Cytoplasmic streaming in the cell model ofNitella

Summary The plasmalemma ofNitella internode was made freely permeable to solutes by treating the cell with detergent and EGTA under plasmolysis. After the treatment, the cytoplasmic streaming was stopped by bathing the cell in a medium lacking ATP. The streaming was reactivated by perfusing the exterior of the permeabilized cell with a medium containing both Mg²⁺ and ATP. The reactivated streaming could be reversibly stopped by depletion of ATP. However, depletion of Mg²⁺ irreversibly inhibited the streaming.Cytochalasin B at 5 g/ml irreversibly inhibited the reactivated streaming within a minute, showing that microfilaments are involved in the streaming.Abbreviations ATP adenosine-5-triphosphoric acid - CB cytochalasin B - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DMSO dimethylsulfooxide - DTT dithiothreitol - EGTA ethyleneglycol-bis(-aminoethylether)-N,N tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl-sulfonylfluoride     

Ca2+ ion reversibly inhibits the cytoplasmic streaming ofNitella

Summary When Ca²⁺, K⁺ or Cl^– was injected iontophoretically into the cytoplasm of intactNitella cell, only Ca²⁺ reversibly inhibited the cytoplasmic streaming. However, when an extremely large current was used, the cytoplasmic streaming was reversibly inhibited irrespective of the ion species. This inhibition may be due to a transient increase of free Ca²⁺.     

Bidirectional flow of endoplasm inPhysarum plasmodium under centrifugal acceleration

Summary The behavior of cytoplasmic streaming in plasmodial strand ofPhysarum polycephalum was studied under centrifugal acceleration using a centrifuge microscope of the stroboscopic type. Cytoplasmic streaming in the plasmodium was greatly affected by changes in the acceleration. The endoplasmic flow in the centrifugal direction was accelerated, while that in the centripetal was retarded, by centrifugal acceleration. The centrifugal acceleration required to stop the endoplasmic flow in the centripetal direction did not cause total cessation of streaming but always induced a bidirectional flow of endoplasm in one and the same strand. Each profile of velocity distribution of the bidirectional flow was both parabola with flattened apex. One possible cause of the bidirectional flow is discussed.Dedicated to Emeritus Professor Noburo Kamiya on the occasion of his 80th birthday     

Cytoplasmic streaming in giant algal cells: A historical survey of experimental approaches

Various methods have been used to study cytoplasmic streaming in giant algal cells during the past three decades. Simple techniques can be used with characean internodal cells to modify the cell constitution in various ways to gain insight into the mechanism of cytoplasmic streaming. Another method involves isolatingin vitro a huge drop of uninjured endoplasm, to examine its physical and dynamic properties. The motive force responsible for streaming has been measured by three different techniques with similar results. Subcortical fibrils consisting of bundles of F-actin with the same polarity are indispensable for streaming. Differential treatment of the endoplasm and ectoplasm has shown that putative characean myosin is localized in the endoplasm. Studies of the roles of ATP, Mg²⁺, Ca²⁺, H⁺ etc. in the streaming have been conducted by cellular perfusion, which allows removal of the tonoplast, or by techniques permeabilizing the protoplasmic membrane. A slow version of the movement can even be artificially reproduced by combining characean actinin situ and exogenous myosin in the presence of Mg-ATP. The findings thus far obtained support the hypothesis that cytoplasmic streaming in characean cells is caused by an active shearing force produced by interaction of the actin filament bundles on the cortex with myosin in the endoplasm.     

Variation in velocity of cytoplasmic streaming and gravity effect in characean internodal cells measured by laser-Doppler-velocimetry

