Plant electrical memory

Electrical signaling, short-term memory and rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus flytrap) have been attracting the attention of researchers since the XIX century. We found that the electrical stimulus between a midrib and a lobe closes the Venus flytrap upper leaf without mechanical stimulation of trigger hairs. The closing time of Venus flytrap by electrical stimulation is the same as mechanically induced closing. Transmission of a single electrical charge between a lobe and the midrib causes closure of the trap and induces an electrical signal propagating between both lobes and midrib. The Venus flytrap can accumulate small subthreshold charges, and when the threshold value is reached, the trap closes. Repeated application of smaller charges demonstrates the summation of stimuli. The cumulative character of electrical stimuli points to the existence of short-term electrical memory in the Venus flytrap.Key words: plant memory, electrophysiology, electrical signaling, venus flytrap, Dionaea muscipula ellisPlants are capable of intelligent responses to complex environmental signals.^1–27 Signaling and memory play fundamental roles in plant responses. The existence of different forms of plant memory is well known.^1–22 Depending on the duration of memory retention, there are three types of memory in plants: sensory memory, short term memory and long term memory. A few examples of studies involving plant memory are: transgeneration memory of stress,^1,6,10 immunological memory of tobacco plants²² and mountain birches,¹⁸ storage and recall functions in seedlings,⁹ chromatin remodelling in plant development,^4,19 vernalization and epigenetic memory of winter,^12,13 induced resistance and susceptibility to herbivory,² memory response in ABA-entrained plants,⁶ memory of stimulus,^16,17 and systematic acquired resistance in plants exposed to a pathogen.²² Cellular memory is an example of long term memory and is a long-term maintenance of a particular pattern of gene expression. Chromatin dynamics including histone modification, histone replacement and chromatin remodeling play key roles in cellular memory.⁴ Plants are intelligent organisms and capable of functions such as learning, individuality, plasticity and memory.⁵ There are a few mathematical models of plant learning and memory.^14,15 Some plants exhibit clues of an electrical memory as well.We found that Venus flytrap has a short term electrical memory^20,21 Rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus flytrap) has been attracting the attention of researchers and as a result its mechanism has been widely investigated. When an insect touches the trigger hairs, these mechanosensors generate an electrical signal that acts as an action potential, which activates the trap closing. Macfarlane²³ found that two mechanical stimuli required for the trap closing should be applied within an interval from 0.75 s to 20 s. Brown and Sharp²⁴ found that at high temperature of 35–40°C usually only one mechanical stimulus is required.The inducement of non-excitability after excitation and the summation of subthreshold irritations were developed in the vegetative and animal kingdoms in protoplasmic structures prior to morphological differentiation of nervous tissues. These protoplasmic structures merged into the organs of a nervous system and adjusted the interfacing of the organism with the environment. Some neuromotoric components include acetylcholine neurotransmitters, cellular messenger calmodulin, cellular motors actin and myosin, voltage-gated channels, and sensors for touch, light, gravity and temperature.^25–27 Although this nerve-like cellular equipment has not reached the same great complexity as in animal nerves, a simple neural network has been formed within the plasma membrane of a phloem or plasmodesmata enabling it to communicate efficiently over long distances.^5,26,27 The reason why plants have developed pathways for electrical signal transmission most probably lies in the necessity to respond rapidly to environmental stress factors. Different environmental stimuli evoke specific responses in living cells, which have the capacity to transmit a signal to the responding region. In contrast to chemical signals such as hormones, electrical signals are able to rapidly transmit information over long distances.²⁷ Electrical potentials have been measured at the tissue and whole plant levels.²⁶Using our new charge injection method,²⁰ it was evident that the application of an electrical stimulus between the midrib (positive potential) and a lobe (negative potential) causes Venus flytrap to close the trap without any mechanical stimulation. The average stimulation pulse voltage sufficient for rapid closure of the Venus flytrap was 1.50 V (standard deviation is 0.01 V, n = 50) for 1 s. The inverted polarity pulse with negative voltage applied to the midrib did not close the plant. Applying impulses in the same voltage range with different polarities for pulses of up to 100 s did not open the plant. It was found that energy for trap closure is generated by ATP hydrolysis. ATP is used by the motor cells for a fast transport of protons. The amount of ATP drops from 950 µM per midrib before mechanical stimulation to 650 µM per midrib after stimulation and closure.²⁸ However, it is not clear if electrical stimulation triggers closing process in the motor cells, or contributes energy to the closing action.The action potential delivers sufficient electrical charge to the midrib,²¹ which can activate the osmotic motor. To check this hypothesis, we measured effects of transmitted charge from the charged capacitors between the lobe and the midrib of Venus flytrap. Transmission of a single electrical charge (mean 13.63 µC, median 14.00 µC, std. dev. 1.51 µC, n = 41) causes trap closure and induces an electrical signal propagating between the lobes and the midrib. The electrical signal in the lobes was not an action potential, because its amplitude depended on the applied voltage from the charged capacitor. Charge induced closing of a trap plant can be repeated 2–3 times on the same Venus flytrap plant after reopening. Transmission of a single electrical charge (mean 13.63 µC, median 14.00 µC, std. dev. 1.51 µC, n = 41) causes the trap to close and induces an electrical signal that propagates between the lobe and the midrib. Figure 1 illustrates that the Venus flytrap can accumulate small charges, and when the threshold value is reached, the trap closes. A summation of stimuli is demonstrated through the repetitive application of smaller charges. If we apply two or more consecutive injections of electrical charge within a period of less than 50 s, the trap will close when a total of 14 µC charge is reached.Open in a separate windowFigure 1Mechanism of the Dionaea trap closure.Repeated application of smaller charges demonstrates a summation of stimuli. If we apply two or more injections of electrical charges within a period of less then 20 s, the Venus flytrap upper leaf closes as soon as the total of 14 µC charge is transmitted. Similar phenomenon was reported by Czaja,²⁹ who determined the intensity of threshold stimuli to be 2.4 µC for a closing electrostimulation of another carnivorous plant Aldrovanda vesiculosa, and 0.91 µC for an opening electrostimulation. Our attempts to open the Venus flytrap upper leaf by changing polarity of injected charge and increasing the charge from 14 µC to 100 µC were not successful. Usually, the trap opens a few days after closing in the same way as after mechanically stimulated closing.Previous work by Brown and Sharp²⁴ indicated that electrical shock between lower and upper leaves can cause the Venus flytrap to close, but in their article, the amplitude and polarity of applied voltage, charge and electrical current were not reported. The trap did not close when we applied the same electrostimulation between the upper and lower leaves as we applied between a midrib and a lobe, even when the injected charge was increased from 14 µC to 750 µC. It is probable that the electroshock induced by Brown and Sharp²⁴ had a very high voltage or electrical current.It is common knowledge that the leaves of the Venus flytrap actively employ turgor pressure and hydrodynamic flow for fast movement and catching insects. In these processes the upper and lower surfaces of the leaf behave quite differently. During the trap closing, the loss of turgor by parenchyma lying beneath the upper epidermis, accompanied by the active expansion of the tissues of the lower layers of parenchyma near the under epidermis, closes the trap. The cells on the inner face of the trap jettison their cargo of water, shrink and allow the trap lobe to fold over. The cells of the lower epidermis expand rapidly, folding the trap lobe over. These anatomical features constitute the basis of the new hydroelastic curvature model.²⁰In terms of electrophysiology, Venus flytrap responses can be considered in three stages: (i) stimulus perception, (ii) signal transmission and (iii) induction of response (Fig. 1).     

Anistropically varying conductivity in irreversible electroporation simulations

Background

One recent area of cancer research is irreversible electroporation (IRE). Irreversible electroporation is a minimally invasive procedure where needle electrodes are inserted into the body to ablate tumor cells with electricity. The aim of this paper is to propose a mathematical model that incorporates a tissue’s conductivity increasing more in the direction of the electrical field as this has been shown to occur in experiments.

Method

It was necessary to mathematically derive a valid form of the conductivity tensor such that it is dependent on the electrical field direction and can be easily implemented into numerical software. The derivation of a conductivity tensor that can take arbitrary functions for the conductivity in the directions tangent and normal to the electrical field is the main contribution of this paper. Numerical simulations were performed for isotropic-varying and anisotropic-varying conductivities to evaluate the importance of including the electrical field’s direction in the formulation for conductivity.

Results

By starting from previously published experimental results, this paper derived a general formulation for an anistropic-varying tensor for implementation into irreversible electroporation modeling software. The anistropic-varying tensor formulation allows the conductivity to take into consideration both electrical field direction and magnitude, as opposed to previous published works that only took into account electrical field magnitude.The anisotropic formulation predicts roughly a five percent decrease in ablation size for the monopolar simulation and approximately a ten percent decrease in ablation size for the bipolar simulations. This is a positive result as previously reported results found the isotropic formulation to overpredict ablation size for both monopolar and bipolar simulations. Furthermore, it was also reported that the isotropic formulation overpredicts the ablation size more for the bipolar case than the monopolar case. Thus, our results are following the experimental trend by having a larger percentage change in volume for the bipolar case than the monopolar case.

Conclusions

The predicted volume of ablated cells decreased, and could be a possible explanation for the slight over-prediction seen by isotropic-varying formulations.

     

Wound currents and wound healing in the newt,Notophthalmus viridescens

Summary Wounded amphibian skin heals initially by a migration of epithelial cells from the cut edge towards the center of the wound. The density of currents leaving wounds made in Notophthalmus viridescens skin was manipulated in order to determine whether electrical fields associated with these currents might have a significant role in promoting this cell migration during wound healing. Wounds were made with either a needle (200 m) or a biopsy punch (500 m). Currents leaving the wounds were measured with a vibrating probe, and the wounds fixed at various times after wounding. When the Na⁺-dependent currents were reduced by blocking Na⁺ channels with benzamil, wound healing, as revealed by scanning electron microscopy and by paraffin histology, was impaired. These results are consistent with the hypothesis that there is an electrical component to wound healing.     

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy ¹. OHCs are able to change shape —elongate, shorten and bend— in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals ².OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization ^{2, 3}. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) ⁴. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins ^{5, 6}. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research ⁷.The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) ⁸. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches ^9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques ¹². For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.Download video file.^{(62M, mov)}     

Natural electrical RF oscillation from cells

Electrical oscillatory rf phenomena are present during the division of cells. These were examined by studying the attraction of cells for polarizable powders. They are understood to occur by a process termed microdielectrophoresis (-DEP), the motion induced by a nonuniform electric field acting on a polarizable body. The suggestion that an electrical oscillatory aspect may also be involved in the contact or density inhibition of cell division and the mechanisms that may cause invasiveness of oncogenic cells are theoretically explored (i.e., changes in either the power level or the frequency of the oscillatory phenomena associated with cell division, or in the degree of electrical insulation of the cell from electrical damping by nearby cells). A number of experiments to test this hypothesis are suggested.     

Lack of homogeneity of receptive fields of visual neurons in the cortical area 18 of the cat

The receptive fields of complex neurons within area 18 of the cerebral cortex of the cat were determined by a computer-assisted method using a moving light bar substantially shorter than the long diameter of the receptive field as a visual stimulus. The visual cells repeatedly generated nerve impulses when the stimulus crossed well-defined active points within their receptive fields. Outside of these active points, the cells remained silent. It is suggested that the receptive fields are formed by a discontinuous accumulation of such active points. When the electrical activities of two neighbouring visual neurons are recorded simultaneously, their active points do not coincide. In addition, some active points were located outside the most prominent excitatory part of the receptive field of the studied cells. Individual visual cells typically differ in the number and distribution of active points. Since these cells best respond to a stimulus moving in a certain direction, it is suggested that they may act as direction of movement and/or velocity detectors. Alternate firing of a number of neighboring cells connected to a distributed pattern of peripheral receptors may form a system which is able to code for velocity and direction of the moving stimulus.     

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)     

Membrane and junctional properties of the isolated frog lens epithelium

Summary The isolated frog lens epithelium can be maintained intact in both appearance and electrical properties for more than 24 hours. The mean resting membrane potential was –80 mV and the cells were depolarized by both high potassium and low calcium Ringer's solution in a manner very similar to that of the whole lens. The epithelial cells were found to be well coupled using both electrical and dye-injection techniques. Electrical coupling was measured using separate current-injection and voltage-measuring electrodes and the relationship between the induced voltage and distance from the current-passing electrode could be well fitted by a Bessel Function solution to the cable equation. The values obtained from the fit for the membrane and internal resistances were 1.95 m² and 25 m, respectively. Exposure to octanol (500m) or low external Ca²⁺ (<1m) failed to disrupt significantly the intercellular flow of current. There was evidence to suggest thatraised intracellular calcium does, however, uncouple the cells. Dye coupling was investigated by microinjecting Lucifer Yellow CH into single epithelial cells. Diffusion into surrounding cells was rapid and, in control medium, occurred in a radially symmetrical manner. In contrast to the electrical coupling data, dye transfer appeared to be blocked by exposure to 500 m octanol and was severely restricted on perfusing with low external calcium. Differences between the electrical and dye-coupling experiments indicate either that there are two types of junction within the cell and only the larger type, permeable to Lucifer Yellow, is capable of being uncoupled or that there is only one large type of junction which can be partially closed by uncoupling agents.     

Electrical signaling and photosynthesis: Can they co-exist together?

Mechanical irritation of trigger hairs and subsequent generation of action potentials have significant impact on photosynthesis and respiration in carnivorous Venus flytrap (Dionaea muscipula). Action potential-mediated inhibition of photosynthesis and stimulation of respiration is confined only to the trap and was not recorded in adjacent photosynthetic lamina. We showed that the main primary target of electrical signals on assimilation is in the dark enzymatic reaction of photosynthesis. Without doubt, the electrical signaling is costly, and the possible co-existence of such type of signals and photosynthesis in plant cell is discussed.Key words: action potential, carnivorous plant, Dionaea muscipula, electrical signaling, photosynthesis, respiration, Venus flytrapTrap closure of the Venus flytrap (Dionaea muscipula) is one of the fastest movements in plant kingdom. Mechanical irritation of trigger hairs protruding from upper leaf epidermis results in generation of action potential. At room temperature, two touches generate two action potentials and activate the trap snap shut in a fraction of second.¹ After the rapid movement secures the prey, struggling results in generation of further action potentials which cease to occur when the prey stops moving.² We documented that trigger hair irritation and subsequent generation of action potentials have significant effect on photosynthesis and respiration. Action potentials propagate in the trap and were not recorded in adjacent lamina (Fig. 1). This is in accordance with the observation that no changes of photosynthetic and respiration rate as well as effective quantum yield of photosystem II photochemistry were recorded in lamina. Detailed analysis of chlorophyll fluorescence kinetics revealed that the main primary target of action potentials is in the dark enzymatic reaction of photosynthesis and changes in quantum yield of primary photochemistry are just a consequence of decreased CO₂ fixation. However, electrical signals have probably also small effect on excitation energy trapping, charge stabilization and recombination reaction in photosystem II as measurements of fast chlorophyll a fluorescence transient indicates. This effect may be explained by repulsion of charges in reaction center of photosystem II.^3,4 The changes of photosynthesis upon impact of electrical signals probably have no benefit for plant and are only a negative consequences caused by the changes of the ionic environment.Open in a separate windowFigure 1Dionaea muscipula with entrapped wasp of the genus Polistes. Action potentials and rate of net assimilation at irradiance 80 µmol m⁻²s⁻ PAR (A_n) in response to 15 s mechanical trigger hair irritation (between 160–175 s) in trap (upper row) and photosynthetic lamina (lower row).These findings may have more consequences for plants in general. The electrical activity of plant cell was for the first time described by Burdon-Sanderson in 1873.⁵ Hence electrical signals do not belong exclusively to animal kingdom however they never develop the same degree of complexity as in animal nerves. Electrical signals are capable of transmitting signals more quickly over long distances when compared with chemical signals (e.g., hormones).^6,7 They are not confined only to the sensitive plants (e.g., Mimosa, Dionaea), but play also an important role in every non-sensitive plants and in both groups have significant effect on photosynthesis and respiration.^8–14 It is not surprising, that if electrical signals are costly in term of consumption of ATP and increased respiration with concurrent inhibition of photosynthesis, the same degree of complexity as in animals could not be developed. If plant growth depends on photosynthesis, this raises the question whether electrical signals and photosynthesis may co-exist together. The continuous electrical activity would inhibit the main source of energy for plants—photosynthetic assimilation. This may also explain why the plants are sessile organisms. For rapid coordinated movements, electrical activity plays an important role in animals. Unlike animals, plants usually rely on slow movements in which the role of plant hormones is indispensable. In this concept, it is not surprising that the more complex electrical activity was recorded in root transition zone—the heterotrophic part of plant body.^15,16 And this may also explain why the more evident electrical activity in the plant world has evolved in the traps of carnivorous plants like Dionaea, Aldrovanda or Drosera.^17–19 In general the traps of carnivorous plants are considered to be less efficient in photosynthesis.²⁰ Any of the action potentials produced by Drosera tentacles or Dionaea trap do not spread to photosynthetic active lamina, thus the main side of CO₂ fixation is protected.²¹ It is possible that such temporal carbon costs associated with insect trapping and retention may be outweighed by the benefits gained later from the prey—increased nitrogen concentration in the leaves stimulates photosynthetic assimilation.²² The possible ecophysiological impact of electrical signals on daily carbon gain in sensitive plants remains to be elucidated. We still do not completely understand the electrical signals in plants, and further research in this area is necessary to understand the full meaning of electrical activity in plants.     

Visualization of Flagella during Bacterial Swarming

When cells of Escherichia coli are grown in broth and suspended at low density in a motility medium, they swim independently, exploring a homogeneous, isotropic environment. Cell trajectories and the way in which these trajectories are determined by flagellar dynamics are well understood. When cells are grown in a rich medium on agar instead, they elongate, produce more flagella, and swarm. They move in coordinated packs within a thin film of fluid, in intimate contact with one another and with two fixed surfaces, a surfactant monolayer above and an agar matrix below: they move in an inhomogeneous, anisotropic environment. Here we examine swarm-cell trajectories and ways in which these trajectories are determined by flagellar motion, visualizing the cell bodies by phase-contrast microscopy and the flagellar filaments by fluorescence microscopy. We distinguish four kinds of tracks, defining stalls, reversals, lateral movement, and forward movement. When cells are stalled at the edge of a colony, they extend their flagellar filaments outwards, moving fluid over the virgin agar; when cells reverse, changes in filament chirality play a crucial role; when cells move laterally, they are pushed sideways by adjacent cells; and when cells move forward, they are pushed by flagellar bundles in the same way as when they are swimming in bulk aqueous media. These maneuvers are described in this report.Swarming is a common yet specialized form of surface translocation exhibited by flagellated bacteria and is distinct from swimming (23). When cells are grown on a moist nutrient-rich surface, they differentiate from a vegetative to a swarm state: they elongate, make more flagella, secrete wetting agents, and move across the surface in coordinated packs. Here we focus on the mechanics of bacterial swarming, as exhibited by the model organism Escherichia coli. Others have worked on swarm-cell differentiation in a variety of organisms, including Proteus, Salmonella, Pseudomonas, Serratia, Bacillus, and Vibrio. For example, screens for genes required for swarming in E. coli or Salmonella have been made by Inoue et al. (25) and Wang et al. (40, 41). Vibrio is a special case, because a single polar flagellum enables cells to swim while multiple lateral flagella promote swarming (32). For general reviews, see the work of Allison and Hughes (1), Shapiro (37), Fraser and Hughes (17), and Fraser et al. (16). Also see the work of Eberl et al. (15), Sharma and Anand (38), Harshey (18), Daniels et al. (11), Kaiser (26), O''Toole (33), and Copeland and Weibel (10).Swarming was first observed with Proteus mirabilis by Hauser (22), who named the genus for a sea god able to change his own form. Proteus is distinctive because cells switch periodically from the vegetative to the swarming state, building terraced colonies (36, 42). This is not observed with E. coli under the conditions used here, where swarms expand at a constant rate propelled by cells swimming vigorously in a monolayer behind a smooth outer boundary.Swarming in E. coli was discovered by Harshey, who found that K-12 strains, which lack the lipopolysaccharide O antigen, swarmed on Eiken agar (from Japan) but not on Difco agar (from the United States), presumably because the former is more wettable (19, 20). Chemotaxis is not required: cells lacking the chemotaxis response regulator CheY swarm perfectly well, provided that mutations in the motor protein FliM enable transitions between clockwise (CW) and counterclockwise (CCW) rotational states (31). It was suggested that these reversals promote wetness by causing cells to shed lipopolysaccharide.How do cells in E. coli swarms move across an agar surface? What are their flagella doing? We sought to answer such questions by performing a global analysis of videotaped data (of phase-contrast images) collected from 5 regions of 2 swarms, plotting body lengths, speeds, propulsion angles, local track curvatures, and temporal and spatial correlations, and we found that cells reorient on a time scale of a few tenths of a second, primarily by colliding with one another (13). Our previous report did not describe analyses of individual tracks or visualization of flagella. This aspect of the work is presented here.Most of the time, cells are driven forwards by a flagellar bundle in the usual way. Flagellar filaments from different cells can intertwine and form common bundles, but this is rare. However, cells in swarms do something not ordinarily seen with swimming cells: they back up. They do this without changing the orientation of the cell body by moving back through the middle of the flagellar bundle. This involves changes in filament shape (in polymorphic form), from normal to curly and back to normal. Polymorphic forms were classified by Calladine (7), on the basis of earlier work by Asakura (3), in terms of the relative lengths of 11 protofilaments, longitudinal arrays of protein subunits that comprise the filament. All polymorphic forms are helical, with some being left-handed (e.g., the normal form) and some being right-handed (e.g., the semicoiled and curly forms, which have half the pitch of the normal filament and half the pitch and half the amplitude, respectively). Transformations from one shape to another can be caused in various ways, e.g., by changes in pH, salinity, or temperature (21, 27, 28) or by application of torque (24). The changes observed with swarm cells are driven by the latter mechanism, when motors switch from CCW to CW rotation. When swimming cells tumble, polymorphic transformations also occur, in the order normal to semicoiled to curly and back to normal (14, 39). But we rarely see the semicoiled form with cells in swarms, and when it appears, it is quite transient. We wonder whether polymorphic transformations evolved to enable cells to escape confined environments when the only way out is to back up, keeping the filaments close to the sides of the cell body.     

Alamethicin-induced electrical long distance signaling in plants

Systemic signals induced by wounding and/or pathogen or herbivore attack may be realized by either chemical or mechanical signals. In plants a variety of electrical phenomena have been described and may be considered as signal-transducing events; such as variation potentials (VPs) and action potentials (APs) which propagate over long distances and hence are able to carry information from organ to organ. In addition, we recently described a new type of electrical long-distance signal that propagates systemically, i.e., from leaf to leaf, the “system potential” (SP). This was possible only by establishing a non-invasive method with micro-electrodes positioned in substomatal cavities of open stomata and recording apoplastic responses. Using this technical approach, we investigated the function of the peptaibole alamethicin (ALA), a channel-forming peptide from Trichoderma viride, which is widely used as agent to induce various physiological and defence responses in eukaryotic cells including plants. Although the ability of ALA to initiate changes in membrane potentials in plants has always been postulated it has never been demonstrated. Here we show that both local and long-distance electrical signals, namely depolarization, can be induced by ALA treatment.Key words: alamethicin, long distance electrical signal, depolarization, non-invasive recordingPeptaibols are linear membrane active peptide antibiotics produced by various fungi.^1,2 They are characterized by the presence of an unusual amino acid, α-aminoisobutyric acid, and a C-terminal hydroxylated amino acid and are generated by non-ribosomal peptide synthetases.^2,3 Due to their amphiphilic nature they self-associate into oligomeric ion-channels that span the width of lipid bilayer membranes.³ Peptaibols exhibit antibiotic activity against bacteria and fungi, tissue damage in insect larvae, as well as cytolytic activity towards mammalian cells.² ALA is a voltage-gated ion channel-forming peptide mixture that consists of at least 12 compounds each containing 20 amino acid residues.^1,2 This peptaibole is often used to elicit typical systemic responses in plants, many of which are in the context of indirect defences, such as the induction and accumulation of secondary metabolites in general, volatile compounds, and the phytohormones jasmonic acid and salicylic acid but also tendril coiling is induced.^4–7 Moreover, ALA has been shown to permeabilize mitochondria and the plasma membrane of tobacco suspension culture cells but not the tonoplast for small molecules.⁸ Thus, ALA is a valuable tool in plant science but its primary biological activity in plant tissues has never been shown experimentally.Because of the channel-forming properties of ALA, effects on the membrane potential of cells are very likely. Therefore, to monitor ALA application-elicited electrical signals, we employed the non-invasive method with microelectrodes placed in the apoplasm of the sub-stomatal cavities of open stomata. Whereas electrical signals in plants such as action potentials and variation potentials have been well documented in literature,⁹ this particular approach has been successfully applied for the detection and characterization of the novel system potentials.^10,11 With an intracellular recording the depolarization of a membrane occurs when the cell interior becomes less negative; whereas for the apoplastic recording used here, the inverse argument holds true. To avoid confusion, we follow the convention and call an apoplastic hyperpolarization a depolarization.^10,11ALA (mixture of several isoforms purchased from Sigma-Aldrich, Germany) was tested on the dicotyledonous lima bean (Phaseolus lunatus L.) and the monocotyledonous barley (Hordeum vulgare L.). As shown in Figure 1, upon a 5 nM ALA stimulus a 40 mV depolarization was induced and detected at a distance of 5 cm on the same leaf in lima bean. Systemic electric propagation was tested and demonstrated in barley (Fig. 2). We used barley for these experiments because we learned from earlier studies that electrical signals are not propagated between the primary leaves of lima bean whereas in barley electrical signals pass nodes and internodia more easily. Application of 25 nM ALA on one leaf (S-leaf) resulted in a 45 mV depolarization response at a distant of 25 cm on a different leaf (T-leaf), indicating that ALA induced a systemic electrical response that moved from one to the other leaf. In both plants the velocity of the propagating depolarization signal was calculated to be 2.5 cm min⁻¹. Whereas these results basically demonstrate the ability of ALA to initiate electrical signals in plant tissues both locally and systemically, the molecular interconnections between these electrical phenomena, the induction and synthesis of phytohormones as well as secondary metabolites, and how the ALA signal is transduced mechanistically, remains to be elucidated.Open in a separate windowFigure 1Local response of Phaseolus lunatus to Alamethicin. (A) Experimental setup for the measurement of apoplastic voltage changes using microelectrodes positioned in the sub-stomatal cavities of P. lunatus.¹¹ Stimuli and measurements of apoplastic voltage changes were performed on the same leaf with a distance of approximately 5 cm. The tip of the leaf was submerged in 5 mM KCl with 0.1 mM CaCl₂, pH 5, and the solution was connected to earth with a reference electrode filled with 0.5 M KCl. (B) The voltage response to 5 nM Alamethicin (ALA) (Sigma-Aldrich, Germany) added to a cut injury of the P. lunatus leaf at the indicated time shows a hyperpolarization of the apoplast (negative shift of the apoplastic potential), suggesting depolarization of the symplast. A typical recording of one out of three independent experiments is presented.Open in a separate windowFigure 2Systemic response of Hordeum vulgare to Alamethicin. (A) Experimental setup for the measurement of apoplastic voltage changes using microelectrodes positioned in the sub-stomatal cavities of H. vulgare.¹¹ Stimuli were applied to one leaf (Stimulus leaf, S-leaf), and measurements of apoplastic voltage changes were performed on a second leaf (Target leaf, T-leaf) with a distance of approximately 25 cm. The tip of the T-leaf was submerged in 5 mM KCl with 0.1 mM CaCl₂, pH 5, and the solution was connected to earth with a reference electrode filled with 0.5 M KCl. (B) The voltage response to 25 nM Alamethicin (ALA) (Sigma-Aldrich, Germany) added to a cut injury of the H. vulgare S-leaf at the indicated time shows a hyperpolarization of the apoplast (negative shift of the apoplastic potential), suggesting depolarization of the symplast. A typical recording of one out of three independent experiments is presented.     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C

