Bioelectricity Buzz

Recent Bioelectricity-Related Articles Selected by Ann M. Rajnicek, Media Editor of Bioelectricity

As spring emerges in the northern hemisphere and bulbs awaken from their winter slumber, it is fitting that this Special Issue of Bioelectricity features the bioelectrical aspects of plants. Fittingly, Bioelectricity Buzz opens with an article about the relationship between plant root surface charge and ion uptake. Other featured topics are migration of ciliated organisms in electric fields (EFs), dissection of molecule-level dynamics of proton pumps in neuron vesicles, and a wireless “smart” electrical bandage that improves wound healing.

“Char”ging at the Root: Biochar Alters Root Surface Charge and Increases Cation Uptake

Sadly, I lack the patience and the “green thumb” required to sustain a lovely garden, but Farhangi-Abriz and Ghassemi-Golezani (2023) suggest that adding biochar to the soil might help plant growth by changing the electrical properties of root surfaces in ways that boost ion uptake.

Optimal nutrient uptake by roots is vital for plant health, and various soil additives target root efficiency to improve crop vigor and productivity. Biochar is a carbon-rich substance produced by controlled combustion of organic material under anaerobic conditions. It is gaining prominence due to its potential to reduce nitrous oxide emissions and to lessen greenhouse gas emissions. It also improves soil quality and nutrient availability to enhance crop yield.

The local soil, root, microbial environment is known collectively as the rhizosphere. Conditions at the root–soil interface are crucial for determining the quality of plant nutrient and ion uptake, especially the surface properties of the root's epidermal cells. Plant root surfaces have a net negative charge due to carboxyl, hydroxyl, and phosphate groups at cell membranes and cell walls. The type and density of functional groups on the cell wall surface regulate uptake of nutrients, water, and ion exchange. Mechanistically, these processes are linked to the surface electrochemical properties of roots, including zeta potential, surface charge, and cation exchange capacity.

Biochar is known to improve the physical and chemical properties of soil and to ensure availability of nutrients in the rhizosphere, but its impact on the bioelectrical properties of roots was unknown. Farhangi-Abriz and Ghassemi-Golezani (2023) therefore tested the hypothesis that biochar addition to the rhizosphere would alter the electrochemical traits of roots by changing the functional groups on their surfaces, with consequences for calcium, magnesium, and iron uptake. They compared safflower (Carthamus tinctorius L.), an annual plant with a deep root system, with mint (Mentha crispa L.), a perennial with a shallow root system.

First, the influence of biochar on soil was quantified, revealing an increase of soil pH and its cation exchange capacity. Next, sterilized seeds sown into standard soil or biochar-enriched soil were grown under greenhouse conditions. After 45 days, biochar treatment had increased plant height, shoot biomass, and root mass for both plant types, but it enhanced root length only for safflower, not mint. Indeed, biochar was most successful in improving root and shoot growth of safflower.

Measurements of the actual content of calcium, magnesium and iron revealed higher levels of each in roots (both plant species) when grown in biochar-supplemented soil. Calcium was elevated by 36%, magnesium was increased in the range of 34–41%, but iron showed the largest increase, at 61% for mint and 63% for safflower. The maximum nutrient absorption capacity of the roots was also tested by placing roots in nutrient-rich solutions. Biochar treatment improved maximum sorption capacity of calcium by 30% and 70% for safflower and mint, respectively. For iron, the improvement was 42% for safflower and 55% for mint, with mint also showing the highest magnesium sorption capacity.

To determine the impact of biochar on the electrochemical properties of roots, their zeta potential was measured using the streaming potential method. Overall, safflower roots had the lowest zeta potential and lowest (most negative) surface charge when compared with mint. Biochar lowered zeta potential values by 31% for safflower and 42% for mint compared with control, and surface charge was also reduced by 30% and 35% for safflower and mint, respectively. When measured using the ion exchange colorimetric method, safflower had the highest cation exchange capacity. Tetrazolium chloride reduction intensity quantified root activity, revealing similar final root activity in both plant types.

Biochar addition increased the content of carboxyl groups of polygalacturonic acids (PGAs), hydroxycinnamic acids (HCAs), and phenolic hydroxyl groups on safflower and mint root walls. Indeed, the addition of biochar to soil increased the total amount of cation-exchangeable groups (PGAs and HCAs together) by about 30% in safflower and by about 32% in mint. Safflower roots had more functional groups, cation exchange capacity, and root activity than mint under both growing conditions, which may relate to the relatively deep roots of safflower, an annual plant.

Collectively, the experiments demonstrate for the first time that biochar impacts the electrochemical properties of plant root directly and is not restricted to influencing properties of soil in the rhizosphere. Biochar's influence on roots increases the number of functional groups on cell walls with consequent improvement of maximum sorption capacity of plant roots. Since cation exchange groups (HCAs and PGAs) are key for control of nutrient uptake, their increase would be expected to increase in plant biomass and crop yield, but this remains to be tested using a variety of plant and soils enriched with biochar.

All Hands to the Pump: Single-Molecule Scale Dynamics of Proton Pumps in Individual Neurotransmitter Vesicles

The proton gradient established across nerve synaptic vesicle (SV) membranes facilitates their filling with the neurotransmitters necessary for their role in synaptic signaling. Kosmidis and coworkers have revealed fascinating insights into the dynamics of proton pumping in individual vesicles at single-molecule resolution.

Vacuolar-type adenosine triphosphatases (V-ATPases) are a family of rotary mechanoenzymes that hydrolyze ATP to provide the energy required to pump protons across cell and organelle membranes. Although V-ATPases are important in many cell types, they serve an especially important role in neurons by facilitating the loading of neurotransmitters into intracellular SVs. This is vital for neuronal synaptic transmission, a neuron's main function, so it is surprising that each SV contains only one or two copies of the crucial V-ATPase.

Consequently, any fluctuation in the activity of the enzyme would affect the functionality of the vesicle, potentially impacting nerve signaling. To fully understand the role of V-ATPases in neurotransmitter function, it is therefore imperative to understand its functional dynamics at the single-molecule level. Employing a lot of patience and a toolbox of sophisticated techniques, Kosmidis and team have (perhaps unexpectedly) found that, rather than pumping continuously, the enzyme switches stochastically between three relatively long-term modes of activity or inactivity.

Kosmidis and team isolated SVs from rat brains. To permit fluorescence microscopy, SVs were loaded with between 16 and 300 copies of a genetically encoded fluorescent pH reporter and the soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAP) receptor (SNARE) acceptor complex, synaxin1 and SNAP25. The resulting single large unilamellar vesicles (LUVs) were coupled to a glass surface at a density consistent with single vesicle observation. Then, the SVs isolated from rat brains were introduced with fusion of one SV per LUV, yielding immobilized hybrid SVs containing the pH reporter and the entire SV proteome.

The addition of ATP to activate proton pumping caused an ATP-specific reduction of lumen pH in about 3–5% of vesicles, reaching an equilibrium plateau at about 15 min. The addition of the V-ATPase-specific inhibitor bafilomycin collapsed this pH gradient indicating that the V-ATPase is solely responsible for lumen acidification.

Acidification was monitored at the single particle level over periods of up to 3 h to reveal the long-term kinetics of vesicle lumen acidification. About 90% of the single vesicles displayed reversible stochastic pauses in acidification that could indicate transitions of the transporter to relatively long-lived inactive modes. Control experiments ruled out the possibility that this was attributable to a leakage event either through the channel or through the membrane. Acidification events were not synchronized between individual vesicles, but two distinct phases were revealed: intrinsic acidification (about 30 s) and mode-switching (about 100 s). The ability to resolve this stochastic switching further supported the notion of a very small number of individual V-ATPase molecules per vesicle, as did the observation that ∼81% of the active vesicles showed a single acidification plateau.

A Bayesian event detection model that explored the dynamics of transitions between modes supported the notion that a single stochastic process was involved and that it was indeed single-molecule activity that was being reported. However, about 30% of the mode-switching events modeled did not plateau because they were faster than the time required for pH equilibration. Information about the event lifetimes permitted an estimation that the pump spends about half the time switched off, indicating that the “off” mode plays an important role in the normal function of the V-ATPase beyond its well established “on” pumping cycle.

