THE EFFECT OF SUDDEN CHANGES OF TEMPERATURE ON PROTOPLASMIC STREAMING

A sudden fall of temperature of 15° to 20° causes a complete cessation of the protoplasmic streaming in Nitella. The recovery of the normal rate follows a definite course and the time of recovery is dependent on the temperature. Evidence is adduced to show that the inhibition of streaming is due to a physical phenomenon.     

Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation inCharaceae internodal cells

Summary The mechanism of the cessation of cytoplasmic streaming upon membrane excitation inCharaceae internodal cells was investigated.Cell fragments containing only cytoplasm were prepared by collecting the endoplasm at one cell end by centrifugation. In such cell fragments lacking the tonoplast, an action potential induced streaming cessation, indicating that an action potential at the plasmalemma alone is enough to stop the streaming.The active rotation of chloroplasts passively flowing together with the endoplasm also stopped simultaneously with the streaming cessation upon excitation. The time lag or interval between the rotation cessation and the electrical stimulation for inducing the action potential increased with the distance of the chloroplasts from the cortex. The time lag was about 1 second/15 m, suggesting that an agent causing the rotation cessation is diffused throughout the endoplasm.Using internodes whose tonoplast was removed by replacing the cell sap with EGTA-containing solution (tonoplast-free cells,Tazawa et al. 1976), we investigated the streaming rate with respect to the internal Ca²⁺ concentration. The rate was roughly identical to that of normal cells at a Ca²⁺ concentration of less than 10^–7 M. It decreased with an increase in the internal Ca²⁺ concentration and was zero at 1 mM Ca²⁺.The above results, together with the two facts that Ca²⁺ reversibly inhibits chloroplast rotation (Hayama andTazawa, unpublished) and the streaming in tonoplast-free cells does not stop upon excitation (Tazawa et al. 1976), lead us to conclude that a transient increase in the Ca²⁺ concentration in the cytoplasm directly stops the cytoplasmic streaming. Both Ca influxes across the resting and active membranes were roughly proportional to the external Ca²⁺ concentration, which did not affect the rate of streaming recovery. Based on these results, several possibilities for the increase in Ca²⁺ concentration in the cytoplasm causing streaming cessation were discussed.     

Studies on cessation of cytoplasmic streaming under K+-induced depolarization inNitella axilliformis

Internodal cells ofNitella axilliformis had a membrane potential of about−120mV and showed active cytoplasmic streaming with a rate of about 90 μm/sec in artificial pond water (APW) at 25C. When APW was replaced with 50 mM KCl solution, the membrane potential depolarized accompanying an action potential, and the cytoplasmic streaming stopped. Soon after this quick cessation, the streaming started again, but its velocity remained very low for at least 60 min. Removal of KCl from the external medium led to repolarization of the membrane and accelerated recovery of the streaming. The change in the concentration of free Ca²⁺ in the cytoplasm ([Ca²⁺]_c) was monitored by light emission from aequorin which had previously been injected into the cytoplasm. Upon application of KCl to the external medium, the light emission, i.e., [Ca²⁺]_c, quickly increased. It then decreased exponentially and reached the original low level within 100 sec. The cause of the long-lasting inhibition of cytoplasmic streaming observed even when [Ca²⁺]_c had returned to its low resting level is discussed based on the mechanism proposed for action potential-induced cessation of cytoplasmic streaming; inactivation of myosin by Ca²⁺-dependent phosphorylation or formation of cross bridge between actin filaments and myosin.     

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.     

Membrane control of cytoplasmic streaming in characean cells

When a characean cell generates an action potential, cytoplasmic streaming transiently stops and then recovers gradually. Calcium ion is one of the most important factors mediating between membrane excitation and cessation of cytoplasmic streaming. When an internode ofNitella flexilis is subjected to transcellular osmosis, both membrane depolarization and cessation of streaming take place at the endoosmotic part of the cell. It was also found that Ca²⁺ plays a key role in mediating between osmosis induced hydration of the cytoplasm and the cessation of cytoplasmic streaming. The present article reviews how Ca²⁺ acts as a second messenger in intracellular signal transduction in controlling the cytoplasmic 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.     