Summary Gap junctional coupling was studied in pairs of murine pancreatic acinar cells using the double whole-cell patch-clamp technique. During stable electrical coupling, addition of OAG (1-oleoyl-2-acetyl-sn-glycerol) induced a progressive reduction of the junctional conductance to the detectable limit (3 pS). Prior to complete electrical uncoupling, varius discrete single channel conductances between 20 and 100 pS could be observed. Polymyxin B, a potent inhibitor of the protein kinase C (PKC) system, completely suppressed OAG-stimulated electrical uncoupling. Dialysis of cell pairs with solutions containing PKC. isolated from rat brain, also caused electrical uncoupling. The presence of 0.1mm dibutyryl cyclic AMP and 5mm ATP in the pipette solution, which serves to stabilize the junctional conductance, did not suppress the effects of OAG or isolated PKC. We conclude that an increase of protein kinase C activity leads to the closure of gap junction channels, presumably via a PKC-dependent phosphorylation of the junctional peptide, and that this mechanism is dominant over cAMP-dependent upregulatory effects in the experimental time range (1 hr). A correlation of the observed single channel conductances with the appearance of channel subconductance states or various channel populations is discussed.     

Morphology and electrophysiological characteristics of caudodorsal cells of Lymnaea stagnalis in dissociated cell culture

• 1.1. The neuroendocrine caudodorsal cells (CDCs) of Lymnaea stagnalis are a network of about 100 electrotonically coupled neurones. The CDCs release multiple peptides, including an ovulation hormone, during a period of electrical activity, the CDC-discharge.
• 2.2. In isolated brains, a similar period of electrical activity (the afterdischarge) can be induced in all CDCs by a period of intracellular repetitive suprathreshold stimulation of one CDC.
• 3.3. In order to study the regulation of this electrical behaviour in the absence of electrical interactions and in a controlled environment, experiments were performed on CDCs in dissociated cell culture.
• 4.4. Methods for isolation and cell culture are described. Cell cultures had long-term viability and outgrowth of neurities occurred under serum-free conditions.
• 5.5. CDCs in cell culture maintained their capability of producing afterdischarges upon electrical stimulation. Cells in culture appeared more excitable than cells in the intact isolated brain.
• 6.6. The characteristic responses of CDCs in intact isolated brains to acetylcholine and FMRFamide were preserved in cultured CDCs. Both agents induced a transient hyperpolarization of the membrane, inexcitability and inhibition of an ongoing discharge.
• 7.7. In experiments where isolated CDCs were closely apposed, but physically separate, it was found that an afterdischarge in one CDC could induce a discharge in the other CDC.
• 8.8. These results confirm previous results which showed that an excitatory factor is released from the brain during the afterdischarge (Ter Maat et al., 1988, Brain Res., 43, 77–82), and demonstrate that this excitatory factor is released from the CDCs themselves.