Modeling proton efflux events after the V-ATPase is switched off revealed the presence of two leakage paths: (the majority) longer duration slow leakage through the membrane, and faster duration leakage through the active ATPases. The authors concluded that this represents a third “leaky” mode of V-ATPase function in addition to the known “active” and “inactive” modes.

The influence of electrochemical proton gradient feedback on regulation of the V-ATPase pump was determined experimentally by quantifying the proton gradients of individual vesicles. A proton-mediated electrochemical membrane potential was established by adding only ATP, and then, chloride was added for charge compensation, effectively removing the electrical component of the electrochemical gradient. These conditions increased the maximal level of acidification, with an estimated 80% of the V-ATPase molecules increasing their pumping rates. This indicates that a positive feedback loop exists because increasing the proton gradient from 0.75 to 1.75 boosts the lifetime of the proton pumping “on” mode by 210%.

The authors are the first to test the effect of ATP on proton pumping at the single-molecule level, which is important for resolving how the speed of ATP hydrolysis and rotation speed of the V-ATPase molecule shaft relate quantitatively to the proton current. The Michaelis–Menten constant (K_m) of V-ATPases for ATP hydrolysis is about 100 μM, so they tested ATP concentrations in the range of 10–1000 μM. An ATP-dependent increase in proton pumping and change in pH was observed. Paradoxically, they found that the single-molecule pumping rate was constant in the range of 30–1000 μM. The interpretation is that at ATP levels much lower than its K_m coupling of shaft rotation to proton movement across the membrane is the main rate-limiting step but increasing ATP further results in futile hydrolysis and rotation cycles. Below 30 μM, ATP hydrolysis is the rate-limiting factor.

Collectively, this work shows that, at least for mammalian brain vesicles, the V-ATPase has a functional repertoire beyond its canonical proton pumping mode. It can switch between on, off, and leaky modes such that SV acidification for a population is asynchronous and stochastic. This has implications for nervous system function since each SV has no more than two V-ATPase molecules, and the V-ATPase-driven proton gradient is responsible for neurotransmitter loading.

This work demonstrates the power of single-molecule imaging for dissecting dynamics of protein function. Hopefully, future work will use similar techniques to explore the roles of other membrane proteins at such fine resolution.

A Model Swimmer: The Dynamics Underpinning Ciliate Microorganism Electrotaxis

When free-swimming ciliated microorganisms (e.g., Paramecium) encounter an EF in their environment, they respond with directional locomotion. Daul and colleagues have explored the spatiotemporal mechanisms by which extracellular voltage gradients control ciliary locomotory machinery responsible for steering.

The prospect of manipulating microrobots with external EFs to carry out tasks (e.g., environmental sensors) is increasingly plausible as miniaturized biomechanics emerge. These automata are often inspired by biology, so their performance would be optimized through understanding how living organisms respond predictably to such EFs. Daul and colleagues described a model of ciliate electrotaxis that integrates membrane potential and ciliary movement.

Coleps hirtus, a single-celled ciliate with distinctive calcified alveolar plates, has wide global distribution in planktonic freshwater and saltwater habitats. Its locomotion is accomplished by coordinated rhythmic beating of cilia aligned in longitudinal rows in the plasma membrane encasing its roughly barrel-shaped surface. Their swimming behavior is characterized by distinctive alternate episodes of linear forward movement in a counterclockwise helical spiral and tumbling circular swimming, with constant movement to facilitate feeding.

Although EF-directed swimming of Paramecium has been reported several times, cathode-directed swimming of C. hirtus was described only once previously (∼130 years ago), with reversal toward the anode recorded at higher EFs. A subsequent mechanistic hypothesis linked spatially graded changes in membrane potential and the resultant shift in voltage-gated ion channel activity with ciliary beating. By that hypothesis, the plasma membrane at the cathode-facing side of the cell becomes depolarized, and the anode-facing side becomes hyperpolarized, impacting behavior of distinct populations of ion channels. For example, at the anode-facing side, voltage-dependent potassium channels open, leading to an efflux of K⁺ ions and increased ciliary beat frequency. By contrast, in the cathode-facing membrane, voltage-dependent calcium channels open, leading to influx of Ca²⁺ ions.

