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.     

Progress in plasmodial differentiation improves regularity of oscillating contractions in Physarum polycephalum

Based on the knowledge about subcellular morphogenetic processes in the acellular slime mold Physarum polycephalum, we hypothesized that during differentiation of undifferentiated endoplasm to the highly differentiated complex structure of the contractile apparatus of this organism, the regularity of oscillating contractions must improve. We measured the endogenous contraction automaticity starting from the de novo generation within minutes after sampling small portions of undifferentiated endoplasm. The standard deviation of the normalized period duration of these samples was compared to the respective values of radial contractions of differentiated protoplasmic plasmodial strands. The mean normalized standard deviation in endoplasmic drops was 28.3+/-12.2%. Respective values in protoplasmic strands were 10.0+/-3.7%. The difference between the experimental groups was highly significant (p<0.0001). We interpret the verification of our hypothesis as an indication that the very regular oscillating contractions in fully differentiated stages of Physarum require the complex structure of the sophisticated contractile apparatus, represented by the circular plasmalemma invagination system of protoplasmic strands, while the regularity is lower in stages, where the differentiation is still in progress. We believe that this is due to deficits in coordination capabilities, which need a directional and spatially oriented protoplasmic streaming as a precondition.     

Rotational streaming in fiber cells and its role in translocation

All visible protoplasmic streaming in sections of various plant stems was reversibly stopped by 2,4-dinitrophenol (DNP). Sections contained epidermal, cortical, and fiber cell types. Cells treated with DNP retained their semipermeability as evidenced by their plasmolysis in sucrose solutions. Washing out the DNP resulted in the rapid resumption of protoplasmic streaming in all 3 cell types. Both the rate of movement of sodium fluorescein and the shape of the advancing dye front were greatly altered by DNP treatment. Dye transport was decreased in the fibers and little affected in cortical cells. The results suggest that rotational streaming accelerates the translocation of soluble substances in fiber cells.     

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.     

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.     

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.     

Explaining the “Pulse of Protoplasm”: The search for molecular mechanisms of protoplasmic streaming

Explanations for protoplasmic streaming began with appeals to contraction in the eighteenth century and ended with appeals to contraction in the twentieth. During the intervening years, biologists prop...     

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 EFFECT OF AUXINS ON PROTOPLASMIC STREAMING. II

1. A further study has been made of the effect of indole-3-acetic acid (auxin) on protoplasmic streaming in the epidermal cells of the Avena coleoptile. 2. The transient nature of the effect of auxin, both in accelerating and retarding streaming, is due to the temporary exhaustion of carbohydrate from the tissues. In presence of 1 per cent fructose or some other sugars the acceleration or retardation of streaming by auxin is not transient, but is maintained for at least 2 hours. 3. The retardation of streaming brought about by concentrations of auxin above 0.5 mg. per liter is due to oxygen deficiency This has been confirmed in several ways. 4. It follows that the effect of auxin is to increase the respiration of the coleoptile tissue. 5. Younger coleoptiles, 3 cm. long, are sensitive to lower concentrations of auxin than those 5 cm. long, and more readily exhibit oxygen deficiency as a result of the action of auxin. However, after decapitation their response to auxin more closely resembles that of 5 cm. coleoptiles. 6. The retardation of streaming in such coleoptiles, resulting from oxygen deficiency, is delayed by very dilute solutions of histidine. On this basis an explanation is suggested for the results of Fitting on streaming in Vallisneria leaves. 7. The mean rate of streaming in control untreated coleoptiles in pure water varies with the time of year, but not with the time of day. 8. The results support the view that auxin accelerates an oxygen-consuming process which controls the rate of protoplasmic streaming, and that the latter controls growth. The substrate for this process is probably sugar. 9. It is suggested that auxin also accelerates another oxygen-consuming process, which may withdraw oxygen from the process which controls streaming rate and hence cause retardation of the latter.     

FIBRILLAR DIFFERENTIATION IN A MICROPLASMODIUM OF THE SLIME MOLD PHYSARUM POLYCEPHALUM

The motility of Physarum polycephalum microplasmodia depends upon the conditions under which they are cultured. To investigate the relation between protoplasmic streaming and filamentous structures observed in the cytoplasm, microplasmodia were collected from shaken cultures, agar plates and shaken cultures of the organism which had previously been plate-cultured.
No sign of streaming could be found in materials in shaken culture, even in those which were shaken after they had once been motile on an agar plate. The immotile microplasmodia in both cases failed to contain any filamentous structures.
Microplasmodia on agar plates were motile, showing vigorous peripheral movements (projection of pseudopods) and inner protoplasmic streaming. In the motile organisms two types of filamentous structures were observed: loose networks just inside the plasma membrane of rounded pseudopods with smooth surfaces; and compact, straight bundles beneath the pseudopods or in much deeper locations.     

