Intercellular communication in Chara: factors affecting transnodal electrical resistance and solute fluxes

The permeability of plasmodesmata in the nodal complex of branch cells of Chara corallina was examined by measuring both the transnodal electrical resistance and transnodal fluxes of ³⁶CI and ¹⁴C-buty-rate. Under normal circumstances, the resistance across the node was low, but increased rapidly in response to metabolic inhibition, pressure gradients across the node or excision of one of the cells. For each of these treatments, there was a substantial reduction in solute transport between the cells. Acidification of the cytoplasm by weak acids or alkalinization by amines did not affect either the electrical resistance or the flux of solutes through the node between whorl cells. The transnodal resistance was significantly higher in older cell pairs, but was unaffected by large transnodal voltage differences or by the passage of action potentials. There was no evidence that short-term increases in cytoplasmic calcium have any effect on plasmodesmatal permeability.     

Intercellular Transport in Plants: II. CYCLOSIS AND THE RATE OF INTERCELLULAR TRANSPORT OF CHLORIDE IN CHARA

The effect of streaming speed on intercellular transport ofchloride has been studied using pairs of internodal cells ofChara. The rate of transport was measured by that fraction ofthe chloride that entered one internode which was transportedout of it into the cells of the node and the next internode.The speed of cytoplasmic streaming was altered by treating thefirst cell with cytochalasin B. The relative rate of intercellular transport depended markedlyon the streaming speed at all speeds up to those found in untreatedcells. The chloride influx into the treated cell did not dependon the streaming speed. It is concluded that the rate of intercellular transport oflow molecular weight solutes in Chara will be normally limitedby the rate at which cytoplasmic streaming brings solute tothe plasmodesmata, rather than by the diffusion permeabilityof the plasmodesmata. This conclusion may well apply to othercharophyte plants, and could in principle apply to higher plants.     

Influence of Cytoplasmic Streaming and Turgor Pressure Gradient on the Transnodal Transport of Rubidium and Electrical Conductance in Chara corallina

In order to study the transnodal transport of Rb⁺ in internodalcells of Chara corallina, a low-temperature loading system wasestablished to separate the loading process from the transportprocess. Tandem cells, consisting of internode-node-internode,were isolated from algal plants. Treatment of a single internodewith 100 mM RbCl at 5°C for 30 min caused an accumulationof 43 mM Rb⁺ in the cytoplasm of this cell (= source cell),but no Rb⁺ was found in the other internode (= sink cell) ofthe tandem cells. In 40 min after a return to 25°C, about12% of the Rb⁺ loaded in the source cell was transported intothe sink cell. The apparent transnodal permeability of Rb⁺ wascalculated to be 4.6 x 10^–7 m.s^–1. Under the assumptionthat the total cross-sectional area of plasmodesmata occupies10% of the nodal area, the diffusion coefficient of RbCl throughplasmodesmata was calculated to be 2.3 x 10^–11 m².s^–1which is about 1% of the free diffusion coefficient in water(2 x 10^–9m².s^–1). The transnodal transport of Rb⁺ was intimately correlated withthe rate of cytoplasmic streaming. The rate of streaming inboth the source and sink cells was varied either by treatingthe cells with cytochalasin B (CB) or by lowering the temperature.The transport rate correlated with the streaming rate irrespectiveof the method used. Since the level of ATP was not influencedby CB or low temperature, the transnodal transport is assumedto be the result of passive diffusion process through plasmodesmata. A turgor pressure gradient across the node decreased both thenodal electrical conductance and the transnodal transport ofRb⁺. By contrast, the exposure of both internodal cells to asolution of sorbitol had no effect on either of them. A turgorpressure gradient of 240 mOsm decreased the transport of Rb⁺in the first hour to 3% of the control, while it decreased thenodal conductance to about 50%. The increase in the electricalresistance occurred on the junction side between the node andthe internode that was treated with sorbitol. Cytochalasin Ehad no effect on the nodal electrical resistance. It is assumedthat plasmodesmata are equipped with a valve-like mechanismwhich is sensitive to the gradient of turgor pressure acrossthe node and is not regulated by an actomyosin system. (Received February 15, 1989; Accepted April 20, 1989)     

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.     