Summary Velocities of cytoplasmic streaming were measured in internodal cells ofNitella flexilis L. andChara corallina Klein ex Willd. by laser-Doppler-velocimetry to investigate the possibility of non-statolith-based perception of gravity. This was recently proposed, based on a report of gravity-dependent polarity of cytoplasmic streaming. Our measurements revealed large spatial and temporal variation in streaming velocity within a cell, independent of the position of the cell with respect to the direction of gravity. In 58% of the horizontally positioned cells the velocities of acropetal and basipetal streaming, measured at opposite locations in the cell, differed significantly. In 45% of these, basipetal streaming was faster than acropetal streaming. In 60% of the vertically positioned cells however the difference was significant, downward streaming was faster in only 61% of these. When cell positions were changed from vertical to horizontal and vice versa the cells reacted variably. A significant difference between velocities in one direction, before and after the change, was observed in approx. 70% of the measurements, but the velocity was faster in the downward direction, as the second position, in only 70% of the significantly different. The ratio of basipetal to acropetal streaming velocities at opposite locations of a cell was quite variable within groups of cells with a particular orientation (horizontal, normal vertical, inverted vertical). On average, however, the ratio was close to 1.00 in the horizontal position and approx. 1.03 in the normal vertical position (basipetal streaming directed downwards), which indicates a small direct effect of gravity on streaming velocity. Individual cells, however, showed an increased, as well as a decreased, ratio when moved from the horizontal to the vertical position. No discernible effect of media (either Ca^{2 +}-buffered medium or 1.2% agar in distilled water) on the streaming velocities was observed. The above mentioned phenomenon of graviperception is not supported by our data.Abbreviations g gravitational acceleration (9.81 m/s²) - LDV laser-Doppler-velocimetry - VR velocity ratio Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

Effect of supraoptimal temperatures on the function of the subcortical fibrils and an endoplasmic factor inNitella internodes

Summary The effect of supraoptimal temperatures onNitella cells was studied with special reference to the function of subcortical fibrils and an endoplasmic factor. Local heat-treatment (50 °C for 1 minute) of an internodal cell ofNitella disclosed that 1. the subcortical fibrils in the treated area remain normal, not affected by the treatment, 2. the subcortical fibrils alone produce no cytoplasmic streaming, 3. the endoplasm contains an extremely heat-labile factor which is indispensable for streaming, and 4. the stagnant endoplasm in the heat-treated area is neither coagulated nor gelated by heat.Preliminary reports appeared in Proc. 37th Annu. Meet. Bot. Soc. Jpn. P. 160 (1972, in Japanese) and in Abst. Annu. Meet. Jpn. Soc. Cell Biol. p. 57 (1975, in Japanese).     

Structural basis of cytoplasmic streaming in Characean internodal cells. A hydrodynamic analysis

Summary Hydrodynamic equations were derived which relate the velocity profile of endoplasmic streaming with the motive force generated by active sliding of endoplasmic organelles in Characean internodal cells, under two implicit assumptions that (1) the sliding velocity of putative organelles is comparable to the streaming velocity of endoplasm, and (2) subcortical endoplasm is far less viscous than bulk endoplasm.The equations were extended so as to calculate the velocity profile in flattened or perfused internodal cells. Calculated profiles were basically consistent with reported patterns of streaming under these conditions.Utilizing published data, we deduce some hydrodynamic parameters of streaming, and predict the dimensions of putative organelles expected to drive entire cytoplasm. A revision for published values of the motive force of streaming is proposed.Hydrodynamic analyses made earlier on the spherical organelles are repeated. The results show that the organelles may generate streaming, depending on the configurationin vivo of fine filaments protruding from the body of the organelles.     

Cytoplasmic streaming inParamecium

Summary Certain species ofParamecium demonstrate rotational cytoplasmic streaming, in which most cytoplasmic particles and organelles flow along permanent route, in a constant direction. By means of novel methods of immobilization, observation and recording, some dynamic properties of cytoplasmic streaming have been described. It was found that the velocity profiles of coaxial layers of cytoplasm have a (parabolic) paraboidal shape and the mean output of cytoplasm flow in different examined zones of streaming is constant. As the consequence of randomly distributed elementary propulsion units within the cytoplasm, particles, which serve as markers of movement, exhibit movements of a saltatory nature; this form of movement is seen inParamecium streaming only in cases of error due to polarization of the saltating particles. Interaction of actin filaments and myosin is likely to occur under specific conditions in microcompartments of cytoplasm where local solations are generated eventually leading to contractions which might propagate on gelated neighbouring areas. Places of elementary contractions are scattered. Therefore the motile effect appears as streaming. Rotational cytoplasmic streaming inParamecium may serve as a convenient model for the study of the dynamics and function of cytoplasmic motility.     

