membranes and is released by the fungus. The presence of the fungus not only induces stalk formation in this stalkless variant, but also increases the rate of sorocarp formation in 3 of 5 additional species of cellular slime molds assayed.     

A fatty acid elongase ELO with novel activity from Dictyostelium discoideum

Fatty acid elongation was examined in the cellular slime mold, Dictyostelium discoideum. Profiling of the total fatty acid content of D. discoideum indicated that fatty acid elongation is active. Orthologs of the fatty acid elongase ELO family were identified in the D. discoideum genome and the cDNA for one, eloA, was cloned and functionally characterized by expression in yeast. EloA is a highly active ELO with strict substrate specificity for monounsaturated fatty acids, in particular 16:1^Δ9 to produce the unusual 18:1^Δ11 fatty acid. This is the first report on fatty acid elongation in a cellular slime mold.     

Vacuolated plant cells as ideal osmometer: reversibility and limits of plasmolysis, and estimation of protoplasm volume in control and water-stress-tolerant cells

Abstract. The osmotic behaviour of vacuolated plant cells (adaxial epidermal cells of Allium cepa bulb scales, and epidermal as well as chloroplast containing subepidermal stem base cells of Pisum sativum) was studied over a wide range of CaCl₂ concentrations. The following results were obtained.

• a. Allium cepa and Pisum sativum plant cells behave as an ideal osmometer as far as plasmolytic contraction of the protoplast is concerned.
• b. The protoplasts of these cells could be plasmolysed to 15–45% of their original volume without the loss of membrane semi-permeability.
• c. Cells plasmolysed in 1.0 kmol m^?3 CaCl₂ could be completely deplasmolysed and upon deplasmolysis the cells resumed protoplasmic streaming.
• d. The above findings (a-c) indicate that during gradual plasmolysis and deplasmolysis membrane semi-permeability is maintained.
• e. At very high plasmolysing concentrations vacuoles covered with the tonoplast separated from the rest of the protoplasm in some cells whereas others showed systrophy. Extruded vacuoles were able to respond to osmotic shrinkage.
• f. The non-solvent space in Allium cells of about 3% also corresponded to the protoplasm volume calculated from the protoplast geometry (mean from results of direct measurement method and subtraction method).
• g. Subepidermal stem base cells of water-stress-tolerant Pisum plants had a 75% greater non-solvent space than the control cells indicating that a water-stress-tolerant cell may contain a larger amount of protoplasm and/or a vacuole with a higher content of colloidal material in the vacuole.
• h. Water-stress-tolerant cells showed greater tolerance to osmotic dehydration (volume reduction) than control cells.