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     

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 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.     

Involvement of Ca2 + and flowing endoplasm in recovery of cytoplasmic streaming after K+-induced cessation

Summary When K⁺ of high concentration (50 mM) was applied toNitella cells, the cytoplasmic streaming stopped instantly as in the case of electrical stimulation. Recovery of the streaming after chemical stimulation was much slower than after electrical stimulation. When the endoplasm content was modified by centrifugation, streaming recovery was accelerated in the centrifugal cell fragments rich in endoplasm and deccelerated in those poor in it. The recovery was also accelerated either by permeabilizing the plasmalemma in the presence of EGTA in the external solution or by removing the tonoplast by vacuolar perfusion with the EGTA-containing medium. We concluded that the streaming was recovered due to decrease of the cytoplasmic Ca²⁺ concentration, which seems to be accelerated by sequestering of Ca²⁺ by endoplasmic components. The slow recovery of the streaming after KCl-stimulated cessation is assumed to be caused by continuous influx of Ca^{2 +} during the prolonged membrane depolarization.Abbreviations ATP adenosine 5-triphosphoric acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

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.     

Primary effect of bromoxynil to induce plant cell death may be cytosol acidification

Bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile, is a commonly used herbicide and is also used as a tool to trigger rapid cell death in basic botany. However, the primary effect inducing cell death is not known. Bromoxynil inhibited the cytoplasmic streaming and killed cells in Chara corallina when it was applied in the acidic external medium. At higher pH, bromoxynil was inert even at high concentrations. It was speculated that bromoxynil in the protonated form enters the cell and acidifies the cytosol by releasing H⁺. Experiments using analogues of bromoxynil supported this possibility. Acidification of the cytosol by bromoxynil was confirmed by experiments using pollen tubes. Based on the acidity of the apoplast, the herbicide action of bromoxynil in higher plants was discussed.     

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     

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.     

Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair 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     

Artificial control of cytoplasmic pH and its bearing on cytoplasmic streaming,electrogenesis and excitability of characeae cells

Effects of changing the cytoplasmic pH on the cytoplasmic streaming, membrane potential and membrane excitability were studied in tonoplast-free cells ofChara australis andNitellopsis obtusa. The cytoplasmic pH was varied by internal perfusion of pH-buffered media.Nitellopsis cells were perfused only once, whileChara cells were perfused twice to control the pH more accurately. In both materials the rate of cytoplasmic streaming was maximum at about pH 7, low at pH 8.5–9 and almost zero at pH 5–5.5. The membrane potential was most negative at about pH 7. InChara the membrane potential supported by Mg·ATP was strongly inhibited at pH 5.5, and almost zero at pH 9, supporting the results obtained by Fujiiet al. (1979) on cells ofChara australis which were perfused once. The action potential could be induced by electrical stimulation inChara at pH 6.0–9.0 and inNitellopsis at pH 6.6–7.9. The membrane resistance ofNitellopsis was high at acidic and neutral pH values and low at alkaline pH, while that ofChara was low at both acidic and alkaline pH values.     

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.     

21-kDa polypeptide,a low-molecular-weight cyclophilin,is released from pollen of higher plants into the extracellular medium in vitro

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca²⁺. This phenomenon was also dependent on pH and on the concentrations of MgCl₂ in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl₂ concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis-trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.Abbreviations CaM Calmodulin - CBB Coomassie-brilliant-blue - CsA Cyclosporin A - CyP Cyclophilin     

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: I. Effects of La3+, verapamil,EGTA, W-7, and TFP on the action potential

Summary The cytoplasmic streaming of the normal internodal cell of giant algaChara stops transiently at about the peak of action potential. Application of La³⁺ or verapamil (a calcium channel blocker) or removal of external Ca²⁺ by EGTA caused a partial depolarization of the resting potential, partial decrease of the membrane conductance and a marked decrease of the amplitude of action potential. Under these conditions, the conductance at the peak of action potential reduced markedly and the streaming of cytoplasm did not cease during action potential (excitation-cessation (EC) uncoupling). The effects of Ca²⁺ channel blockers could not be removed by addition of CaCl₂ to the external medium. In contrast, the effect of EGTA on the excitability could be removed to a greater extent and the cytoplasmic streaming ceased at about the peak of action potential by the addition of Ca²⁺ externally. Application of calmodulin antagonists W-7 or TFP caused similar effects on the action potential and on the cytoplasmic streaming.     

