Cytoplasmic streaming in internodal cells ofNitella under centrifugal acceleration: a study done with a newly constructed centrifuge microscope

Summary We constructed a new centrifuge microscope of the stroboscopic type, with which the cytoplasmic streaming inNitella internodal cells under centrifugal acceleration was studied. Under moderate centrifugal acceleration (ca. 50–100×g), the direction of cytoplasmic streaming in an internodal cell ofNitella is parallel to the direction of the subcortical fibrils. The speed of endoplasm flowing contiguous to the subcortical fibrils is neither accelerated nor retarded by moderate centrifugal acceleration. The endoplasmic flow, however, stops suddenly following an electrical stimulus. The endoplasm contiguous to the subcortical fibrils is immobilized transiently at the time of streaming cessation induced by an electrical stimulus under centrifugal acceleration at 50–100×g, even at 900×g. It is suggested that transitory cross bridges between the immobilized endoplasm and the subcortical fibrils are formed at the time of streaming cessation. The bulk endoplasm flows as a whole in the direction parallel to that of the subcortical fibrils and stops promptly upon electrical stimulation. Soon after the stoppage the bulk endoplasm starts to flow passively in the direction parallel to that of the centrifugal acceleration as a result of the centrifugal force.Abbreviations APW artificial pond water - CMS centrifuge microscope     

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⁺.     

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.     

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.     

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     

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     

The differential effects of cytochalasin B on microfilament populations and cytoplasmic streaming

Summary Two distinguishable populations of microfilaments (mfs) can be identified in the radish root hair. Bundles of mfs are found throughout the cytoplasm, excluding the tip region of the hair. Single mfs occur only as a cortical array, specifically associated with the microtubules. Both mf populations are oriented parallel to the direction of streaming. Hairs grown in 5 g/ml cytochalasin B (CB) exhibit site-specific differential responses to the drug in both their streaming pattern and sensitivity of their mfs. Cytochalasin B elicits the following responses: 1. cytoplasmic streaming is reduced in all regions of the hair; 2. small particles (<1 m in diameter) still stream, whereas large particles (>1 m in diameter) no longer stream but exhibit an oscillatory or rotational motion; 3. filament bundles show increasing sensitivity to CB along the length of the hair; 4. single mfs show decreasing sensitivity to CB along the hair length. The effects of CB on cytoplasmic streaming can be related to its effects on both mf populations, thus suggesting that although mf bundles are probably involved in streaming in the sub apical and basal regions of the hair, single mfs are most likely involved in generating the slower, more irregular streaming patterns exhibited in the hair tip and CB-treated hair base.     

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.     

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     

Differential treatment ofAcetabularia with cytochalasin B and N-Ethylmaleimide with special reference to their effects on cytoplasmic streaming

Summary Cytoplasmic streaming in the stalk ofAcetabularia, ryukyuensis at the vegetative stage was reversibly inhibited by cytochalasin B (cB) of 50 g/ml and irreversibly by N-Ethylmaleimide (NEM) above concentrations of 0.25 mM.After the endoplasm and the chloroplasts were pushed forward one end of the stalk by gentle centrifugation at about 500 × g for 3 minutes, numerous ectoplasmic striations remainedin situ in the stalk cortex. The striations ran in parallel with the longitudinal axis of the stalk at unequal intervals. The endoplasm streamed back only along these striations.By combining centrifugation and a double chamber technique, the endoplasm and the cortex of the stalk were treated separately with CB or NEM. CB treatment of the cortex arrested streaming; when treatment was restricted to the endoplasm, streaming continued at an normal rate. NEM treatment restricted to the cortex permitted normal streaming rates. Treatment restricted to the moving endoplasm inhibited streaming.These results suggest that microfilaments and a moiety, possibly myosin, play an active role in the streaming. Microfilaments must reside in the cortex, especially in the ectoplasmic striations, while the putative myosin must reside in the moving endoplasm.     

Cytoplasmic streaming in the root cortex and its role in the delivery of potassium to the shoot

The influence of cytochalasin B (CB), a potent inhibitor of cytoplasmic streaming, on ⁸⁶Rb-labelled K⁺ translocation by detopped Lycopersicon esculentum Mill., Cucumis sativus L. and Zea mays L. plants was examined by measuring the radioactivity in xylem exudate before and after the addition of CB to the medium bathing the roots. CB caused complete cessation of cytoplasmic streaming in root segments within 15 min but was without effect on either total ⁸⁶Rb uptake or exudation. Thus factors other than cytoplasmic streaming limit the movement of K⁺ across the symplast of the root of higher plants.     

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.     

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     

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     

Roles of actin filaments in cytoplasmic streaming and organization of transvacuolar strands in root hair cells ofHydrocharis

Summary Effects of cytochalasin B and mycalolide-B on cytoplasmic streaming, organizations of actin filaments and the transvacuolar strand were studied in root hair cells ofHydrocharis, which shows reverse fountain streaming. Both toxins inhibited cytoplasmic streaming and destroyed the organizations of actin filaments and transvacuolar strands. However, we found a great difference between these toxins with respect to reversibility. The effects of cytochalasin B were reversible but not those of mycalolide B. The present results suggest that actin filaments work as a track of cytoplasmic streaming and as a cytoskeleton to maintain the transvacuolar strand. The usefulness of root hair cells ofHydrocharis in studying the dynamic organization of actin filaments of plant is discussed.Abbreviations CB cytochalasin B - DMSO dimethylsulfoxide - ML-B mycalolide B     

Two separate zones of helicoidally orientated microfibrils are present in the walls ofNitella internodes during growth

Summary The dynamic changes in microfibril architecture in the internode cell walls of the giant unicellular algaNitella translucens were studied during cell expansion. Thin section electron microscopy in conjunction with mild matrix polysaccharide extraction techniques revealed three distinct architectural zones in the walls of fully grown cells. These zones were related to distinct phases of growth by monitoring changes in cell wall architecture of internodes during active cell expansion. The initial microfibril deposition before the onset of active cell growth is helicoidal. A helicoid is a structurally complex but ordered arrangement of microfibrils that has been detected increasingly often in higher plant cell walls. During active cell elongation microfibrils are deposited transversely to the direction of cell elongation as shown in earlier studies by birefringence measurements in the polarizing microscope. The gradual decline in cell elongation corresponds with a final helicoidal deposition which continues after cell expansion ceases entirely.The continual presence of the initial helicoidal zone in the outer wall region during the whole growth process suggests that these microfibrils do not experience strain reorientation and are continually reorganized, or maintained, in a well ordered helicoidal arrangement.     

The sliding theory of cytoplasmic streaming: fifty years of progress

Fifty years ago, an important paper appeared in Botanical Magazine Tokyo. Kamiya and Kuroda proposed a sliding theory for the mechanism of cytoplasmic streaming. This pioneering study laid the basis for elucidation of the molecular mechanism of cytoplasmic streaming—the motive force is generated by the sliding of myosin XI associated with organelles along actin filaments, using the hydrolysis energy of ATP. The role of the actin–myosin system in various plant cell functions is becoming evident. The present article reviews progress in studies on cytoplasmic streaming over the past 50 years.     

Detection of gravity-induced polarity of cytoplasmic streaming inChara

Summary Gravity induces a polarity of cytoplasmic streaming in vertically-oriented internodal cells of characean algae. The motive force that powers cytoplasmic streaming is generated at the ectoplasmic/endoplasmic interface. The velocity of streaming, which is about 100 m/s at this interface, decreases with distance from the interface on either side of the cell to 0 m/s near the middle. Therefore, when discussing streaming velocity it is necessary to specify the tangential plane through the cell in which streaming is being measured. This is easily done with a moderate resolution light microscope (which has a lateral resolution of 0.6 m and a depth of field of 1.4 m), but is obscured when using any low resolution technique, such as low magnification light microscopy or laser Doppler spectroscopy. In addition, the effect of gravity on the polarity of cytoplasmic streaming declines with increasing physiological age of isolated cells. Using a classical mechanical analysis, we show that the effect of gravity on the polarity of cytoplasmic streaming cannot result from the effect of gravity acting directly on individual cytoplasmic particles. We suggest that gravity may best be perceived by the entire cell at the plasma membrane-extracellular matrix junction.