Arrest of cytoplasmic streaming induces algal proliferation in green paramecia

A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.     

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     

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.     

Protoplasmic streaming inAcetabularia

Summary A compilation of characteristics of the two different systems of intracellular transport inAcetabularia (Koop andKiermayer 1980 a and b) is given.The presence of microfilaments-presumably F-actin-in the cytoplasm ofAcetabularia is demonstrated by electron microscopy.The evidence for an involvement of microtubules in streaming is strengthened by the induction of birefringent vinblastine crystals in the stalk of vegetative cells.Isolated portions of cytoplasm formin vitro more than 100 m long filopodium-like processes, which are highly birefringent. The processes show intensive immunofluorescent staining with both, anti-actin and anti-tubulin as a primary antibody.A perfusion buffer is presented, which after replacing the vacuolar sap does not lead to a change in cytoplasmic morphology or streaming pattern and velocities.     

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)     

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.     

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.     

NaCl-induced changes in protoplasmic characteristics of Hordeum vulgare cultivars differing in salt tolerance

Water permeability and cytoplasmic viscosity and streaming were investigated in seedlings of two Hordeum vulgare cultivars differing in salt tolerance. Six-day-old seedlings were grown for 4 additional days in Hoagland solution with and without 100 m M NaCl added.
Observations and measurements were made in subepidermal cells of the coleoptile using plasmolytic and centrifugation methods and recordings of the speed of movement of microsomes.
Water permeability was about the same in controls of both cultivars, and was decreased by NaCl stress, but decreased less in the tolerant cultivar. Cells from control plants of the stress tolerant variety had a higher cytoplasmic viscosity than cells from the moderately sensitive cultivar. Cytoplasmic viscosity in both cultivars decreased due to NaCl stress, and more so in the sensitive one. Cytoplasmic streaming was faster in the controls of the salt sensitive cultivar than in controls of the salt tolerant cultivar; NaCl had no significant effect on cytoplasmic streaming in both cultivars.
The specific responses of the cytoplasm of the sensitive and tolerant cultivars to the salt treatment reflect differences in its structure and composition. These differences in the cytoplasm already exist before exposure to salt stress but some alterations of cytoplasmic parameters (e.g. water permeability) were induced by the saline environment.     

The role of cytoplasmic streaming in symplastic transport

The distributing of materials throughout a symplastic domain must involve at least two classes of transport steps: plasmodesmatal and cytoplasmic. To underpin the latter, the most obvious candidate mechanisms are cytoplasmic streaming and diffusion. The thesis will be here advanced that, although both candidates clearly do transport cytoplasmic entities, the cytoplasmic streaming per se is not of primary importance in symplastic transport but that its underlying molecular motor activity (of which the streaming is a readily visible consequence) is. Following brief tutorials on low Reynolds number flow, diffusion, and targeted intracytoplasmic transport, the hypothesis is broached that macromolecular and vesicular transport along actin trackways is both the cause of visible streaming and the essential metabolically driven cytoplasmic step in symplastic transport. The concluding discussion highlights four underdeveloped aspects of the active cytoplasmic step: (i) visualization of the real‐time transport of messages and metabolites; (ii) enumeration of the entities trafficked; (iii) elucidation of the routing of the messages and metabolites within the cytoplasm; and (iv) transference of the trafficked entities from cytoplasm into plasmodesmata.     

Ultrastructure of wheat seedling mitochondria under anoxia and postanoxia

Summary To characterize the cytoplasmic streaming in characean cells, tonoplast-free cells were prepared and the chemical composition of the cytoplasm was modified by intracellular perfusion. Perfusion media containing sulfate inhibited the cytoplasmic streaming. The extent of inhibition was dependent on the concentration of ATP: the lower the concentration of ATP, the stronger the inhibition. It was suggested that inhibition by sulfate is competitive with respect to ATP. The effects of other anions were also examined.Abbreviations ATP adenosine-5-triphosphate - [ATP] ATP concentration - ADP adenosine-5-diphosphate - EGTA ethylene glycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MES 2-(N-morforino) ethanesulfonic acid - PEP phosphoenol pyruvate - Tris tris(hydroxymethyl) aminomethane - v velocity of cytoplasmic streaming     

Entamoeba Motility: Dynamics of Cytoplasmic Streaming, Locomotion and Translocation of Surface-Bound Particles, and Organization of the Actin Cytoskeleton in Entamoeba invadens

ABSTRACT. The dynamics of cytoplasmic streaming, retrograde translocation of externally bound particles and locomotion by Entamoeba invadens were compared. Locomoting amoebae were monopodial, exhibited fountain flow cytoplasmic streaming and translocated externally bound erythrocytes to the rear of cells. The rates of rearward flow of peripheral cytoplasmic vacuoles and of the externally bound particles were equal to the rate of cell forward locomotion. Rhodamine-phalloidin staining revealed a distinct cortical polymerized actin cytoskeleton. This was least evident about the periphery of the advancing pseudopod, increased in density toward the rear of the cell and was most concentrated in the uroid. A monoclonal anti-eucaryotic actin antibody, which recognized monomeric Entamoeba actin on immunoblots, stained trophozoites by indirect immunofluorescence throughout the cytoplasm, but not in the cortical regions stained by rhodamine-phalloidin. This and other evidence implied that the antibody recognized only unpolymerized actin in Entamoeba . We propose that locomotion, cytoplasmic streaming and translocation of externally bound particles are driven by a common actin-based mechanism in Entamoeba , possibly involving retrograde cortical actin flow and recycling.     