     

Inhibitor effects on the accumulation and efflux of nickel ions in plasmid pMOL28-harboring strains of Alcaligenes eutrophus

The accumulation and efflux of ⁶³Ni²⁺ ions were studied using two strains of the strictly respiratory bacterium Alcaligenes eutrophus, the wild type strain N9A and its transconjugant N9A-M243 which harbors plasmid pMOL28.1 encoding constitutive resistance to nickel. When 1 M ⁶³Ni²⁺ is added to respiring cells, strain N9A accumulates high, but M243 only negligibly small amounts of nickel. When the cells were preincubated for about 20 h under anoxic conditions and were then exposed to 1 M ⁶³Ni²⁺ anaerobically, both strains accumulated approximately the same amounts of nickel. Aeration of these preloaded cells resulted in rapid efflux by strain M243 but renewed uptake of nickel by N9A. ⁶³Ni²⁺ uptake and efflux are highly sensitive to protonophores such as CCCP, FCCP and TCS but insensitive to DCCD (each at 20 M concentrations). Measurements on the effects of the inhibitors on biosynthetic processes requiring ATP either from substrate phosphorylation or from electron transport phosphorylation made sure that at the concentrations used the inhibitor effects were specific. Thus the results suggest that in the nickelresistant strain M243 under normal aerobic conditions two constitutive energy-dependent nickel transport systems can function concomitantly, a chromosomally-determined specific uptake system and a plasmid-mediated specific nickel efflux system.     

A scalable life cycle inventory of an electrical automotive traction machine—Part I: design and composition

Purpose

A scalable life cycle inventory (LCI) model of a permanent magnet electrical machine, containing both design and production data, has been established. The purpose is to contribute with new and easy-to-use data for LCA of electric vehicles by providing a scalable mass estimation and manufacturing inventory for a typical electrical automotive traction machine. The aim of this article (part I of two publications) is to present the machine design, the model structure, and an evaluation of the models’ mass estimations.

Methods

Data for design and production of electrical machines has been compiled from books, scientific papers, benchmarking literature, expert interviews, various specifications, factory records, and a factory site visit. For the design part, one small and one large reference machine were constructed in a software tool, which linked the machines’ maximum ability to deliver torque to the mass of its electromagnetically active parts. Additional data for remaining parts was then gathered separately to make the design complete. The two datasets were combined into one model, which calculates the mass of all motor subparts from an input of maximum power and torque. The range of the model is 20–200 kW and 48–477 Nm. The validity of the model was evaluated through comparison with seven permanent magnet electrical traction machines from established brands.

Results and discussion

The LCI model was successfully implemented to calculate the mass content of 20 different materials in the motor. The models’ mass estimations deviate up to 21% from the examples of real motors, which still falls within expectations for a good result, considering a noticeable variability in design, even for the same machine type and similar requirements. The model results form a rough and reasonable median in comparison to the pattern created by all data points. Also, the reference motors were assessed for performance, showing that the electromagnetic efficiency reaches 96–97%.

Conclusions

The LCI model relies on thorough design data collection and fundamental electromagnetic theory. The selected design has a high efficiency, and the motor is suitable for electric propulsion of vehicles. Furthermore, the LCI model generates representative mass estimations when compared with recently published data for electrical traction machines. Hence, for permanent magnet-type machines, the LCI model may be used as a generic component estimation for LCA of electric vehicles, when specific data is lacking.

     

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.Apoptosis is an important regulatory mechanism of tissue homeostasis. It is triggered by the extrinsic pathway through the activation of proapoptotic receptors or by the intrinsic pathway through the destabilization of mitochondria in response to various forms of cell injury or stress.¹ Notably, stressed cells are also strongly influenced by intercellular communicative networks. This includes diffusible growth factors, cytokines and other small molecules secreted from neighbouring cells, which can modulate the fate of distressed cells. For example, stem cells release growth factors to protect dysfunctional neurons in the brain.² In tumour stroma, activated fibroblasts are thought to promote tumour progression by secreting growth factors that act in a paracrine manner.³ Moreover, contact-dependent signalling, for example, via adhesion molecules, can trigger contact inhibition or protection of endothelial cells.⁴ In addition, gap junctions have been shown to be involved in the transfer of death or survival molecules in different cell types.⁵ Therefore, the signals transferred from neighbouring cells influence the viability of target cells through different pathways.In 2004, our group described a previously unrecognized form of cell-to-cell interaction based on nanoscaled, F-actin-containing membrane tubes.^{6, 7} These tubes, referred to as membrane or tunneling nanotubes (TNTs), were subsequently found in numerous cell types in culture and in tissues.^{8, 9, 10, 11} Importantly, TNTs facilitate the intercellular exchange of diverse cellular signals and components ranging from electrical signalling to organelles.^{12, 13, 14, 15} Moreover, pathogens such as human immunodeficiency virus (HIV) and prions can spread between cells along TNTs.^{16, 17} Consistent with the model that TNTs are involved in cell-to-cell communication, apoptosis regulators may be transferred via TNTs between apoptotic and healthy cells to alter the fate of recipient cells. Indeed, it has been shown that TNTs can propagate the death signal Fas ligand between T lymphocytes to induce cell death.^{18, 19} TNTs have been also proposed to participate in the rescue of injured cardiomyoblasts or endothelial cells by mesenchymal stem cells (MSCs) through transferred mitochondria.^{20 ,21} However, the rescue mechanism by how and when this event was accomplished remains elusive.In this study, we found that PC12 cells stressed by ultraviolet (UV) radiation were rescued from apoptosis when cocultured with untreated, healthy PC12 cells. Single-cell analysis showed that stressed cells in the early stages of apoptosis form a new type of TNT to interact with untreated cells. These TNTs have a distinct cytoskeletal composition and biophysical properties when compared with TNTs interconnecting normal PC12 cells. We also observed the presence and transport of mitochondria in the TNTs formed by stressed cells. Notably, the rescue effect was inhibited when the formation of TNTs were impaired by incubating with an F-actin-depolymerizing drug, or when the mitochondria of rescuer cells were damaged. Our results suggest that the delivery of functional mitochondria via TNTs mediates the recovery of PC12 cells in the early stages of apoptosis.     