Cessation of cytoplasmic streaming of Chara internodes during action potential

Cytoplasmic streaming of the Chara internode stops temporarilyat the peak of the action potential. Use of the technique ofvacuolar perfusion established that the sudden cessation ofcytoplasmic streaming is caused mainly by a temporary disappearanceof its motive force. Recovery of the rate of cytoplasmic streamingoccurs in parallel with that of the motive force. The ‘viscosity’of the cytoplasm remains almost unchanged during the whole periodof excitation except at the peak of the action potential. (Received February 1, 1968; )     

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.     

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: I. Effects of La3+, verapamil,EGTA, W-7, and TFP on the action potential

Summary The cytoplasmic streaming of the normal internodal cell of giant algaChara stops transiently at about the peak of action potential. Application of La³⁺ or verapamil (a calcium channel blocker) or removal of external Ca²⁺ by EGTA caused a partial depolarization of the resting potential, partial decrease of the membrane conductance and a marked decrease of the amplitude of action potential. Under these conditions, the conductance at the peak of action potential reduced markedly and the streaming of cytoplasm did not cease during action potential (excitation-cessation (EC) uncoupling). The effects of Ca²⁺ channel blockers could not be removed by addition of CaCl₂ to the external medium. In contrast, the effect of EGTA on the excitability could be removed to a greater extent and the cytoplasmic streaming ceased at about the peak of action potential by the addition of Ca²⁺ externally. Application of calmodulin antagonists W-7 or TFP caused similar effects on the action potential and on the cytoplasmic streaming.     

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.     

Cessation of cytoplasmic streaming follows an increase of cytoplasmic Ca2+ during action potential inNitella

Summary Taking advantage of prolonged action potential under low temperature, we studied temporal relationship among the action potential, increase of cytoplasmic Ca²⁺ concentration and cessation of cytoplasmic streaming inNitella. The Ca²⁺ concentration began to increase at a very early stage of the action potential and the cessation of streaming followed that increase.Abbreviations APW artificial pond water     

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.     

Voltage Clamp Studies on the Slow Inward Current during the Excitation of Nitellopsis obtusa

The ionic currents across the plasmalemma of Nitellopsis obtusawere measured in voltage clamp experiments. Depolarization ofthe cell by 30–100 mV from the level of the resting potentialresulted in (1) a rapid inward current, (2) a subsequent slowinward current, and (3) a stationary outward current. The firstcurrent component changed sign at –20 to –30 mV.The second component decreased to a minimum at this clampedlevel. With increasing depolarizing steps some slow transientcurrent component reappeared without changing sign. This transientinward current occurred also when the potential was clampedeither at large depolarizing (+80 mV) or at large hyperpolarizing(–300 mV) potentials. In cases when the slow inward currentcomponent was evident cessation of protoplasmic streaming wasobserved. The ATPase inhibitor dicyclohexylcarbodiimide (DCCD)at a concentration of 2 x 10^–5 M in the external mediuminhibited the slow transient inward current without affectingthe first rapid current component. It is suggested that theirreversible slow transient current component reflects the onsetof some active ion-transport system in the plasmalemma duringcell excitation.     

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.     

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).     

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).     