Calcium levels are key for directing C. hirtus movement because as Ca²⁺ levels increase inside cilia their beat direction rotates, ultimately reversing completely. The reversed and amplified ciliary beating yields competing rearward and forward forces, with the dominant force depending on the orientation of the elongated cell relative to the field vector. Asymmetric ciliary beating in distinct cell regions results in torque, which orients the cell toward the cathode, so it moves in that direction.

Daul and colleagues revisited this hypothesis, modeling the dynamic motility behavior of C. hirtus as a function of the extracellular EF strength. Cells were exposed to a direct current EF of up to 11 V/cm using a custom-made rectangular chamber and live imaging with subsequent cell tracking at 20-s intervals. As expected, in the absence of an applied external EF, the cell migration paths were not oriented in an organized path; some displayed erratic helical swimming patterns, and some cells would merely rotate on the spot. In contrast, the number of cells showing stabilization of swimming helicity and directed electrotaxis toward the cathode increased with increasing EF strength (mean at about 2.1 V/cm).

Cells were subjected to reversal of the EF (3 V/cm) polarity at 1-min intervals for 20 min to further explore the dynamics of electrotaxis. The cells were monitored without an EF, and the same population was subjected to the EF reversal intervals, with observations made during the initial 20 s after reversal. Without an EF, about 10% of the cells moved toward the cathode, but once the EF was switched on, about 80% of the cells migrated to the cathode. However, after 6 min, the percentage began to decrease, dropping to 60% by 20 min. The number of cells in the observation window fell significantly over several minutes, possibility due to cells becoming exhausted. Therefore, the remaining experiments documenting swimming dynamics were restricted to a 20-s observation period.

To determine the threshold for cathodal migration, the EF was increased by 0.2 V/cm increments up to 3 V/cm and then by 1 V/cm increments until 11 V/cm. The threshold EF intensity or cathode direction was identified at 0.4 V/cm, but at EFs >3 V/cm, about 1–2% of cells moved toward the anode, increasing to about 5% at 11 V/cm. The authors proposed that the reversed electrotaxis of this subpopulation represents cells in which the depolarization of the membrane causes the reverse beat of the cilia to dominate, leading to slower, but anode-directed locomotion.

Although no relationship was identified for mean velocity of cells and EF strength, the EF reduced the variability of swimming velocity so that the monotonic directionality was increased. The result was that cells would move more efficiently toward the cathode side of the observation chamber under EF stimulation compared with controls.

The influence of calcium on EF-directed migration was tested due to its key role in ciliary beating. Extracellular calcium was manipulated over the range of 0–30 μM using 20 s observation and a 3 V/cm EF (and no EF control). Without extracellular calcium in the medium, most cells did not move, demonstrating its importance for the ciliary beating that underpins locomotion. The threshold extracellular calcium level that yielded cathode-directed swimming was 5 μM. At that concentration, the responses mimicked the baseline responses described above in which medium contains 250 μM CaCl₂.

Collectively, the data extend understanding of electrotaxis by identifying key EF threshold values and adding response times for C. hirtus electrotaxis. However, further work is still needed. A clear advantage of the method used by Daul's team is that it allows for simultaneous automated tracking of many cells (high throughput) and therefore generates the large population data required for useful mathematical modeling. But a disadvantage in the context of this study is that it does not permit exploration of the spatiotemporal dynamics of individual cells as they respond to the initial EF stimulus, or even more intriguingly, to its reversal.

I look forward to future direct evidence for the role of spatially distinct membrane potentials in driving the ciliary beating mechanism in individual C. hirtus undergoing electrotaxis. Specifically, its potential (pun intended) role in mechanisms underpinning the reversal of swimming at higher EFs. Perhaps, this could be performed using the new generation of membrane potential reporters and, considering their constant motility, a fast camera!

Smart Wound Dressing: A Wireless Electroactive Bandage with Built in Feedback Sensors

Unfortunately, wound care management for chronic nonhealing wounds still has huge business potential due to the global scale and persistence of the clinical problem. Jiang and a collection of cross-disciplinary colleagues presented a clever smart bandage that addresses several of the key limitations inherent in most “electro-dressings.”