Visualization of peroxisomes in living plant cells reveals acto-myosin-dependent cytoplasmic streaming and peroxisome budding

Here we examine peroxisomes in living plant cells using transgenic Arabidopsis thaliana plants expressing the green fluorescent protein (GFP) fused to the peroxisome targeting signal 1 (PTS1). Using time-lapse laser scanning confocal microscopy we find that plant peroxisomes exhibit fast directional movement with peak velocities approaching 10 microm s(-1). Unlike mammalian peroxisomes which move on microtubules, plant peroxisome movement is dependent on actin microfilaments and myosin motors, since it is blocked by treatment with latrunculin B and butanedione monoxime, respectively. In contrast, microtubule-disrupting drugs have no effect on peroxisome streaming. Peroxisomes were further shown to associate with the actin cytoskeleton by the simultaneous visualization of actin filaments and peroxisomes in living cells using GFP-talin and GFP-PTS1 fusion proteins, respectively. In addition, peroxisome budding was observed, suggesting a possible mechanism of plant peroxisome proliferation. The strong signal associated with the GFP-PTS1 marker also allowed us to survey cytoplasmic streaming in different cell types. Peroxisome movement is most intense in elongated cells and those involved in long distance transport, suggesting that higher plants use cytoplasmic streaming to help transport vesicles and organelles over long distances.     

A gradient model of cardiac pacemaker myocytes

We have formulated a spatial-gradient model of action potential heterogeneity within the rabbit sinoatrial node (SAN), based on cell-specific ionic models of electrical activity from its central and peripheral regions. The ionic models are derived from a generic cell model, incorporating five background and exchange currents, and seven time-dependent currents based on three- or four-state Markov schemes. State transition rates are given by non-linear sigmoid functions of membrane potential.

By appropriate selection of parameters, the generic model is able to accurately reproduce a wide range of action potential waveforms observed experimentally. Specifically, the model can fit recordings from central and peripheral regions of the SAN with RMS errors of 0.3987 and 0.7628 mV, respectively. Using a custom least squares parameter optimisation routine, we have constructed a spatially-varying gradient model that exhibits a smooth transition in action potential characteristics from the central to the peripheral region, whilst ensuring individual membrane currents remain physiologically accurate. Smooth transition action potential characteristics include maximum diastolic potential, overshoot potential, upstroke velocity, action potential duration and cycle length. The gradient model is suitable for developing higher dimensional models of the right atrium, in which action potential heterogeneity within nodal tissue may be readily incorporated.     

Cyclosis-mediated transfer of H2O2 elicited by localized illumination of Chara cells and its relevance to the formation of pH bands

Cytoplasmic streaming occurs in most plant cells and is vitally important for large cells as a means of long-distance intracellular transport of metabolites and messengers. In internodal cells of characean algae, cyclosis participates in formation of light-dependent patterns of surface pH and photosynthetic activity, but lateral transport of regulatory metabolites has not been visualized yet. Hydrogen peroxide, being a signaling molecule and a stress factor, is known to accumulate under excessive irradiance. This study was aimed to examine whether H₂O₂ produced in chloroplasts under high light conditions is released into streaming fluid and transported downstream by cytoplasmic flow. To this end, internodes of Chara corallina were loaded with the fluorogenic probe dihydrodichlorofluorescein diacetate and illuminated locally by a narrow light beam through a thin optic fiber. Fluorescence of dihydrodichlorofluorescein (DCF), produced upon oxidation of the probe by H₂O₂, was measured within and around the illuminated cell region. In cells exhibiting active streaming, H₂O₂ first accumulated in the illuminated region and then entered into the streaming cytoplasm, giving rise to the expansion of DCF fluorescence downstream of the illuminated area. Inhibition of cyclosis by cytochalasin B prevented the spreading of DCF fluorescence along the internode. The results suggest that H₂O₂ released from chloroplasts under high light is transported along the cell with the cytoplasmic flow. It is proposed that the shift of cytoplasmic redox poise and light-induced elevation of cytoplasmic pH facilitate the opening of H⁺/OH^?-permeable channels in the plasma membrane.     

Actin in living and fixed characean internodal cells: identification of a cortical array of fine actin strands and chloroplast actin rings

Summary We report on the novel features of the actin cytoskeleton and its development in characean internodal cells. Images obtained by confocal laser scanning microscopy after microinjection of living cells with fluorescent derivatives of F-actin-specific phallotoxins, and by modified immunofluorescence methods using fixed cells, were mutually confirmatory at all stages of internodal cell growth. The microinjection method allowed capture of 3-dimensional images of high quality even though photobleaching and apparent loss of the probes through degradation and uptake into the vacuole made it difficult to record phallotoxin-labelled actin over long periods of time. When injected at appropriate concentrations, phallotoxins affected neither the rate of cytoplasmic streaming nor the long-term viability of cells. Recently formed internodal cells have relatively disorganized actin bundles that become oriented in the subcortical cytoplasm approximately parallel to the newly established long axis and traverse the cell through transvacuolar strands. In older cells with central vacuoles not traversed by cytoplasmic strands, subcortical bundles are organized in parallel groups that associate closely with stationary chloroplasts, now in files. The parallel arrangement and continuity of actin bundles is maintained where they pass round nodal regions of the cell, even in the absence of chloroplast files. This study reports on two novel structural features of the characean internodal actin cytoskeleton: a distinct array of actin strands near the plasma membrane that is oriented transversely during cell growth and rings of actin around the chloroplasts bordering the neutral line, the zone that separates opposing flows of endoplasm.     

Nodes of Ranvier act as barriers to restrict invasion of flanking paranodal domains in myelinated axons

Accumulation of voltage-gated sodium (Na(v)) channels at nodes of Ranvier is paramount for action potential propagation along myelinated fibers, yet the mechanisms governing nodal development, organization, and stabilization remain unresolved. Here, we report that genetic ablation of the neuron-specific isoform of Neurofascin (Nfasc(NF1??)) in vivo results in nodal disorganization, including loss of Na(v) channel and ankyrin-G (AnkG) enrichment at nodes in the peripheral nervous system (PNS) and central nervous system (CNS). Interestingly, the presence of paranodal domains failed to rescue nodal organization in the PNS and the CNS. Most importantly, using ultrastructural analysis, we demonstrate that the paranodal domains invade the nodal space in Nfasc(NF1??) mutant axons and occlude node formation. Our results suggest that Nfasc(NF1??)-dependent assembly of the nodal complex acts as a molecular boundary to restrict the movement of flanking paranodal domains into the nodal area, thereby facilitating the stereotypic axonal domain organization and saltatory conduction along myelinated axons.     

Investigation of the temperature dependence of the cross bridge parameters for attachment,force generation and detachment as deduced from mechano-chemical studies in glycerinated single fibres from the dorsal longitudinal muscle of Lethocerus maximus

Birefringence change during excitation was studied by using Nitellopsis obtusa. The velocity change of cytoplasmic streaming during an action potential was measured simultaneously by fluctuation analysis of transmitted light intensity. The origin of the retardation change was discussed by comparing optical retardation change to the time course of the action potential, the cytoplasmic streaming velocity change and the cell contraction.By the time course analysis of retardation change, we concluded that the change of the birefringence might be the sum of the changes of cytoplasmic flow and that of the size of length and diameter of the cell. But it is still difficult to separate the change to its components.     

Long-distance signal transmission and regulation of photosynthesis in characean cells

Photosynthetic electron transport in an intact cell is finely regulated by the structural flexibility of thylakoid membranes, existence of alternative electron-transport pathways, generation of electrochemical proton gradient, and continuous exchange of ions and metabolites between cell organelles and the cytoplasm. Long-distance interactions underlying reversible transitions of photosynthetic activity between uniform and spatially heterogeneous distributions are of particular interest. Microfluorometric studies of characean cells with the use of saturating light pulses and in combination with electrode micromethods revealed three mechanisms of distant regulation ensuring functional coordination of cell domains and signal transmission over long distances. These include: (1) circulation of electric currents between functionally distinct cell domains, (2) propagation of action potential along the cell length, and (3) continuous cyclical cytoplasmic streaming. This review considers how photosynthetic activity depends on membrane transport of protons and cytoplasmic pH, on ion fluxes associated with the electrical excitation of the plasmalemma, and on the transmission of photoinduced signals with streaming cytoplasm. Because of signal transmission with cytoplasmic flow, dynamic changes in photosynthetic activity can develop far from the point of photostimulus application and with a long delay (up to 100 s) after a light pulse stimulus is extinguished.     

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.     

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.     