Cytoplasmic streaming inChara rhizoids

Summary In-vivo videomicroscopy ofChara rhizoids under 10^–4g demonstrated that gravity affected the velocities of cytoplasmic streaming. Both, the acropetal and basipetal streaming velocities increased on the change to microgravity. The endogenous difference in the velocities of the oppositely directed cytoplasmic streams was maintained under microgravity, yet the difference was diminished as the basipetal streaming velocity increased more than the acropetal streaming velocity. Direction and structure of microfilaments labeled by rhodamine-phalloidin had not changed after 6 min of microgravity.Abbreviations g gravitational acceleration - Nizemi slow rotating centrifuge microscope - Texus technological experiments under reduced gravity     

Immobilization of endoplasm flowing contiguous to the actin cables upon electrical stimulus inNitella internodes

Summary Using a stroboscopic centrifuge microscope, we demonstrated that, when actin cables (=bundles of F-actin) had been previously removed locally from the cell cortex ofNitella internodes, the passively flowing endoplasm found there under centrifugal force did not stop at all upon electrical stimulus, while the actively flowing endoplasm contiguous to the actin cables at the normal cell cortex promptly stopped following the stimulus and was immobilized for several seconds. The results present evidence that, upon electrical stimulus, the presence of actin cables is required to immobilize the endoplasm flowing contiguous to the actin cables in a state that resists displacement by centrifugal force.Abbreviations APW artificial pond water - CMS centrifuge microscope     

Reconstitution of cytoplasmic streaming inCharaceae

Summary The active sites of actin of oneCharaceae species were found to interact with the endoplasmic factor from a different species. Protoplasm was suqueezed out of cells ofChara australis with vacuoles that had been perfused beforehand with a medium containing EGTA and Mg · ATP. Centrifugation of this protoplasmic mixture divided it into the supernatant composed of endoplasmic granules and the precipitate composed of chloroplasts and nuclei. When the endoplasmic granular aggregates were introduced into a tonoplast-freeNitella axilliformis cell treated with NEM to inactivate the endoplasmic factor, they became attached to theNitella gel and streamed longitudinally with the polarity. Treatment of the endoplasmic granules with the strong Mg²⁺chelator CyDTA (1,2-cyclohexane diamineN, N-tetraacetic acid) irreversibly inhibited reconstitution of the cytoplasmic streaming.Abbreviations APW artificial pond water - ATP adenosine-5-triphosphoric acid - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N-tetraacetic acid - HMM heavy meromyosin - NEM N-ethylmaleimide - PEP phosphoenolypyruvate - PIPES piperazine-N,N-bis-(2-ethane-sulfonic acid) - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride     

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     

Measurement of motive force for cytoplasmic streaming in characean internodes

Summary With an attempt to measure the motive force responsible for cytoplasmic streaming in characean internodal cells, the difference between densities of cytoplasm and vacuolar sap was heightened by about 10 times (density of vacuolar sap was made larger than that of cytoplasm) by replacing the natural vacuolar sap ofChara corallina with an artificial one of higher density. Endoplasmic flow contiguous to the peripheral actin cables (peripheral flow of endoplasm) in the centrifugal direction was not influenced at all by the application of centrifugal acceleration up to 1400 g. We thus concluded that the motive force for the peripheral flow should be much larger than 12dyn/cm², a figure more than 10 times larger than that for bulk endop lasmic flow so far reported.Dedicated to Emeritus Professor Noburo Kamiya on the occasion of his 80th birthday     

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.     

Actin-microtubule interactions in the algaNitella: analysis of the mechanism by which microtubule depolymerization potentiates cytochalasin's effects on streaming

Summary In the characean algaNitella, depolymerization of microtubules potentiates the inhibitory effects of cytochalasins on cytoplasmic streaming. Microtubule depolymerization lowers the cytochalasin B and D concentrations required to inhibit streaming, accelerates inhibition and delays streaming recovery. Because microtubule depolymerization does not significantly alter³H-cytochalasin B uptake and release, elevated intracellular cytochalasin concentrations are not the basis for potentiation. Instead, microtubule depolymerization causes actin to become more sensitive to cytochalasin. This increased sensitivity of actin is unlikely to be due to direct stabilization of actin by microtubules, however, because very few microtubules colocalize with the subcortical actin bundles that generate streaming. Furthermore, microtubule reassembly, but not recovery of former transverse alignment, is sufficient for restoring the normal cellular responses to cytochalasin D. We hypothesize that either tubulin or microtubule-associated proteins, released when microtubules depolymerize, interact with the actin cytoskeleton and sensitize it to cytochalasin.Abbreviations APW artificial pond water - Ca_c cytoplasraic free calcium concentration - DMSO dimethyl sulfoxide - MT microtubule-minus - MT+ microtubule-plus.     

