The Effect of ATP (Disodium Salt) upon Rotational Streaming

Through the continuous treatment with various solutions of ATP disodium salt the rotational streaming of the cytoplasma in barley root hairs has been stimulated about 1.2–1.7 times. With the concentrations employed the stimulation of the streaming did not depend on the external ATP supply, but on the initial rate of streaming. It is assumed that the main source of energy supporting the protoplasmic streaming is ATP. Therefore, the results obtained may be interpreted on the basis of variations in ATP content and its degradation products. The differences between initial rates of streaming are supposed to be due to variations of the endogenous ATP level. The ATP taken up probably stimulates the rotational streaming both through the supply of delivered energy and by lowering the cytoplasm viscosity. On the contrary, products of ATP hydrolysis increase the cytoplasm viscosity and induce a lowering or even cessation of the streaming.     

Intracellular Mobilization of Ca2+ and Inhibition of Cytoplasmic Streaming Induced by Transcellular Osmosis in Internodal Cells of Nitella flexilis

When transcellular osmosis was induced in internodal cells ofNitella flexilis that had been rendered inexcitable by treatmentwith KCl or EGTA, the rate of cytoplasmic streaming was reducedand the membrane was depolarized. In both KCl- and EGTA-treatedcells, the endoosmosis induced a significant increase in theconcentration of Ca²⁺ in the cytoplasm, which was demonstratedby monitoring the emission of light from aequorin that had beeninjected into the cytoplasm. When transcellular osmosis was induced in tonoplast-free cells,in which the intracellular Ca²⁺ concentration had been stabilizedat a very low level by treatment with the Ca²⁺-chelating agentEGTA, no change in the rate of cytoplasmic streaming on theendoosmosis side was observed. Hydration of the cytoplasm in the absence of endoosmosis wasinduced by direct introduction of a hypotonic medium into thevacuole by intracellular perfusion. The results mimicked theinhibition of streaming induced by transcellular osmosis. During transcellular osmosis, chloroplasts on the endoosmosisside swelled as a result of dilution of the cell sap. Swellingof chloroplasts was demonstrated to be unrelated to the inhibitionof streaming, since streaming was retarded at sites from whichchloroplasts had been removed. It is suggested that hydration of the cytoplasm triggers themobilization of Ca²⁺ from internal stores and causes an increasein the level of cytoplasmic Ca²⁺ that is responsible for theinhibition of streaming. Possible mechanisms for the osmosis-inducedincreases in the level of Ca²⁺ in the cytoplasm are discussed. (Received January 11, 1993; Accepted November 8, 1993)     

The Relationship between myo-Inositol, ATP and Rotational Streaming

The rotational streaming of cytoplasm in barley root hairs has been stimulated about 1.3–1.8 times through continuous treatment with various solutions of myo-inositol. The stimulation attained the same level as after ATP administration and was dependent on the external myo-inositol supply with the employed concentrations. The stimulation was cut off by simultaneous treatment with myo-inositol and uranyl salts. By using uranyl acetate the rate of streaming was maintained about the value of the control. The uranyl chloride caused an inhibition in the rotational streaming and later made it to cease altogether. The simultaneous treatment with myo-inositol and 2.4-DNP (dinitrophenol) induced a quick inhibition in 60% of the root hairs. consecutively stopping rotational streaming. It is assumed that the stimulation of rotational streaming is not due to the direct effect of myo-inositol but to ATP formed in the reaction: inositol hexaphosphate ++ ADP ? ATP + inositol. According to the results obtained by testing with uranyl salts, the phosphorylation of inositol probably takes place at the cell surface. The effect of 2,4-nNP points to the presence of two competitive metabolic processes involved in ATP consumption: the upkeep of rotational streaming and the uptake of substances by the plant cell.     

Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes

Mass movements of cytoplasm, known as cytoplasmic streaming, occur in some large eukaryotic cells. In Drosophila oocytes there are two forms of microtubule-based streaming. Slow, poorly ordered streaming occurs during stages 8-10A, while pattern formation determinants such as oskar mRNA are being localized and anchored at specific sites on the cortex. Then fast well-ordered streaming begins during stage 10B, just before nurse cell cytoplasm is dumped into the oocyte. We report that the plus-end-directed microtubule motor kinesin-1 is required for all streaming and is constitutively capable of driving fast streaming. Khc mutations that reduce the velocity of kinesin-1 transport in vitro blocked streaming yet still supported posterior localization of oskar mRNA, suggesting that streaming is not essential for the oskar localization mechanism. Inhibitory antibodies indicated that the minus-end-directed motor dynein is required to prevent premature fast streaming, suggesting that slow streaming is the product of a novel dynein-kinesin competition. As F-actin and some associated proteins are also required to prevent premature fast streaming, our observations support a model in which the actin cytoskeleton triggers the shift from slow to fast streaming by inhibiting dynein. This allows a cooperative self-amplifying loop of plus-end-directed organelle motion and parallel microtubule orientation that drives vigorous streaming currents and thorough mixing of oocyte and nurse-cell cytoplasm.     

