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.     

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 RELATION OF THE STABILITY OF PROTOPLASMIC FILMS IN NOCTILUCA TO THE DURATION AND INTENSITY OF AN APPLIED ELECTRIC POTENTIAL

1. The experiments demonstrate that when a constant electric potential of sufficient intensity is applied to Noctiluca, the protoplasmic films which represent a part of the visible continuous phase of the cytoplasm and plasma membrane at the surface of the cell, become unstable and break down, thus releasing the acid contents of one of the internal discontinuous phases present in the cytoplasm of Noctiluca. This process which occurs first at anode then at the cathode side of the cell, appears to be a selective deemulsification or coalescence similar to that at the surface of an emulsion having a viscous continuous phase. 2. The experiments demonstrate that Nernst''s equation See PDF for Equation which expresses approximately the relation of duration and intensity of a constant electric current to threshold stimulation of striated muscle, applies equally well to the process of anodal coalescence in Noctiluca. 3. Anodal and cathodal coalescence have different thresholds, due to the fact that the semipermeable plasma film at the surface of the cell is asymmetric with respect to the direction of the applied current. Attention is called to the possible relation between this phenomenon and the conditions occurring at the synapse between neurons. 4. The stability of the protoplasmic films in relation to the applied electric potential is greater in young cells than in old cells, or in other words the threshold intensity of the stimulus is higher for young than for old cells. 5. Attention is called to the occurrence in the same cell of different receptor-affector mechanisms having a corresponding difference in intensity threshold when an electric current is acting as a stimulus.     

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.     

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.     

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.     

Interactions of the circadian and circatidal rhythms of the littoral gastropod melanerita atramentosa Reeve

Melanerita atramentosa Reeve is active during high water and for a period after the ebb, until the substratum dries. A circatidal rhythm with an endogenous inequality between the diurnal and nocturnal high water activity is present in freshly collected snails placed under constant conditions (LL, 30 lux, temperature 21 °C, water level constant). The free-running period of the circatidal rhythm was approximately 24.3 h, slightly less than the tidal period of 24.8 h. This rhythm faded out after 4–6 days of constant conditions. Snails acclimated to non-tidal conditions were active at night. This rhythm persisted under constant conditions for 5–10 days, after which it too faded out. Shock-freezing re-initiated the circatidal rhythm, supporting a ‘multiple-clock’ hypothesis of control. Its limits entrainment are probably narrow, but an entrained periodicity persists for a number of cycles. A model of clock interaction and environmental influence is advanced.     

The influence of neurohumoral transmitter substances on protoplasmic streaming in the myxomycete Physarella oblonga

The authors studied the influence of adrenaline-like substances and acetylcholine on the protoplasmic streaming in Physarella oblonga, investigating the effect on the duration of the rhythmic period. Adrenaline and noradrenaline proved to cause a shortening, whereas acetylcholine appeared to be capable of causing an extension of the period duration. The authors present and discuss critically the possibility that the adrenergic and cholinergic transmitter substances, which influence the visceral (mechano-) effectors in the higher animal organism often antagonistically, may also affect an elementary “functional” effector system responsible for protoplasmic streaming, and that in so doing they have comparable effects. The results of experiments with eserine, acetylcholine and a mixture of these two substances render it probable that an acetylcholine esterase and acetylcholine occur in the plasmodium. The mean normal value of the period duration was, calculated from 976 measurements and expressed in seconds: 284.183 ± 1.486 (S.E. of the mean). The average duration of the progressive and the regressive phase was found to be almost the same.     

Does the difference in the timing of eclosion of the fruit fly Drosophila melanogaster reflect differences in the circadian organization?

The eclosion rhythm of a laboratory population of Drosophila melanogaster was studied under 12h light, 12h dark (LD 12:12) cycles. Although most of the flies were found to eclose just after “lights on” in LD 12:12, termed within gate (WG) flies, a few flies were found to eclose nearly 10h after peak eclosion, termed outside gate (OG) flies. The circadian parameters of the clocks controlling oviposition rhythms in the WG and the OG flies were estimated to understand the cause of such differences in the timing of eclosion. The distribution of the fraction of individual flies exhibiting single, multiple, and no significant period in the WG flies was significantly different from distribution in the OG flies. Compared to the WG flies, more OG flies were found to exhibit oviposition rhythm with multiple periodicity, whereas more WG flies exhibited an oviposition rhythm with a single significant period. The fraction of flies with arrhythmic oviposition was similar in both the WG and the OG flies. Free-running period τ in constant darkness (DD) and the phase angle difference ψ in LD 12:12 for the oviposition rhythm of WG and OG flies were significantly different. These results suggest that the differences in the time of eclosion between the flies eclosing within the gate and outside the gate of eclosion are probably due to differences in the circadian system controlling eclosion, which is reflected by the differences in their oviposition rhythm. (Chronobiology International, 18(4), 601-612, 2001)     