Which phase of the contraction-relaxation cycle of cytoplasmic actomyosin in Physarum is modulated by blue light?

The influence of blue-green light (496 nm) and blue light (450 nm) on contraction behaviour of protoplasmic strands, endoplasmic veins and “de novo” generation of contraction activity in endoplasmic drops of Physarum polycephalum was investigated. The contraction-relaxation cycle was divided into contraction (t_c) and relaxation (t_r) and separately analysed. Both wavelengths prolongated only the duration of contraction, whereas the duration of relaxation remained unaffected. The effect is quantitatively the same for endoplasmic veins and protoplasmic strands and is more pronounced in the case of blue light (450 nm).Blue light shows not only an influence on the duration of contraction but also on the time point of “de novo” generation of oscillatory activity of endoplasmic drops. Since blue light reversibly inhibits both glucose consumption and acidification of the medium, the obtained results point to a modification of the contraction phase via energy metabolism. However, the influence of a plasmalemmal proton pump has also to be taken into account.     

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.     

Effect of heat shock on the chilling sensitivity of trichomes and petioles of African violet (Saintpaulia ionantha)

Chilling at 6°C caused an immediate cessation of protoplasmic streaming in trichomes from African violets ( Saintpaulia ionantha ), and a slower aggregation of chloroplasts in the cells. Streaming slowly recovered upon warming to 20°C, reaching fairly stable rates after 4, 15, 25 and 35 min for tissue chilled for 2 min and for 2, 14 and 24 h, respectively. The rate of ion leakage from excised petioles into an isotonic 0.2  M mannitol solution increased after 12 h of chilling and reached a maximum after 3 days of chilling. A heat shock at 45°C for 6 min reduced chilling-induced rates of ion leakage from excised 1-cm petiole segments by over 50%, namely to levels near that from non-chilled control tissue. Heat-shock treatments themselves had no effect on the rate of ion leakage from non-chilled petiole segments. Protoplasmic streaming was stopped by 1 min of heat shock at 45°C, but slowly recovered to normal levels after about 30 min Chloroplasts aggregation was prevented by a 1 or 2 min 45°C heat-shock treatment administered 1.5 h before chilling, but heat-shock treatments up to 6 min only slightly delayed the reduction in protoplasmic streaming caused by chilling. Tradescantia virginiana did not exhibit symptoms associated with chilling injury in sensitive species (i.e. cessation of protoplasmic streaming in stamen hairs and increased ion leakage from leaf tissue).     

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 EFFECT OF AUXINS ON PROTOPLASMIC STREAMING. III

1. A new method is described which gives a continuous record of the absolute rate of protoplasmic streaming in epidermal cells of the Avena coleoptile. 2. With this method a study was made of the influence of malate and iodoacetate on streaming velocity, in order to make correlations with the previously established effects of these substances on growth and respiration. 3. In the presence of optimum concentrations of indole-3-acetic acid in freshly cut sections, malate had no effect on streaming. In the presence of very low concentrations of the auxin, however, malate increased the range of response, so that the threshold of auxin sensitivity was lowered some ten times by the malate. Malate alone had no effect on streaming. 4. In coleoptile sections, soaked overnight in sugar solution or in water, the acceleration of streaming normally caused by auxin almost disappears, but the presence of malate causes large accelerations of streaming by the auxin. 5. Similarly, in sections from old coleoptiles, which no longer show acceleration of streaming by auxin, the acceleration is restored when malate is added together with the auxin. 6. Malate does not enter the cell as rapidly as does auxin, but easily detectable amounts penetrate within 30 minutes. 7. Iodoacetate in the concentration which inhibits growth (5 x 10^–5 M) completely inhibits the acceleration of streaming by auxin. In still lower concentrations iodoacetate slightly accelerates streaming. Higher concentrations, up to 2 x 10^–4 M, did not reduce the rate of streaming below that of controls without auxin. The effect of iodoacetate is therefore to inhibit the acceleration caused by auxin and not to affect the basal streaming rate. 8. It is concluded that, just as for growth and respiration, malate is necessary for the response to auxin shown by acceleration of streaming. This further strengthens the triple parallel between the effects of auxin on streaming, growth, and respiration, all of which are apparently mediated by the 4-carbon acid system.     

A study of protoplasmic streaming in Nitella by laser Doppler spectroscopy.