Cyclosis-related asymmetry of chloroplast–plasma membrane interactions at the margins of illuminated area in <Emphasis Type="Italic">Chara corallina</Emphasis> cells

Cytoplasmic streaming in plant cells is an effective means of intracellular transport. The cycling of ions and metabolites between the cytosol and chloroplasts in illuminated cell regions may alter the cytoplasm composition, while directional flow of this modified cytoplasm may affect the plasma membrane and chloroplast activities in cell regions residing downstream of the illumination area. The impact of local illumination is predicted to be asymmetric because the cell regions located downstream and upstream in the cytoplasmic flow with respect to illumination area would be exposed to flowing cytoplasm whose solute composition was influenced by photosynthetic or dark metabolism. This hypothesis was checked by measuring H⁺-transporting activity of plasmalemma and chlorophyll fluorescence of chloroplasts in shaded regions of Chara corallina internodal cells near opposite borders of illuminated region (white light, beam width 2 mm). Both the apoplastic pH and chlorophyll fluorescence, recorded in shade regions at equal distances from illuminated area, exhibited asymmetric light-on responses depending on orientation of cytoplasmic streaming at the light–shade boundary. In the region where the cytoplasm flowed from illuminated area to the measurement area, the alkaline zone (a zone with high plasma membrane conductance) was formed within 4-min illumination, whereas no alkaline zone was observed in the area where cytoplasm approached the boundary from darkened regions. The results emphasize significance of cyclosis in lateral distribution of a functionally active intermediate capable of affecting the membrane transport across the plasmalemma, the functional activity of chloroplasts, and pattern formation in the plant cell.     

A Translocation Hypothesis based on the Structure of Plant Cytoplasm

Two types of linear structures have been seen in plant cytoplasm,largely by phase-contrast microscopy. Microscopic fine threads,o.Iµ to Iµ in diameter, were revealed in hair cells,where they formed endoplasmic systems along streaming pathwaysin the parietal cytoplasm and in transvacuolar strands. Duringcirculation streaming, small plastids and mitochondria-likeparticles were observed moving along the fine threads. Similarfine threads, together with small plastids and mitochondria-likeparticles occurred in phloem exudate and transcellular microscopicstrands. The transcellular strands, Iµ to 7µ indiameter, were seen in sieve-tube elements, phloem parenchyma,border parenchyma, and cortical cells. The movement of small plastids across end walls in border-parenchymacells, the appearance of the same structures within strandsin phloem cells, and the undiminished occurrence of small plastidsin successive drops of phloem exudate are collectively takenas evidence for their participation in translocation. Particlemovement is thought to occur through transcellular strands inassociation with fine threads, and to be motivated by a transcellularform of protoplasmic streaming.     

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.     

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.     

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.     

Applications of ultrasound streaming and radiation force in biosensors

Direct radiation force (DRF) and acoustic streaming provide the main influences on the behaviour of particles in aqueous suspension in an ultrasound standing wave (USW). The direct radiation force, which drives suspended particles towards and concentrates them in acoustic pressure node planes, has been applied to rapidly transfer cells in small scale analytical separators. The DRF also significantly increased the sensitivity of latex agglutination test (LAT) by concentrating the particles of an analytical sample in the pressure node positions and hence greatly increasing the antibody-antigen encounter rate. Capture of biotinylated particles and spores on a coated acoustic reflector in a quarter wavelength USW resonator was DRF-enhanced by 70- and 100-fold, respectively compared to the situation in the absence of ultrasound. Acoustic streaming has been successfully employed for mixing small analytical samples. Cavitation micro-streaming substantially enhanced, through mixing, DNA hybridization and the capture efficiency of Escherichia coli K12 on the surface of immunomagnetic beads. Acoustic streaming induced in longitudinal standing wave and flexural plate wave immuno-sensors increased the detection of antigens by a factor of five and three times, respectively. Combined DRF and acoustic streaming effects enhanced the rate of the reaction between suspended mixture of cells and retroviruses. The examples of a biochip and an ultrasonic immuno-sensor implementing the DRF and acoustic streaming effects are also described in the review.     

The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.     

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.     