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.     

Cytoplasmic streaming affects gravity-induced amyloplast sedimentation in maize coleoptiles

Living maize (Zea mays L.) coleoptile cells were observed using a horizontal microscope to determine the interaction between cytoplasmic streaming and gravity-induced amyloplast sedimentation. Sedimentation is heavily influenced by streaming which may (1) hasten or slow the velocity of amyloplast movement and (2) displace the plastid laterally or even upwards before or after sedimentation. Amyloplasts may move through transvacuolar strands or through the peripheral cytoplasm which may be divided into fine cytoplasmic strands of much smaller diameter than the plastids. The results indicate that streaming may contribute to the dynamics of graviperception by influencing amyloplast movement.     

Cytoplasmic streaming does not drive intercellular passage in staminal hairs ofSetcreasea purpurea

Summary The effect of inhibition of cytoplasmic streaming on intercellular passage of carboxyfluorescein (CF) in staminal hairs ofS. purpurea was examined. Tip cells of staminal hairs were microinjected with buffered-CF. Cytoplasmic streaming was then inhibited by addition of KCN or NaN₃ to the external bathing solution. In separate experiments, cytoplasmic streaming was inhibited by microinjection of cytochalasin D along with the buffered-CF. CF passage over a 5 minutes treatment period was monitored by video fluorescence microscopy and video intensity analysis. Cytoplasmic streaming ceased within 1 minute of inhibitor agent treatment, however, little change in the kinetics of intercellular passage was noted over the 5 minute experimental period. Th us, cytoplasmic streaming plays no major role in the regulation of intercellular passage of the hydrophilic, negatively charged molecule CF.The work is dedicated to professor Saal Zalik, Department of Plant Science, University of Alberta, on his 65th birthday.     

Visualization of a rapid,red/far-red light-dependent reaction by centrifuge microscopy

Summary Using a centrifuge microscope with stroboscopic illumination, we examined the effects of light irradiation on the passive movement of chloroplasts in dark-adapted mesophyll cells ofVallisneria gigantea. While irradiation with red light accelerates the passive gliding of chloroplasts produced by centrifugal force, irradiation with far-red light negates this effect. Irradiation with blue light does not accelerate the passive gliding, while red light is completely effective even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis. An apparently active movement of chloroplasts can be induced by irradiation with red or blue light only in the presence of the far-red light-absorbing form of phytochrome. The significance of the reaction in the light with respect to the regulation of cytoplasmic streaming is discussed.Abbreviations APW artificial pond water - CMS centrifuge microscope of the stroboscopic type - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr phytochrome, far-red light-absorbing form - Pr phytochrome, red light-absorbing form     

Motive force and rate of cytoplasmic streaming inCharaceae cells in relation to osmotic pressure and ionic strength of the vacuole and the cytoplasm

Summary The effect of osmolarity of the vacuolar sap ofChara australis on cytoplasmic streaming was analyzed using the vacuolar perfusion technique. The osmolarity was varied between 0.3 M, which is normal and 1.2 M. The streaming rate decreased markedly with an increase in sap osmolarity, while the motive force increased significantly. This may be explained in terms of an increase in the sliding resistance at the sol-gel interface where active shearing occurs. Increase in the resistance is assumed to be caused by osmotic dehydration of the cytoplasm. This assumption was verified by the fact that in tonoplast-free cells, no significant inhibition of the streaming was observed by heightening the osmolarity of the cytoplasm with sorbitol. Heightening it with K⁺ salts inhibited the streaming to a greater extent than with sorbitol. The inhibition differed according to the anion species. Potassium methanesulfonate at 0.3 M and KCl at 0.6 M stopped the streaming almost completely, while 0.59 M K₂SO₄ was less inhibitory. Actin filaments were observed even in the presence of 0.6 M KCl.