The 28th Conference of the International Society for Chronobiology (ISC) will be held in Bucharest (Romania), at Ramada Parc Hotel on 4–8 June 2014

The eclosion rhythm of a laboratory population of Drosophila melanogaster was studied under 12h light, 12h dark (LD 12:12) cycles. Although most of the flies were found to eclose just after “lights on” in LD 12:12, termed within gate (WG) flies, a few flies were found to eclose nearly 10h after peak eclosion, termed outside gate (OG) flies. The circadian parameters of the clocks controlling oviposition rhythms in the WG and the OG flies were estimated to understand the cause of such differences in the timing of eclosion. The distribution of the fraction of individual flies exhibiting single, multiple, and no significant period in the WG flies was significantly different from distribution in the OG flies. Compared to the WG flies, more OG flies were found to exhibit oviposition rhythm with multiple periodicity, whereas more WG flies exhibited an oviposition rhythm with a single significant period. The fraction of flies with arrhythmic oviposition was similar in both the WG and the OG flies. Free-running period τ in constant darkness (DD) and the phase angle difference ψ in LD 12:12 for the oviposition rhythm of WG and OG flies were significantly different. These results suggest that the differences in the time of eclosion between the flies eclosing within the gate and outside the gate of eclosion are probably due to differences in the circadian system controlling eclosion, which is reflected by the differences in their oviposition rhythm. (Chronobiology International, 18(4), 601–612, 2001)     

Studies on contraction rhythm of the plasmodial strand III. Role of endoplasmic streaming in synchronization of local rhythms

Summary Two separate segments of plasmodial strands (Physarum polycephalum) generally contract and relax with different periods, but if the two are bridged with another small strand segment to make into a single system, the contraction cycles of the two previously separate segments become gradually unified under isometric as well as isotonic conditions. To clarify the possible role of the streaming endoplasm as the information carrier for synchronization, we stopped the streaming between two halves of a single strand either by cutting it or by using the double-chamber method without cutting it. When the endoplasm is prevented from flowing between the two halves of the same continuous system, which had been in good synchrony, their contraction-relaxation rhythms become out of phase with each other. After the endoplasm in the strand is allowed to stream freely again, the synchrony of their cyclic contraction is reestablished. It was concluded that endoplasm flowing back and forth in a plasmodial strand must carry a factor(s) which coordinates the period and phase of the contraction-relaxation cycle but does not control the amplitude of the cycle.The present work was supported in part by grants-in-aid from the Mitsubishi Foundation and the Japanese Ministry of Education, Science, and Culture.     

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.     

Responsiveness to light of the circadian clock controlling eclosion in the blowfly, Lucilia cuprina

ABSTRACT. Eclosion in Lucilia cuprina (Wiedemann) occurs near dawn. The rhythm of eclosion persists in both darkness and constant light of high intensity (490μW cm^-2) with a period close to 24h. The sensitivity to light of the circadian clock controlling eclosion varies greatly according to the stage of the life cycle. During larval life the free running rhythm in darkness can be phase shifted by light pulses of 100μW cm^-2 intensity, with the transition from a Type 1 phase response curve to a Type 0, occurring with pulses of between 1 and 8h. Extending the last light period of LD to 24 h followed by constant darkness resets the phase of the rhythm by 12h, a transition from constant light to constant darkness initiates rhythmicity in flies made arrhythmic by being reared from eggs collected from adults maintained in constant light. After pupariation, the rhythm is relatively insensitive to light. Rhythmicity is sometimes induced by a transition from constant light to constant darkness, but the phase of the rhythm is not shifted by extending the last light period of LD before entering constant darkness. Repeated LD cycles applied after pupariation initiate and entrain the rhythm.     

Circadian periodicity in cockroaches altered by high frequency light-dark cycles

Summary The circadian period of the freerunning activity rhythm in the cockroach is systematically altered by high frequency light-dark cycles (HF-LD) according to the ratio of light to dark within each cycle. With a standard 10 min cycle time, brief (e.g., 0.5 min) exposure to light each cycle causes the free-running period to shorten significantly in comparison to its steady-state value in constant darkness. As the ratio of light to dark in HF-LD is increased, the period of the rhythm is progressively lengthened. These findings are discussed in terms of the general proposition that light, applied throughout the circadian cycle, predictably modifies periodicity according to the asymmetrical shape of the circadian phase response curve.Abbreviations LD light-dark cycles in which cycle length is in hours - HF-LD light-dark cycles in which cycle length is in min; period of the activity rhythm; change in period of the activity rhythm - PRC phase response curve - LL constant light     

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.     