Entamoeba motility: dynamics of cytoplasmic streaming, locomotion and translocation of surface-bound particles, and organization of the actin cytoskeleton in Entamoeba invadens.

The dynamics of cytoplasmic streaming, retrograde translocation of externally bound particles and locomotion by Entamoeba invadens were compared. Locomoting amoebae were monopodial, exhibited fountain flow cytoplasmic streaming and translocated externally bound erythrocytes to the rear of cells. The rates of rearward flow of peripheral cytoplasmic vacuoles and of the externally bound particles were equal to the rate of cell forward locomotion. Rhodamine-phalloidin staining revealed a distinct cortical polymerized actin cytoskelton. This was least evident about the periphery of the advancing pseudopod, increased in density toward the rear of the cell and was most concentrated in the uroid. A monoclonal anti-eucaryotic actin antibody, which recognized monomeric Entamoeba actin on immunoblots, stained trophozoites by indirect immunofluorescence throughout the cytoplasm, but not in the cortical regions stained by rhodamine-phalloidin. This and other evidence implied that the antibody recognized only unpolymerized actin in Entamoeba. We propose that locomotion, cytoplasmic streaming and translocation of externally bound particles are driven by a common actin-based mechanism in Entamoeba, possibly involving retrograde cortical actin flow and recycling.     

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.     

Extranuclear DNA and the Endosymbionts of <Emphasis Type="Italic">Paramecium aurelia</Emphasis>

THE basis of cytoplasmic inheritance in the killer system of Paramecium aurelia has been located to endosymbionts* in the cytoplasm. Breeding experiments have shown the maintenance and replication of some of the endosymbionts in their cellular environment to depend on nuclear genes of the Paramecium host cell¹. There are indications of a specific adaptation between the endosymbionts and certain strains or syngens (≡sibling species)².     

Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.     

Actin cytoskeleton is responsible for the change of cytoplasmic organization in root hair cells induced by a protein phosphatase inhibitor, calyculin A

Summary In root hair cells ofLimnobium stoloniferum, a protein phosphatase inhibitor, calyculin A (CA), at concentrations higher than 50 nM inhibits cytoplasmic streaming and induces remarkable morphological changes in the cytoplasm: the transvacuolar strands disperse and spherical cytoplasmic bodies emerge. The mechanism of the morphological changes of the cytoplasm induced by CA was studied by pharmacological analyses. The formation of spherical bodies in cells treated with CA was suppressed by the actin-depolymerizing and -fragmenting drugs latrunculin B and cytochalasin D at concentrations higher than 100 nM and 5 M, respectively. In contrast, 100 M propyzamide, a microtubule-depolymerizing drug, did not affect the formation of spherical bodies by CA. Interestingly, 60 mM 2,3-butanedione monoxime, an inhibitor of myosin, also suppressed the CA-induced formation of cytoplasmic spherical bodies. These results indicate that the actin cytoskeleton is intimately involved in the morphological changes of the cytoplasm induced by CA.Abbreviations APW artificial pond water - BDM 2,3-butanedione monoxime - CD cytochalasin D - DMSO dimethylsulfoxide - LB latrunculin B - Pro propyzamide     

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.     

Movement of vesicles in cytoplasmic streaming in plasmodium

The moving velocities of vesicles in the cytoplasmic streaming of a slime mold were measured, in which all of the vesicles passing through a designated window were counted. Vesicles in the streaming are distributed in their moving velocities and the distribution itself varies with time. The mean velocity of vesicles and its standard deviation were found to exhibit a linear relationship, suggesting a possibility that vesicles in the cytoplasm would also be involved in force generation.     

Influence of minerals on cytoplasmic streaming in root hairs of intact wheat seedlings (Triticum aestivum L.)

The calcium dependency of the cytoplasmic streaming of wheat root hairs was demonstrated by adding the Ca-Ionophore A 23187. Within three minutes the streaming velocity was decreased dramactically. The influence of ammonium on the cytoplasmic streaming is highly pH-dependent. While at a pH of 9.0 an inhibitory effect was observed even at low ammonium concentrations (0.5 mM) no effect could be measured at a pH of 6.5. Nitrate, independently of medium pH had no effect on the cytoplasmic streaming. The same is true for aluminium. It is suggested that at pH 9 ammonium permiates the plasmalemma as NH₃. Due to higher cytoplasmic pH ( 7.5), NH₃ is protonated leading to an increase in cytoplasmic pH. Ammonium may displace sorbed calcium leading to an increase in the free cytoplasmic calcium responsible for the cessation of the streaming. Alternative explanations are discussed.Abbreviations HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid