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     

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.     

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.     

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 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     

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     

Reversible inhibition of cytoplasmic streaming by intracellular Ca2+ in tonoplast-free cells ofChara australis

Summary The effect of the intracellular concentration of Ca²⁺ on the cytoplasmic streaming of tonoplast-free cells ofChara australis was studied by intracellular perfusion. The perfusion media contained 1 mM Mg · ATP. Both cell ends were cut and left open. Media of different Ca²⁺ concentrations were perfused through the cell and the rate of the cytoplasmic streaming just after perfusion was measured. The critical concentration of Ca²⁺ for inhibiting the streaming was 5 × 10^–4M, which was substantially higher than that found earlier byWilliamson (1975) andHayama et al. (1979). Recovery from the inhibition occurred, though not completely, by removing Ca²⁺.In tonoplast-free cells the Ca²⁺ sensitivity differed according to the culture conditions. Cells cultured indoors exhibited a higher sensitivity than those cultured outdoors. Theformer cells contained granule-rich endoplasm aggregates after loss of the tonoplast, while the latter cells did no such aggregates. The aggregates were fixed to the cortical gel with a high dosage of Ca²⁺ and freed by removing it.     

Gravity-dependent polarity of cytoplasmic streaming inNitellopsis

Summary The internodal cells of the characean algaNitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downwardstreaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR<1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K⁺ channels. The K⁺ channel blocker TEA Cl^– inhibits the NR effect.External Ca²⁺ is required for normal graviresponsivness. The [Ca²⁺] of the medium determines the polarity of cytoplasmic streaming. Less than 1 M Ca²⁺ resulted in a PR<1 while greater than 1 M Ca²⁺ resulted in the normal gravity response. The voltage-dependent Ca²⁺ -channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl₃ resulted in a PR<1, indicating the presence of two types of Ca²⁺ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR ofNitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.     

Analysis of temperature dependence of cytoplasmic streaming using tonoplast-free cells of Characeae

Summary The temperature dependence of cytoplasmic streaming in intact and tonoplast-free cells ofNitellopsis obtusa was studied using a cryomicroscope. The streaming velocity decreases linearly with decrease in the temperature in well-buffered tonoplast-free cells but non-linearly in some intact cells. These results suggest that low temperature causes a disturbance in the homeostasis of calcium and protons, which inhibit cytoplasmic streaming in intact cells.Abbreviations ADP adenosine 5-diphosphate - APW artificial pond water - ATP adenosine 5-triphosphate - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N-tetraacetic acid - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - PIPES piperazine-N, N-bis(2-ethanesulfonic acid) - Tris tris(hydroxymethyl)aminoethane     

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⁺.     

Physarum myosin light chain one: A potential regulatory factor in cytoplasmic streaming

Summary Physarum myosin is composed of a heavy chain of about 225,000 daltons and two small polypeptides of 17,700 and 16,100 daltons, called light chain one (LC 1) and two (LC 2). Light chain one is shown to belong to the general class of regulating light chains by two independent criteria. After denaturation, purification and renaturation of thePhysarum light chains only LC 1 will combine with scallop myofibrils in which one myosin regulatory light chain has been removed. This LC 1 can restore inhibition of the ATPase activity of the myofibrils at 10^–8 M Ca⁺⁺ just as well as light chains from rabbit skeletal myosin. Secondly, this LC 1 is the only component of the myosin that is significantly phosphorylated by an endogenous kinase present in crude actomyosin. An active phosphatase is also present. Preliminary results could not detect calcium sensitivity for either kinase or phosphatase, nevertheless the importance of phosphorylation in affecting activity of biological systems suggests that LC 1 may serve some regulating function for plasmodial actomyosin.     

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.     

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     

Protease-sensitive anchoring of microfilament bundles provides tracks for cytoplasmic streaming inVallisneria

Summary In mesophyll cells of the aquatic angiospermVallisneria gigantea Graebner, the endoplasm streams rotationally along the cell walls normal to the leaf surface in situ. Bundles of microfilaments anchored in the ectoplasm serve as tracks for the cytoplasmic streaming. In single mesophyll cells isolated by enzymatic digestion, hypertonic treatment induces abnormal streaming concomitant with plasmolysis, specifically at one or both of the shorter sides of an approximate rectangle. The disorderly arrangement of microfilaments in such cells has been confirmed by fluorescence microscopy of cells stained with FITC-phalloidin. While inhibitors of proteases added to the enzyme solution used for isolation of cells suppress the disturbance of rotational streaming, exogenously applied protease promotes it. The results suggest that bundles of microfilaments in the ectoplasm are stabilized by protease-sensitive factor(s) in the presence of the cell wall.     

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.     

The endogenous difference in the rates of acropetal and basipetal cytoplasmic streaming inChara rhizoids is enhanced by gravity

Summary Measurements of cytoplasmic streaming inChara rhizoids were made by a streak-photography method combined with dark-field illumination. The velocity of cytoplasmic streaming in the acropetal direction was faster than in the basipetal direction. The difference in the streaming velocities in both morphological directions was apparently due to endogenous forces. In addition to this, a small difference attributable to gravity was superimposed if the rhizoid was oriented parallel to the gravity vector. The difference in the endogenous forces underlying the oppositely directed streams may be as high as about 12-fold the force imposed by gravity but, on average, it is about 5-fold the gravity force. In the normal vertical position of the rhizoid, this endogenously generated difference is enhanced by the gravity effect. In contrast to the variability of streaming rate due to endogenous forces, the effect of the gravity force is relatively uniform. The difference between acropetal and basipetal streaming velocities is perpetuated over the whole range of lowered velocities after treatment with cytochalasin B. After prolonged incubation in cytochalasin B, the basipetal streaming stops earlier than the acropetal streaming. A difference in the length of filaments on both sides of the streaming machinery in rhizoids is proposed as the structural basis for the difference in velocities.     

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.     

Phosphorylation-dephosphorylation is involved in Ca2+-controlled cytoplasmic streaming of characean cells

Summary The mechanism of Ca²⁺ regulation of the cytoplasmic streaming in characean cells was studied in relation to protein phosphorylation and dephosphorylation. A tonoplast-free cell model was developed which was sensitive to Ca²⁺. Protein phosphatase-1 and its inhibitor-1 were applied into the tonoplast-free cells. A synthetic inhibitor of protein phosphatase, -naphthylphosphate, was applied either to tonoplast-free cells from inside or to the outside of plasmalemma-permeabilized cells which are known to be very sensitive to Ca²⁺. ATP--S applied to permeabilized cells strongly inhibited the recovery of the streaming which had been stopped by 10 M Ca²⁺. Both inhibitor-l and -naphthylphosphate inhibited the streaming even in the absence of Ca²⁺. On the other hand, protein phosphatase-l recovered the streaming even in the presence of Ca²⁺.The results indicate that characean streaming is regulated by the phosphorylation state of a regulatory and/or motile protein component. Streaming is activated when the component is dephosphorylated and inactivated when the component is phosphorylated. Ca²⁺ is assumed to stimulate both phosphorylation and dephosphorylation of the component. Involvement of Ca²⁺/calmodulin in the streaming recovery was discussed in terms of the stimulation of dephosphorylation.Abbreviations ATP -S, Adenosine-5-O-(3-thiotriphosphate) - -NP -naphthylphosphate - EGTA ethylenglycol-bis-(-aminoethylether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

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 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