Mechanism of inhibition of cytoplasmic streaming by myrmicacin (Β-hydroxydecanoic acid) inChara andSpirogyra

Summary Myrmicacin (-hydroxydecanoic acid) applied externally inhibited the cytoplasmic streaming ofChara cells in acidic (pH 4.5) but not in neutral (pH 7.5) solution. Application by intracellular perfusion at neutral pH also did not inhibit the streaming. Other carboxylic acids could inhibit the streaming when given as an acidic external solution. Inhibition of the cytoplasmic streaming was concluded to occur due to acidification of the intracellular pH upon application of an acidic external medium of these carboxylic acids. The cytoplasmic streaming ofSpirogyra was also inhibited by myrmicacin and acetic acid in acidic solution.Abbreviations APW artificial pond water - CB cytochalasin B - DMGA 3.3-dimethylglutaric acid - DMSO dimethylsulfoxide - EGTA ethyleneglycol-bis-(\-aminoethyl ether)N - N tetraacetic acid - HEPES 2-hydroxyethylpiperazine - N ethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - pH _i intracellular pH - Tris trishydroxyaminomethane     

Studies on the tonoplast action potential ofNitella flexilis

Summary The origins of the two peaks of the action potential inNitella flexilis were analyzed by inserting two microelectrodes. one into the vacuole and the other into the cytoplasm. It was unequivocally demonstrated that the rapid first peak was generated at the plasmalemma and the slow second peak at the tonoplast. MnCl₂ applied in the external medium abolished the second, tonoplast, peak but not the first, plasmalemma, peak, MnCl₂ also inhibited the cessation of the cytoplasmic streaming accompanying the action potential. CaCl₂ added in MnCl₂-containing medium recovered generation of the tonoplast action potential and the streaming cessation. Since it has been established that the cessation of cytoplasmic streaming on membrane excitation is caused by an increase in cytoplasmic free Ca^2– (Williamson, R.E., Ashley, C.C., 1982.Nature (London) 296:647–651: Tominaga, Y., Shimmen, T., Tazawa, M., 1983,Protoplasma 116:75–77), it is suggested that the tonoplast action potential is also induced by an increase in cytoplasmic Ca²⁺ resulting from the plasmalemma excitation. When vacuolar Cl was replaced with SO ₄ ² by vacuolar perfusion, the polarity of the second, slow peak was reversed from vacuolar positive to vacuolar negative with respect to the cytoplasm, supporting the previous report that the tonoplast action potential is caused by increase in Cl permeability (Kikuyama, M., Tazawa, M., 1976.J. Membrane Biol.29:95–110).     

p-CMBS Modifies Extrafacial Sulfhydryl Groups at the Chara Plasma Membrane: Activation of Ca2+ Influx and Inhibition of Two Different K+ Currents

The effect of the membrane impermeant sulfhydryl group (SH) reagent, p-chloromercuribenzenesulfonic acid (p-CMBS), on electrical membrane transport properties of the giant alga, Chara corallina, was determined. In an external medium with a high K⁺ concentration (5 mM) cells typically exhibited stable membrane potentials close to the K⁺equilibrium potential. The steady-state current-voltage (I-V) relation could be dissected into two distinct components: an almost linear ohmic leak current and a voltage-dependent K⁺ current. Adding 0.5 mM p-CMBS to the external medium resulted in an immediate, short depolarization transient (resembling the time course of an action potential) and was associated with a slow down of the cytoplasmic streaming velocity. The depolarization, as well as the streaming inhibition, could be abolished by pretreating cells with the Ca²⁺ channel inhibitor, LaCl₃. This suggests that the depolarization transient reflected a p-CMBS induced Ca²⁺ influx, a scenario known to trigger membrane excitation and slow down of cytoplasmic streaming. From the I-V analysis it appeared that p-CMBS also caused a reversible inhibition of two additional transmembrane currents: (1) a reduction of a leak current and (2) a modification of the deactivation kinetics of the voltage-dependent K⁺ channels. From the I-V difference analysis, the inhibited leak current was identified as a K⁺ current, because the reversal potential was close to the estimated K⁺ equilibrium potential. Control experiments have furthermore shown that the mercapto reagent, dithiothreitol, partly reversed the effect of p-CMBS. This strengthens the view that the action of the mercurial is related to a specific and direct modification of SH groups. The p-CMBS-evoked inhibition of K⁺ currents was not abolished by the LaCl₃ pretreatment, which suggests that the effect of the SH reagent is not induced indirectly by p-CMBS-triggered Ca²⁺ influx. Therefore, it is suggested that the mercurial interacts direcly with the K⁺ transport protein.     