STIMULATION BY COLD IN NITELLA

Sudden local chilling causes action currents to be set up in Nitella and in Chara, an effect which does not follow gradual local chilling. This may be due to a partial solidification of the non-aqueous protoplasmic surfaces which makes them susceptible to rupture by the protoplasmic streaming. This movement continues usually for several minutes after the chilling, whereas if stimulation occurs at all it occurs immediately on chilling. It is found that a chilled spot is much more sensitive to mechanical stimulation than is a spot at room temperature. Chilling is accompanied by a rise of resistance, a lowered rate of recovery following stimulation, and usually by a falling off in the magnitude of the action curve.     

The Circadian Rhythm of Leaf Movement of Coleus blumei x C. frederici, a Short Day Plant. II. The Effects of Light and Temperature Signals

The phase response curve for the circadian rhythm of leaf movement of Coleus blumei x C. frederici, a short day plant, is generally similar to those reported for other organisms. An increase in the duration of the light signal caused an increase in the extreme values of the phase response curve and shortened the time for transition from maximum delays to maximum advances. Experiments with 2 light signals showed that the overt rhythm of leaf movement represents the rhythm of the light sensitive oscillator even during the transient period that followed the first light signal. A temperature decrease of 7° for 8 hr caused only a transient phase shift in the following 2 cycles but not in the steady state. The combination of such a temperature decrease and a light signal showed that only the overt rhythm of leaf movement was disturbed by the temperature decrease whereas the light sensitive oscillator was free running. A temperature decrease of 11° for 10 hr caused a steady state phase shift and affected the light sensitive oscillator as well.     

A Semidian Rhythm in the Flowering Response of Pharbitis nil to Far-Red Light: I. Phasing in Relation to the Light-Off Signal

Evidence is presented of an endogenous rhythm in flowering response to far-red (FR) irradiation, with a period of about 12 h (hence semidian rhythm), which persists through at least three cycles in constant conditions of continuous light at 27°C and has a marked influence on the flowering response in Pharbitis nil to a subsequent inductive dark period. The phase of the rhythm is not influenced by real time nor by the time from imbibition or from the beginning of the light period. Rather, it is fed forward from the beginning of the FR interruption to the beginning of the inductive dark period. The period of the rhythm is not affected by irradiance but is longer at cooler temperature. When there are two FR interruptions during the preceding light period, it is primarily the later one which determines the phase of the rhythm, although some interactions are evident. There appears to be an abrupt rephasing of the rhythm at the beginning of the inductive dark period. No overt rhythms which could be used as “clock hands” for the semidian rhythm were detected in photosynthesis, stomatal opening, or translocation.     

Circadian rhythm of sex pheromone-releasing behaviour in females of the dermestid beetle,Trogoderma glabrum: Regulation by photoperiod

The effects on sex pheromone-releasing, or calling behaviour, of diel photoperiods of varying daylength, of light cycle phase shifts, and of continuous illumination were investigated in Trogoderma glabrum females. On light régimes with 8 to 20 hr daylengths, calling maxima tended to centre close to photophase midpoints. Although influencing the time of day at which calling occurred, daylength had little effect on the amount of activity or the length of the calling period. When 16 : 8 LD light cycles were advanced or delayed by 4 hr, the time of day at which calling peaks were observed shifted within 2 to 4 cycles so that a constant phase relationship with photoperiod was maintained. Daily calling peaks were evident in groups of females exposed to between 1 and 5 days of continuous illumination, but mean calling time occurred earlier in the day as light exposures were lengthened. It was concluded that a circadian rhythm of calling behaviour exists in T. glabrum females. and that the rhythm can be entrained to 24 hr periodicity by photoperiod.     

A continuum model of contraction waves and protoplasm streaming in strands of Physarum plasmodium

We present a mathematical model for continuously distributed mechanochemical autooscillations (autowaves) in a protoplasmic strand of Physarum polycephalum. The model is based on a hypothesis of local positive feedback between deformation and contraction of the contractile apparatus. This feedback is mediated through a cell regulatory system whose kinetics involves coupling to mechanical strain. Mathematical analysis and computer simulations have demonstrated that the solutions of the model agree quantitatively with the available experimental data. In particular, hydrodynamic interaction alone, between different sections of the strand via the streaming endoplasm, is capable of inducing the characteristic contractile behavior.