A comment on ATPase driven streaming in a reconstituted contractile system

The production of chemo-mechanical effects in a reconstituted system of purified muscle proteins had been studied. It was found that the "streaming" effects observed (by previous workers) in the circular slit of a stream cell were observed under conditions where the absence of artefacts could not be guaranteed. In particular, convective motion motion, due to small temperature gradients is shown to be present, resulting in streaming even in the absence of essential components of the system such as ATP. Temperature inhomogeneities of the order of only 0.1 K . mm-1 are sufficient to cause motion, and any future experiments on streaming must be thermally homogeneous to a greater accuracy than this if valid conclusions are to be drawn.     

The Excystment Process in the Ciliate Nassula ornata Ehrbg.

SYNOPSIS. The ciliate was cultured in natural lake water on Oscillatoria curviceps. Encystment was induced by lack of food. The resting cyst has 2 membranes—a faceted, lamellate ectocyst and a thin endocyst. The kinetosomal rows and the cytopharynx are retained in the cyst, and the position of the cytopharynx with respect to the rows remains unchanged. Thus, the usual polarity of the organism is preserved. Distilled water and plant infusions were relatively ineffective excystment-inducing agents, but a 0.1% (w/v) aqueous solution of peptone, pre-inoculated with wild bacteria, was highly effective. At 21 C it induced the excystment of 97% of the cysts within 2-3.5 hr. During excystment, the contractile vacuole enlarges, thereby producing pressure which ruptures the membranes. The ectocyst tears first—always at the posterior end of the cyst—the endocyst somewhat later. Emergence is largely by cytoplasmic streaming through a relatively small slit in the ectocyst, and the posterior end of the ciliate always emerges first.     

Studies of the chemo-mechanical conversion in artificially produced streamings. I. Reconstruction of a chemo-mechanical system from acto-HMM of rabbit skeletal muscle.

Steady and uniform streamings (SUS) of HMM solutions were set up in the presence of Mg-ATP in a circular slit, on both side-walls of which a Millipore filter was fixed; F-actin filaments from rabbit skeletal muscle were bound onto the Millipore filter by cyanogen bromide in the flow. The direction of the SUS was specificially determined by that of the flow during the fixing of F-actin and was independent of the direction of the initial velocity applied externally to the HMM solutions. The SUS continued for about 90 min with a velocity of about 20 mum/s at 20 degrees C. There was a strong correlation between the acto-HMM ATPase activity and the velocity of SUS when the salt concentration was varied. Moreover, this was also the case when the ATPase activity was controlled by Ca2+, when native tropomyosin was bound to F-actin in the circular slit. Careful examination led to the conclusions that F-actin filaments are fixed on the Millipore filter with a specific polarity and that a chemo-mechanical system had been successfully reconstituted in our "stream cells," in which chemical energy from ATP is converted to the mechanical energy of streaming.     

On the active streaming experiment of actomyosin solutions in glass microcapillaries.

The active-streaming experiments of Oplatka et al. (Oplatka, A. and Tirosh, R. (1973) Biochim. Biophys. Acta 305, 684-688 and Oplatka, A. Gadasi, H., Tirosh, R. Lamed, Y., Muhlrad, A. and Liron, N. (1974) J. Mechanochem. Cell Motil. 2, 295-306) with actomyosin solution in a glass microcapillary is reexamined under various conditions with several kinds of reference material. It is found that vigorous streaming took place in the actomyosin solution as reported by Oplatka et al. However, streaming which is indistinguishable from that observed in the actomyosin solution in the presence of actomyosin ATPase activity also occurred, even when the ATPase activity was blocked. The streaming also cannot be confirmed as being active when using acto-heavy meromyosin solution. There is a possibility that the streaming experiment provides interesting information on the microscopic state of solutions which is not directly related to the chemo-mechanical conversion.     

Generation of the streaming potential by liposomes in cylindrical capillary. Experimental data

A model of creation a streaming potential U as a result of colloidal particle movement in flow in a capillary has been described previously (Zawada 1996) as well as the systems for measurement (Zawada 1990, 1991). The filling of capillary with a solution of liposomes results in a labile adsorbance of liposomes on a capillary glass and changes the measured streaming potential. In order to minimalize these adverse effects, the capillary was covered with phospholipid layer of different composition. Some concentrations of stearylamine as a component of the phospholipid layer may fully compensate the surface charge of the glass capillary and can reduce the liposomes adsorption. The streaming potential of the liposomes solution depends on the ionic strength of the electrolyte and is smaller than the zeta potential for similar liposomes. This suggests that only a part of ions of the liposome ion atmosphere participate in creating of the streaming potential. These are the ions from the hydrodynamic slipping layer. The regression analysis of the relationships between streaming potential U and concentration of liposomes and next ionic strength of the electrolyte gave the value of the surface potential psi0 and the thickness of the hydrodynamic slipping layer d, that is independent of the ionic strength.     

Evidences for direct involvement of microtubules in cleavage furrow formation in newt eggs

This paper aims at examining the effect of colchicine, a microtubular poison, on the process of furrow formation in whole eggs and egg fragments as well as the process of artificial induction of furrow-like dents, in eggs of the newt, Cynops pyrrhogaster. To apply colchicine locally to eggs, the eggs were slit across or along a furrow in a colchicine solution during first cleavage. When a slit was made across or in front of a growing furrow at the onset of its growth, the furrow quickly ceased growing and often regressed. Cortices containing an entire growing furrow were isolated along with a thin layer of subcortical cytoplasm immediately after the start of the first cleavage. Furrows in the cortices degenerated when the cortices were cultured in a colchicine solution, whereas they continued growing when they were cultured in Holtfreter's saline. Furrow-inducing cytoplasm was injected to a site beneath the cortex in the animal half of the egg during first cleavage. When a small slit was made close to the site of the injection in a colchicine solution, no furrow-like dent was induced. These results imply that microtubules are directly involved in the generation and growth of cleavage furrows.     

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.     

Artifical rearrangements of insect ooplasm caused by fixation,and their microkymographic recording

Summary Time-lapse photomicrographs of eggs from the Ichneumonid waspPimpla and from the gall midgeWachtliella reveal that during fixation the ooplasm performs excessive streaming movements when conventional fluids such as Bouin's or Dubosq-Brasil's are used. InWachtliella, intravitelline cleavage may continue for two mitotic cycles during fixation. The cytoplasm of individual egg regions may become displaced up to 45% of the egg's length inPimpla, and up to 14% of the egg's length inWachtliella. Therefore fixed preparations may grossly misrepresent the distribution of egg components in the living state. The changes can be traced back by microkymographic registration of movements during fixation. Microkymograms for this purpose were obtained by projecting a time-lapse film through a slit onto photosensitive material mounted on a rotating drum.The shifting of egg components and the mitotic activity of nuclei fail to occur when the egg is treated with a buffered solution of OsO₄ (1%, 1 min). Subsequent fixation with glutaraldehyde results in satisfactory preservation of microtubules.     

Studies of the chemo-mechanical conversion in artificially produced streamings. III. Dynamic cooperativity--a new cooperativity in actomyosin systems with a polarized arrangement of F-actin.

The order--disorder phase transition in the chemo-mechanical process in our streaming system, which was reported in the preceding paper [Yano, M. & Shimizu, H., J. Biochem, 84, 1087--1092], was studied in detail. Starting from the hypothesis that a new type of cooperativity, dynamic cooperativity, is present in the elementary cycles of the chemo-mechanical conversion, quantitative and consistent agreement was obtained between the theoretical and experimental data on the temperature dependences of the streaming velocity and the ATPase activity, including the presence of the phase transition. The hypothesis was also supported by observations of the temperature dependence of the ATPase activity in resisted streaming. The physical and physiological significance of dynamic cooperativity is considered. It is shown that dynamic cooperativity arises in actomyosin system which have a polarized arrangement of F-actin by directed streaming of the solution, because the rate of the elementary cycle can be accelerated by the streaming. Furthermore, dynamic cooperativity is the key mechanism of self-organization of a dynamical order such that in ordered motions, and is one of the essential conditions for the direct conversion of the chemical energy of ATP to the mechanical energy of ordered and directed motion.     

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.     

THE EFFECT OF AUXINS ON PROTOPLASMIC STREAMING. III

1. A new method is described which gives a continuous record of the absolute rate of protoplasmic streaming in epidermal cells of the Avena coleoptile. 2. With this method a study was made of the influence of malate and iodoacetate on streaming velocity, in order to make correlations with the previously established effects of these substances on growth and respiration. 3. In the presence of optimum concentrations of indole-3-acetic acid in freshly cut sections, malate had no effect on streaming. In the presence of very low concentrations of the auxin, however, malate increased the range of response, so that the threshold of auxin sensitivity was lowered some ten times by the malate. Malate alone had no effect on streaming. 4. In coleoptile sections, soaked overnight in sugar solution or in water, the acceleration of streaming normally caused by auxin almost disappears, but the presence of malate causes large accelerations of streaming by the auxin. 5. Similarly, in sections from old coleoptiles, which no longer show acceleration of streaming by auxin, the acceleration is restored when malate is added together with the auxin. 6. Malate does not enter the cell as rapidly as does auxin, but easily detectable amounts penetrate within 30 minutes. 7. Iodoacetate in the concentration which inhibits growth (5 x 10^–5 M) completely inhibits the acceleration of streaming by auxin. In still lower concentrations iodoacetate slightly accelerates streaming. Higher concentrations, up to 2 x 10^–4 M, did not reduce the rate of streaming below that of controls without auxin. The effect of iodoacetate is therefore to inhibit the acceleration caused by auxin and not to affect the basal streaming rate. 8. It is concluded that, just as for growth and respiration, malate is necessary for the response to auxin shown by acceleration of streaming. This further strengthens the triple parallel between the effects of auxin on streaming, growth, and respiration, all of which are apparently mediated by the 4-carbon acid system.     