Nonhealing wounds cause significant sustained personal suffering for patients, and they represent a huge burden on health care systems. The problem is compounded by increasing global incidence of diabetes and its related risk of significant foot ulcers, combined with an aging population that tends to heal less efficiently.

A wide range of wound dressing therapies incorporate vacuum systems, drugs, or materials aimed at improving wound closure and preventing infection, but these are usually passive systems, with no feedback to permit altering their characteristics as wound conditions change. Although “smart” bandages provide promise because they can sense wound conditions including pH, oxygenation, and electrical impedance, they seldom integrate a responsive treatment reacting to the wound environment.

Several factors need to be incorporated into an optimal smart bandage designed to apply an electrical healing stimulus. It needs to be flexible to permit movement of the underlying tissues without compromising signal detection and delivery. It needs to be portable and wireless, so signals can be sent without the need for cumbersome wires (a potential infection route) and battery packs that are cumbersome to wear. It needs to integrate hardware that is sensitive enough to detect the desired signals in real time, but it must incorporate electrodes that are safe to use near wounds for prolonged periods without decay of performance or excessive adhesion to tissues (removing the dressing could exacerbate injury). Importantly, it needs to be cost-effective.

Jiang and coworkers have developed a smart bandage that meets many of these requirements. The design incorporates a thin (∼100 μm) miniaturized, flexible printed circuit board with an energy harvesting antenna, a microcontroller unit, a crystal oscillator, and filter sensor circuits for continuous sensing of temperature (an indicator of infection) and wound impedance (a measure of wound closure). A parallel circuit delivers programmed electrical stimulation using an adhesive hydrogel electrode based on poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) that delivers both electrical and ionic current. The high charge injection capacity of the hydrogel is maintained even after 10,000 cycles of charge injection. A clever innovation is that the hydrogel backbone can change phase on demand rapidly response to temperature elevation exceeding 40°C. This means the adhesive electrodes peel away easily on demand (but not before), preventing secondary skin damage.

The dressing was tested in a preclinical mouse model. Initial studies proved that the adhesive and electrodes did not cause any adverse skin reactions and that the mice could move freely without discomfort while wearing it.

In a splinted excisional wound model, control mice received sterile dressings without electrical stimulation, but experimental mice received continuous electrical pulses. Compared with the control group, the electrically treated wounds healed significantly faster, as evidenced both by faster elevation of wound impedance reported by the dressing and by a faster decrease in wound area. Additionally, dermal thickness and collagen deposition were improved in the treated group. Neovascularization was also improved in the stimulated group, assessed by microvessel counts and smooth muscle actin. Wound infection was also significantly lower in the treated group, with lower bacterial counts compared with controls.

Diabetic skin is notoriously poor at wound healing, so Jiang and colleagues used a streptozotocin mouse model resembling type 1 diabetes to test the ability of the dressing to improve diabetic wound healing. At the cellular level, they found that the electrical stimulation promoted the expected directional EF responses.

The influence of the electric stimulation on circulating immune cells was tested using a parabiosis model in which a wild-type (WT) mouse was paired with a mouse expressing green fluorescent protein. The WT mouse was wounded and either left untreated (control) or fitted with an active stimulator bandage. After 5 days, the wound tissues were excised from each mouse and subjected to single-cell RNA sequencing. Among circulating inflammatory cells, macrophages and monocytes had the highest number of differentially expressed genes in both treated and unstimulated wounds. Further analysis of gene profiles revealed activation of a variety of genes consistent with a proregenerative phenotype upon electrical stimulation and that the transcriptional changes were confirmed at the protein level. Together, the data suggest that electrical stimulation may drive macrophages toward a more regenerative phenotype conducive to healing.

The innovative smart plaster reported here provides solutions to many of the issues that have prevented such strategies moving successfully to the clinic. However, the next important challenge is to scale the system up, so it is suitable for large, deep clinically significant wounds, such as severe diabetic foot ulcers or deep bed sores that are typically recalcitrant to healing.

Bacteria Having a Ball: Collective Cell Motion and Condensation in an Alternating EF

The ability to coalesce bacteria into densely packed clusters on demand has potential utility in biosensing applications and for systems to detect very early stages of bacterial infection. Toward that end, Ba and colleagues demonstrated dynamic aggregation and separation of ball-shaped bacterial clusters under the influence of an oscillating EF.