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; )     

FURTHER OBSERVATIONS ON THE PHENOMENON OF SECONDARY VACUOLATION IN LIVING CELLS

Invagination of the plasma membrane in plant cells forms peripheral or endocytic structures which often contain a complement of membrane-bound vesicles. These structures, or secondary vacuoles, move with the streaming cytoplasm although their velocities are somewhat slower than that for the various organelles within the cytoplasm. They glide over the nucleus or flow from the peripheral cytoplasm onto a transvacuolar strand and continue unabated along the length of a strand. These structures may detach from the plasma membrane as sacs to become positioned in the cytoplasm directly under the tonoplast and project into the primary vacuole. Some endocytic vacuoles may separate from the peripheral cytoplasm and remain free within the primary vacuole; subsequently they can re-associate with the cytoplasm. While the content and function of these vacuoles are yet to be determined, indirect evidence indicates that they are pinocytic in character since the content of an invagination is confined to the sac upon its detachment from the plasma membrane and is subsequently transported throughout the cell by cyclosis.     

不同波段电磁辐射致大鼠睾丸支持细胞的损伤效应

为探讨不同波段电磁辐射对大鼠睾丸支持细胞(Sertoli细胞)损伤效应的异同.将原代培养的Sertoli细胞经场强6×104V/m的电磁脉冲(electromagnetic pulse,EMP)、平均功率密度为100mW/cm2的S-波段高功率微波(S-band high power microwave,S-HPM)和...     

Proton flows across the plasma membrane in microperforated characean internodes: tonoplast injury and involvement of cytoplasmic streaming

Microperforation of characean cell wall with a glass micropipette in the absence of the tonoplast impalement was found to cause rapid alkalinization of the apoplast by 2–3 pH units, which may rigidify the cell wall structure, thus protecting the cell from further injury. A similar but a deeper insertion of a microneedle, associated with piercing the tonoplast and with an action potential generation, led to a considerable delay in the apoplast alkalinization without affecting the amplitude of the eventual increase in pH. The retardation by the mechanically elicited action potential of the incision-mediated pH transients in the apoplast contrasted sharply to the enhancement of these pH transients by the action potential triggered electrically before the microperforation. Hence, the delay of the apoplast alkalinization was not related to basic ionic mechanisms of plant action potentials. Measurements of the vacuolar pH after mechanical elicitation of an action potential indicate that the tonoplast piercing was accompanied by leakage of protons from the vacuole into the cytoplasm, which may strongly acidify the cytoplasm around the wounded area, thus collapsing the driving force for H⁺ influx from the medium into the cytoplasm. The lag period preceding the onset of external alkalinization was found linearly related to the duration of temporal cessation of cytoplasmic streaming. The results suggest that the delayed alkalinization of the apoplast in the region of tonoplast wounding reflects the localized recovery of the proton motive force across the plasmalemma during replacement of the acidic cytoplasm with fresh portions of unimpaired cytoplasm upon restoration of cytoplasmic streaming.     

Possible involvement of protein phosphorylation in the regulation of cytoplasmic organization and streaming in root hair cells as revealed by a protein phosphatase inhibitor, calyculin A

Summary The effects of a protein phosphatase inhibitor, calyculin A (CA), on cytoplasmic streaming and cytoplasmic organization were examined in root hair cells ofLimnobium stoloniferum. CA at concentrations higher than 50 nM inhibited cytoplasmic streaming and also induced remarkable morphological changes in the cytoplasm. The transvacuolar strands, in which actin filament bundles were oriented parallel to the long axis, disappeared and spherical cytoplasmic bodies emerged in the CA-treated cells. In these spherical bodies, actin filaments were present and the spherical bodies were connected to each other by thin strands of actin filaments. Upon CA removal, transvacuolar strands, in which actin filament bundles were aligned, and cytoplasmic streaming reappeared. A nonselective inhibitor for protein kinases, K-252a, delayed the inhibitory effect of CA on cytoplasmic streaming and suppressed the CA-induced formation of the spherical bodies. From these results, it is suggested that phosphatases sensitive to CA regulate cytoplasmic streaming and are involved in the organization of the cytoplasm in root hair cells.Abbreviations APW artificial pond water - CA calyculin A     

PHASE CINEMICROGRAPHIC OBSERVATIONS ON CULTURED CELLS. II. MASS MOVEMENT OF CYTOPLASM IN EUPHORBIA MARGINATA

Mass movement is a form of streaming in which distinct quantities of cytoplasm flow as entities along a transvacuolar strand or cytoplasmic striations of the peripheral cytoplasm. An individual mass can move at variable velocities during a brief period of time or change its direction of flow. Two masses, when moving at different velocities in the same or different directions along a strand, can be observed to collide. This can occur repeatedly, resulting in the formation of a mass of considerable size. Many organelles can be observed to move at velocities differing from that of the mass; some can be observed to change directions during their movement. A mass may represent a dilation of one or more microstreams within the cytoplasm. Folding of the microstreams within a mass may explain the changes in the direction of movement observed for some organelles. Several levels of movement are associated with streaming, including those of the ground plasm, of the organelles, of the transvacuolar strands and of the cytoplasm masses. These, and possibly more subtle aspects of the streaming phenomenon, must be incorporated into any theory of streaming.     

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.