The identification of F actin in the pollen tube and protoplast of Amaryllis belladonna

The production of protoplasts from the pollen of Amaryllis belladonna has facilitated a more direct investigation of the physiological and mechano-chemical basis of streaming. Cytoplasm is removed from an actively streaming protoplast or intact pollen tube and layered on a coated grid in a solution containing a low free calcium ion concentration. Under these conditions 6 nm thin filaments are observed. The thin filaments are morphologically identical with F actin and bind rabbit muscle HMM, forming characteristic arrowhead complexes that are dissociated by subsequent treatment with MgATP.     

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     

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.     

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.     

Simple photometric auxanometers of high sensitivity

The elongation phase of growth of plant parts, as 1-cm segments excised from wheat coleoptiles here, is very simply recorded photometrically. One or more aligned segments submerged in aerated buffer pushed an Al foil shutter over a slit of light incident on a photodetector such as a solar or photronic cell, connected directly to a recorder, or a photomultiplier tube in a commercial photometer. Convenient kinetic records were obtained at 5 mm per min chart speed when one segment was combined with a 1-mm long slit, or more segments and longer slits, for which a 1-mm chart unit usually exceeded noise and was equivalent to 4 mum growth per cm coleoptile. In the presence of 10 cm of coleoptile segments no replenishment of solution was necessary during kinetic measurements of response in a 50-ml reservoir of IAA more concentrated than 30 nm.     

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.     

Effect of heat shock on the chilling sensitivity of trichomes and petioles of African violet (Saintpaulia ionantha)

Chilling at 6°C caused an immediate cessation of protoplasmic streaming in trichomes from African violets ( Saintpaulia ionantha ), and a slower aggregation of chloroplasts in the cells. Streaming slowly recovered upon warming to 20°C, reaching fairly stable rates after 4, 15, 25 and 35 min for tissue chilled for 2 min and for 2, 14 and 24 h, respectively. The rate of ion leakage from excised petioles into an isotonic 0.2  M mannitol solution increased after 12 h of chilling and reached a maximum after 3 days of chilling. A heat shock at 45°C for 6 min reduced chilling-induced rates of ion leakage from excised 1-cm petiole segments by over 50%, namely to levels near that from non-chilled control tissue. Heat-shock treatments themselves had no effect on the rate of ion leakage from non-chilled petiole segments. Protoplasmic streaming was stopped by 1 min of heat shock at 45°C, but slowly recovered to normal levels after about 30 min Chloroplasts aggregation was prevented by a 1 or 2 min 45°C heat-shock treatment administered 1.5 h before chilling, but heat-shock treatments up to 6 min only slightly delayed the reduction in protoplasmic streaming caused by chilling. Tradescantia virginiana did not exhibit symptoms associated with chilling injury in sensitive species (i.e. cessation of protoplasmic streaming in stamen hairs and increased ion leakage from leaf tissue).     

VMCast: A VM-Assisted Stability Enhancing Solution for Tree-Based Overlay Multicast

Tree-based overlay multicast is an effective group communication method for media streaming applications. However, a group member’s departure causes all of its descendants to be disconnected from the multicast tree for some time, which results in poor performance. The above problem is difficult to be addressed because overlay multicast tree is intrinsically instable. In this paper, we proposed a novel stability enhancing solution, VMCast, for tree-based overlay multicast. This solution uses two types of on-demand cloud virtual machines (VMs), i.e., multicast VMs (MVMs) and compensation VMs (CVMs). MVMs are used to disseminate the multicast data, whereas CVMs are used to offer streaming compensation. The used VMs in the same cloud datacenter constitute a VM cluster. Each VM cluster is responsible for a service domain (VMSD), and each group member belongs to a specific VMSD. The data source delivers the multicast data to MVMs through a reliable path, and MVMs further disseminate the data to group members along domain overlay multicast trees. The above approach structurally improves the stability of the overlay multicast tree. We further utilized CVM-based streaming compensation to enhance the stability of the data distribution in the VMSDs. VMCast can be used as an extension to existing tree-based overlay multicast solutions, to provide better services for media streaming applications. We applied VMCast to two application instances (i.e., HMTP and HCcast). The results show that it can obviously enhance the stability of the data distribution.     

流动电位法对L-色氨酸水溶液结晶过程的表征

以流动电位法研究L-色氨酸饱和水溶液降温结晶过程。考察溶液酸度和起始温度对结晶过程中流动电位(υ)结晶器温度(t)曲线的影响。L-色氨酸饱和溶液在起始温度25和35℃下成核能力较差,在较低起始温度20℃下成核能力较强。随着L-色氨酸饱和溶液酸度的变化,相同起始温度的υ-t曲线表明成核能力有较大差异,酸度pH=5.0和6.0的条件下成核能力较差。运用生长基元理论进行了分析。