Motile bacteria can swarm and develop complex collective movements when influenced by external factors such as hydrodynamic fluid flow or a magnetic field (for magnetotactic species). External EFs can direct the movement of nonmagnetotactic motile bacteria too. For example, Escherichia coli move along the EF vector of a direct current EF, but the dynamics of collective bacterial movement in an EF is not well understood.

Ba and colleagues encased a suspension of E. coli bacteria between two parallel glass electrode plates coated with conductive indium tin oxide spaced 180 μm apart. Crucially, the suspension density was below that known to permit the spontaneous flocking and swarming induced by electrostatic cell–cell interactions in the absence of any other external factors. An oscillating EF was used to prevent directed movement along the EF vector.

As anticipated, the bacteria moved independently in random directions before the EF was switched on, but in EFs above 0.05 V/μm, the bacteria accumulated at the electrodes with toxic effects on the cells. Therefore, EFs of less than 0.05 V/μm were used to influence bacterial aggregation dynamics.

At frequencies in the range of 1–100 Hz, the system is characterized spatially by a “spare phase,” with relatively few bacteria and a “dense phase” in which bacteria tend to accumulate at higher density. These are not completely distinct; the dense phases are interconnected, and individual bacteria move back and forth between both phases. At frequencies below 1 Hz, the dense domains collapse inwardly, forming compact oscillating bacterial clusters. Collapse is rapid, occurring within ∼90 s under EF conditions of 0.035 V/μm and 0.2 Hz.

Confocal microscopy confirmed that the clusters are suspended in the aqueous phase (rather than being attached to the electrode plates) and are spherical, with individual bacteria moving freely within the spherical structures. Upon cessation of the EF, the structures dissolve, with bacteria dissociating away gradually to be free swimming individuals once again. This indicates that the bacteria are alive within the structures and that the EF conditions are not toxic.

Ba and coworkers theorized that the spherical assemblies are the result of electroconvective rolls within the dielectric liquid supporting the bacterial suspension. By this notion, the bacteria act as charged colloidal particles suspended within a bulk liquid experiencing electroconvective flow. Charged colloid particles with negligible influence of gravity would be attracted toward the electrodes, but with significant gravitational influence, the colloid particles would be suspended in the bulk liquid due to a balance between electrophoretic forces and gravity. However, bacteria are alive, so they are not merely influenced passively by gravity.

To test whether biological activity was important for the formation of the spherical structures, polystyrene (PS) particles 2 μm in diameter were used as proxy bacteria. PS particles are charged in an aqueous environment, and they approximate the buoyant density of water, therefore balancing the influence of gravity on the system. When subjected to conditions of an EF of 0.035 V/μm at 0.3 Hz, dynamic clusters of PS spheres are formed, resembling the behaviors observed in living bacterial suspensions. This indicates that the dynamic spheres composed of bacteria emerge due to their charged colloid properties and not as a direct result of any biological function.

By balancing the polarity of the EF (oscillation frequency) with its magnitude, it is possible to create stable suspended dynamic clusters. However, there is a size limit (critical radius) beyond which the clusters split, forming smaller dynamic clusters. For a given EF magnitude, the critical radius was found experimentally to depend on the frequency of the oscillating EF. In general, as the frequency increased, the critical radius at which the clusters split decreased, but the critical radius did not depend on the magnitude of the EF (V/μm). The authors suggested that this is a consequence of the need for sufficient response time to reestablish the steady electroconvective rolls that underpin sphere formation within the dielectric fluid. The critical radius also depends on bacterial density in the suspension; at high densities, the critical radius is higher, and below a critical bacterial density, the number of clusters is very low.

The study suggests that the behavior of bacteria, and indeed other charged particles (colloids), can be modulated in a dielectric liquid by manipulating the electroconductive flow within it. Better understanding of the underlying principles as they apply to specific charged nonbiological nanoparticles and biological species (e.g., viruses, bacteria, protein aggregates) would permit innovative early detection systems and sensors by creating conditions to aggregate them, even when present at low concentrations.

That's all the Buzz until next issue. I hope you found something there to inspire you. I'm off to the shops now to get some biochar for my garden, just in time for spring planting!