Manipulating cytoplasmic pH under anoxia: A critical test of the role of pH in the switch from aerobic to anaerobic metabolism

Ethanol production by maize (Zea mays L.) root tips, measured by an enzymic assay of the suspending medium, was correlated with changes in the cytoplasmic pH, determined by in-vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy, following the onset of anoxia. Strong evidence for the role of the cytoplasmic pH in triggering the switch to ethanol production under anoxia was obtained by: (i) varying the pH of the suspending medium between pH 4 and pH 10; and (ii) using the permeant weak base methylamine to combat the acidification of the cytoplasm induced by the anoxic conditions. Experimentally, it proved to be much easier to manipulate the cytoplasmic pH under anoxia after the pH had stabilised, rather than during the initial rapid acidification that occurred following the onset of anoxia, and in the presence of methylamine, it was possible to impose a normal aerobic cytoplasmic pH value on tissue that was metabolising anaerobically. By this means it was possible to demonstrate the reversibility of the pH effect on ethanol production under anoxia and thus to provide good evidence in support of the biochemical pH-stat model of the anoxic response. The NMR measurement of the cytoplasmic pH in the presence of methylamine was achieved by using a manganese pretreatment technique to eliminate interference between the cytoplasmic and vacuolar P_i signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.Abbreviations Caps 3-(cyclohexylamino)-1-propane sulphonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - NMR nuclear magnetic resonance - Pi inorganic phosphate We acknowledge the financial support of the Agricultural and Food Research Council and G.G.F. acknowledges the receipt of a Research Fellowship from the Royal Commission for the Exhibition of 1851.     

Mitochondrial sequestration of BCECF after ester loading in the giant alga Chara australis

Summary. Ratiometric fluorescent dyes are often used to monitor free ion concentrations in vivo, especially in cells that are recalcitrant to transformation with genetically encoded fluorescent markers. Although intracellular dye distributions are often found to be cytosolic, dye localisation has often not been examined in detail. We began exploring the use of BCECF (2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to monitor pH in the giant alga Chara australis and discovered that younger leaf cells could be loaded using the acetoxymethyl ester of BCECF. However, we were puzzled to find in microphotometric measurements that the fluorescence ratio appeared insensitive to manipulations affecting cytosolic pH. Confocal imaging of C. australis cells loaded with BCECF showed an accumulation of the dye in two locations: (1) on the outside of the chloroplasts in irregularly shaped stationary bodies; (2) within 1–1.5 μm structures that moved rapidly with the pericellular cytoplasmic streaming. Together with the streaming cytoplasm, these organelles were rendered stationary with 50 μM cytochalasin D. Rhodamine 123, a mitochondrionspecific dye, highlighted organelles outside of the chloroplasts, similar to those shown by BCECF in location 1. We conclude that in the cytoplasmic compartment, BCECF was sequestered within cytoplasmic mitochondria in immature and fast-growing cells and within the cortical mitochondrial system in older and slowly growing cells. Thus, BCECF-AM is unsuitable for reporting changes in cytosolic pH in C. australis but might be employed in future to study pH changes in the mitochondria. Correspondence: M. J. Beilby, Biophysics, School of Physics, University of New South Wales, Sydney 2052, Australia.     

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.