The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis

When two different strains of swarming Proteus mirabilis encounter one another on an agar plate, swarming ceases and a visible line of demarcation forms. This boundary region is known as the Dienes line and is associated with the formation of rounded cells. While the Dienes line appears to be the product of distinction between self and nonself, many aspects of its formation and function are unclear. In this work, we studied Dienes line formation using clinical isolates labeled with fluorescent proteins. We show that round cells in the Dienes line originate exclusively from one of the swarms involved and that these round cells have decreased viability. In this sense one of the swarms involved is dominant over the other. Close cell proximity is required for Dienes line formation, and when strains initiate swarming in close proximity, the dominant Dienes type has a significant competitive advantage. When one strain is killed by UV irradiation, a Dienes line does not form. Killing of the dominant strain limits the induction of round cells. We suggest that both strains are actively involved in boundary formation and that round cell formation is the result of a short-range killing mechanism that mediates a competitive advantage, an advantage highly specific to the swarming state. Dienes line formation has implications for the physiology of swarming and social recognition in bacteria.The gram-negative bacterium Proteus mirabilis is well known for its ability to differentiate into hyperflagellated, motile, and elongated swarmer cells that rapidly spread over a surface. When cultured on a nutrient agar plate, a strain of P. mirabilis typically is able to colonize the whole plate within 24 h. This phenomenon is both of interest in terms of the differentiation and survival strategy of the organism and of practical importance, as contamination of agar plates by swarming P. mirabilis is a common problem in diagnostic microbiology. When two different strains of P. mirabilis swarm on the same agar plate, a visible demarcation line with lower cell density forms at the intersection, and this line is known as a Dienes line (5, 6). A Dienes line is seen when both strains are swarming; it is not a property of the smaller vegetative cells (5). When two identical isolates meet, the swarming edges merge without formation of a Dienes line. This phenomenon has been used in epidemiological typing of clinical isolates (4, 20, 23, 28) and raises interesting questions concerning its mechanism and biological importance. Dienes typing in the clinical environment suggests that the number of Dienes types is large; 81 types were found in one study alone (25). Incompatibility between swarming strains may not be unique to P. mirabilis; a comparable process also appears to occur in Pseudomonas aeruginosa (19). Like many other bacteria, some P. mirabilis strains produce bacteriocins, termed proticines (3). It has been shown that Dienes line formation is not directly caused by a proticine, nor has any other secreted substance or cellular lysate been found to contribute to this phenomenon (27). There does, however, seem to be a circumstantial link between proticine production and Dienes line formation. In the 1970s Senior typed strains of P. mirabilis based on their proticine production and sensitivity (23, 24). He found that proticine production is not related to proticine sensitivity (apart from strains being resistant to their own proticine) but that there is a good correlation between a combined proticine production-sensitivity (P/S) type and Dienes line formation. Strains with the same P/S type do not form a Dienes line, whereas strains with different P/S types do. The more closely related the P/S types of two strains, the less clearly defined the Dienes line is, suggesting that relatedness of strains plays a central role. In contrast, a strain of Proteus vulgaris has been shown to be Dienes compatible with a strain of P. mirabilis with the same P/S type (24). Furthermore, Dienes incompatibility between otherwise identical strains can be triggered by phage lysogeny (2). In contrast to P/S type, the polysaccharide (O) or flagellar (H) serotype has been shown to have no relationship to Dienes line formation (25, 27).Research into the mechanism governing Dienes line formation in Proteus has revealed some important features. The Dienes line region contains many large and often rounded cells (5). The nature of these cells remains controversial. Dienes suggested that round cells originated from both strains but that viable round cells always originated from one of the two intersecting strains. He concluded from this that nonviable round cells should therefore be cells of the other strain (6). In contrast, Wolstenhome suggested that the round cells originated mostly from one of the two swarms involved and that only 50% of these cells were viable (32). It has also been noted that round cells occasionally seem to develop into stable L forms lacking a full cell wall and that they can grow to form tiny L-type colonies (5). Additionally, extracellular DNase has been found at the site of the Dienes line (1). The presence of this enzyme has been interpreted to be a result of cell lysis, as P. mirabilis is known to contain large amounts of DNase (22, 26). Recently, a cluster of six genes, termed ids (identification of self), has been linked to the incompatibility in interpenetration between two strains (and therefore formation of the visible demarcation line), although the function and expression of these genes are not understood yet (8).Despite the fact that Dienes line formation has been known for over 50 years, many questions concerning this phenomenon remain unanswered. Recent advances in imaging, molecular biology, and genomics offer new ways of investigating it. In this work, clinical isolates expressing fluorescent proteins were used to observe the cells involved in Dienes line formation in real time, to evaluate the fate of the round cells, and to test the role of extracellular material and direct cell-cell contact in this phenomenon. Furthermore, the biological role of the Dienes phenomenon in the competition between strains in different situations was investigated.