Laser light scattered from particles in the streaming protoplasm of a living cell is shifted in frequency by the Doppler effect. The spectrum of the scattered light can be measured and interpreted to infer details of the velocity distribution in the protoplasm. We have developed this approach to study the protoplasmic streaming in the fresh-water alga Nitella. Our results indicate a characteristic flow pattern to which diffusion makes a negligible contribution. No difference in the velocity of particles of different size is indicated. The streaming velocity linearly with temperature with a supraoptimal temperature of 34 degrees C, and the velocity distribution becomes narrower at high temperatures. The protoplasmic streaming can be inhibited by laser light, and this effect has been used to study the photoresponse of the algae. Using beam diameters of about 50 mum, we have shown that the inhibition is very local, becoming minimal at a displacement of about 200 mum in the upstream direction and 400 mum in the downstream direction. Prolonged exposure produces a bleached area free of chloroplasts, which is three orders of magnitude less sensitive to photoinhibition.     

Permeability of the Plasmalemma as Compared with that of the Tonoplast

In recent years there has been an increasing tendency to regard the tonoplast as the decisive diffusion barrier of the protoplast. The plasmalemma has been assumed to be more or less freely permeable, especially to ions (Brooks 4, Arisz 1, Epstein 8, Briggs and Robertson 3, Sutcliffe 12). This view is, however, based on observations which are far from unequivocal.
In the following we shall try to elucidate the question of the relative permeability and susceptibility of the two plasma membranes towards sodium hydroxide and sodium carbonate. The tests were made on internodal cells of Nitellopsis obtusa , staminal hairs of Tradescantia virginiana and T. zcbrina and epidermal cells of Allium cepa var. sanguinetim. These cells show protoplasmic streaming and either contain anthocyanins or were stained with neutral red.
In these experiments the plasmalemma, or some other layer outside the streaming part of the protoplasm, is assumed to be more or less impermeable towards sodium hydroxide as long as protoplasmic streaming is going on in the cells lying in the strongly alkaline solution. On the other hand, by the time the colour of the cell sap changes a considerable amount of NaOH must have passed through the whole protoplast, including both plasmalemma and tonoplast. The principal object of the experiments was, therefore, to compare (a) the time necessary to stop the protoplasmic streaming, irreversibly, with (b) the time required for the colour of the vacuole to change either to yellow (neutral red) or to blue (anthocyanin).     

Effect of Chilling Temperatures on the Protoplasmic Streaming of Plants from Different Climates

A microscope mount was designed so that specimen temperaturescould be monitored and controlled without impairing phase contrastoptics and used to measure rates of protoplasmic streaming between0 and 25 ?C in trichome cells of Lycopersicon esculentum, Lycopersiconhirsutum, Citrullus vulgaris, Tradescantia albiflora, Digitalispurpurea, and Veronica persica. Between 10 and 20 ?C the rates of streaming varied from 2–6µm s^–1 depending on the temperature, and differencesbetween the species were small. The temperature coefficientof streaming rates was found to increase as the temperaturewas lowered so that the plot of log rate against temperaturehad a steeper slope at the lower temperatures. The largest temperature cofficients were for the warmth-requiringL. esculentum (tomato) and C. vulgaris (water melon), and thesmallest for the temperate-zone plants V. persica (speedwell)and D. purpurea (foxglove). The changes in rate always occurredover a range of temperature; no ‘critical temperature’wasobserved below which streaming abruptly stopped and above whichit was active, although the amount of streaming as well as therate decreased as the lowest temperatures were approached. The temperatures experienced by the specimens during the experimentdid not affect the recovery of normal streaming rates betweenabout 10 and 20 ?C. In a population of a wild tomato, Lycopersicon hirsutum Humb.and Bonpl., collected from different altitudes in Peru and Ecuador,i.e. from locations of different environmental temperature,the rate of protoplasmic streaming at 5 ?C was greatest in thevarieties collected from the highest altitudes. The resultssuggest that streaming rates correlate with genetic adaptationto low temperature in the species examined.     

Morphological anomalies induced by antimicrotubule agents inBryopsis plumosa

Summary Antimicrotubule agents, colchicine, vinblastine, and griseofulvin, induced conspicuous morphological anomalies inBryopsis plumosa. First, following cessation of protoplasmic streaming within 15 minutes, elongation stopped in a few hours. Second, innumerable protrusions or new growth points generated over the cell flank in a few days. Similar phenomena were observed in the cells which were subjected to high pressure or low temperature both of which are known to disrupt microtubule.These phenomena were investigated with light and electron microscopy. It is suggested that inhibition of microtubule dependent protoplasmic streaming which may function as an intracellular transport system causes such morphological anomalies.     

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.