Alterations in membrane transport properties by freezing injury in herbaceous plants:

Leakage of ions from a thawed tissue is a common phenomenon of freezing injury. This leakage is usually assumed to be due to loss of membrane semipermeability or membrane rupture by freezing injury. Freeze injured, yet living, onion (Allium cepa L.) epidermal cells were used to study alterations in cell membranes that result in leakage of ions. In spite of a large efflux of ions, freeze injured cells could be plasmolysed and they remained plasmolysed for several days just like the unfrozen control cells. Injured cells also exhibited protoplasmic streaming. Passive transport of KCl, urea and methyl urea across the cell membranes of injured and control cells was also studied. No difference could be detected for the transport rates of urea and methyl urea between control and injured cells. However, a dramatic increase in the transport rate of KCl was found for the injured cells. Depending upon the extent of initial freezing injury, an increase or a decrease in injury symptoms was found in the post-thaw period. During the progress of freezing injury, 10 days after thawing, a swelling of the protoplasm was seen in the irreversibly injured cells. In spite of this swelling, these cells could be plasmolysed. It appears that the high amount of K⁺ that leaks out into the extracellular water, due to freezing injury, causes protoplasmic swelling by replacing Ca²⁺ in the plasma membrane. We conclude that protoplasmic swelling is a sign of secondary injury. The results presented in this study show that membrane semipermeability is not completely lost and membrane rupture does not occur during the initial stage of freezing injury. In fact, the cells have the ability to repair damage depending upon the degree of injury. Our results show there are specific alterations in membrane semipermeability (e.g., transport of K⁺) which could be repaired completely depending on the degree of injury. These findings suggest that ion leakage due to freezing injury is due to alteration in the membrane proteins and not in the membrane lipids.     

Changes in membrane potential and resistance caused by transient increase of potassium conductance in the unicellular green alga Eremosphaera viridis

The electrophysiological membrane parameters of the unicellular green alga Eremosphaera viridis were determined using an improved computer-supported single-microelectrode technique. These cells developed an average membrane potential of-150 mV in the light and a specific resistance of 1 Ω m² with an external potassium concentration of 1.1 mM and pH 5.5. In the dark, many cells showed a less polarized potential of 30–40 mV and a smaller membrane resistance. At potassium concentrations in the external medium higher than 1 mM, the membrane potential strongly depends on the external potassium content apart from a small electrogenic component. At concentrations lower than 1 mM K⁺, a dependence of the membrane potential upon external potassium concentrations could not be verified. Inserting the internal ion activities in the Goldmann equation shows that, in this range, the proton conductance seems to be predominant over the potassium conductance. Transient changes in the membrane potential and in the membrane resistance were observed after switching off the light, after addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or N,N′-dicyclohexylcarbodiimide, after a sudden decrease in temperature, and after current pulses. These changes resemble the action potentials (AP) found in other plant cells (Chara, Acetabularia). On average, the AP has a delay period of 5.1 s and a duration of 43.8 s showing a sudden decrease and a slower regeneration. The voltage peak during an AP followed exactly the Nernst potential of potassium over a range of external potassium concentrations from 5 μM to 0.2 M. This is true for depolarization or hyperpolarization, depending on the external K⁺-concentration. Tetraethylammonium-hydrogensulphate, a rather specific inhibitor of K⁺ channels in nervous cells, suppressed the AP. The correlation of the appearance of the AP with a short-term opening of potassium channels in the membrane of Eremosphaera is discussed.     

Coupling of excitation and cessation of cyclois in Nitella: role of divalent cations

The effect of external divalent cation salt solutions upon the association of an action potential and cessation of cytoplasmic streaming in Nitella was studied. Nitella cells remained excitable when immersed in solutions of CaCl₂, MgCl₂, BaCl₂, and SrCl₂. Cessation of streaming coincident with excitation occurred in solutions of CaCl₂ or SrCl₂ but not in solutions of MgCl₂ or BaCl₂. In cells exposed to solutions containing mixtures of MgCl₂ and CaCl₂, or MgCl₂ and SrCl₂, it was the [Ca]/[Mg] or [Sr]/[Mg] which determined the effect of an action potential upon the rate of streaming, rather than the absolute concentrations Ca⁺⁺ or Sr⁺⁺. The implications of these data are discussed with respect to the structure involved in the generation of cytoplasmic streaming and the relation of streaming to other types of biological motion.