One-Dimensional Mathematical Model of the Atrioventricular Node Including Atrio-Nodal, Nodal, and Nodal-His Cells

Mathematical models are a repository of knowledge as well as research and teaching tools. Although action potential models have been developed for most regions of the heart, there is no model for the atrioventricular node (AVN). We have developed action potential models for single atrio-nodal, nodal, and nodal-His cells. The models have the same action potential shapes and refractoriness as observed in experiments. Using these models, together with models for the sinoatrial node (SAN) and atrial muscle, we have developed a one-dimensional (1D) multicellular model including the SAN and AVN. The multicellular model has slow and fast pathways into the AVN and using it we have analyzed the rich behavior of the AVN. Under normal conditions, action potentials were initiated in the SAN center and then propagated through the atrium and AVN. The relationship between the AVN conduction time and the timing of a premature stimulus (conduction curve) is consistent with experimental data. After premature stimulation, atrioventricular nodal reentry could occur. After slow pathway ablation or block of the L-type Ca²⁺ current, atrioventricular nodal reentry was abolished. During atrial fibrillation, the AVN limited the number of action potentials transmitted to the ventricle. In the absence of SAN pacemaking, the inferior nodal extension acted as the pacemaker. In conclusion, we have developed what we believe is the first detailed mathematical model of the AVN and it shows the typical physiological and pathophysiological characteristics of the tissue. The model can be used as a tool to analyze the complex structure and behavior of the AVN.     

Nodal flow and the generation of left-right asymmetry

The establishment of left-right asymmetry in mammals is a good example of how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of cilia that are tilted toward the posterior on cells of the ventral node. These cilia are built by transport via the KIF3 motor complex. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of membrane-sheathed particles, called nodal vesicular parcels (NVPs), may result in the activation of the non-canonical Hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.     

The mechanistic basis for noncompetitive ibogaine inhibition of serotonin and dopamine transporters

Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.     

A mixture theory for charged-hydrated soft tissues containing multi-electrolytes: passive transport and swelling behaviors.

A new mixture theory was developed to model the mechano-electrochemical behaviors of charged-hydrated soft tissues containing multi-electrolytes. The mixture is composed of n + 2 constituents (1 charged solid phase, 1 noncharged solvent phase, and n ion species). Results from this theory show that three types of force are involved in the transport of ions and solvent through such materials: (1) a mechanochemical force (including hydraulic and osmotic pressures); (2) an electrochemical force; and (3) an electrical force. Our results also show that three types of material coefficients are required to characterize the transport rates of these ions and solvent: (1) a hydraulic permeability; (2) mechano-electrochemical coupling coefficients; and (3) an ionic conductance matrix. Specifically, we derived the fundamental governing relationships between these forces and material coefficients to describe such mechano-electrochemical transduction effects as streaming potential, streaming current, diffusion (membrane) potential, electro-osmosis, and anomalous (negative) osmosis. As an example, we showed that the well-known formula for the resting cell membrane potential (Hodgkin and Huxley, 1952a, b) could be derived using our new n + 2 mixture model (a generalized triphasic theory). In general, the n + 2 mixture theory is consistent with and subsumes all previous theories pertaining to specific aspects of charged-hydrated tissues. In addition, our results provided the stress, strain, and fluid velocity fields within a tissue of finite thickness during a one-dimensional steady diffusion process. Numerical results were provided for the exchange of Na+ and Ca++ through the tissue. These numerical results support our hypothesis that tissue fixed charge density (CF) plays a significant role in modulating kinetics of ions and solvent transport through charged-hydrated soft tissues.     

Propagation through electrically coupled cells. How a small SA node drives a large atrium.

Each normal cardiac cycle is started by an action potential that is initiated in the sino-atrial (SA) node by automaticity of the SA nodal cells. This action potential then propagates from the SA node into the surrounding atrial cells. We have done numerical simulations of electrically coupled cells to understand how a small SA node can be spontaneously active and yet be sufficiently electrically coupled to the surrounding quiescent atrial cells to initiate an action potential in the atrial cells. Our results with a simple model of two coupled cells and a more complex model of a two-dimensional sheet of cells suggest that some degree of electrical uncoupling of the cells within the SA node may be an essential design feature of the normal SA-atrial system.     

Cessation of cytoplasmic streaming follows an increase of cytoplasmic Ca2+ during action potential inNitella

Summary Taking advantage of prolonged action potential under low temperature, we studied temporal relationship among the action potential, increase of cytoplasmic Ca²⁺ concentration and cessation of cytoplasmic streaming inNitella. The Ca²⁺ concentration began to increase at a very early stage of the action potential and the cessation of streaming followed that increase.Abbreviations APW artificial pond water     

Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells

Background

Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood.

Methodology/Principal Findings

Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed.

Conclusions and Significance

These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.