Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity.

Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and intercellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cellular proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane-potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namely Bacillus subtilis and Escherichia coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5-second electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K+ and the deletion of the K+ channel suggested that the hyperpolarization response is caused by the K+ efflux through the channel. When cells are preexposed to 400 ± 8 nm wavelength light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh-Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore, or alcohol. Therefore, electrically induced membrane-potential dynamics offer a reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level.

Ion channels enable electrical communication in bacterial communities.

The study of bacterial ion channels has provided fundamental insights into the structural basis of neuronal signalling; however, the native role of ion channels in bacteria has remained elusive. Here we show that ion channels conduct long-range electrical signals within bacterial biofilm communities through spatially propagating waves of potassium. These waves result from a positive feedback loop, in which a metabolic trigger induces release of intracellular potassium, which in turn depolarizes neighbouring cells. Propagating through the biofilm, this wave of depolarization coordinates metabolic states among cells in the interior and periphery of the biofilm. Deletion of the potassium channel abolishes this response. As predicted by a mathematical model, we further show that spatial propagation can be hindered by specific genetic perturbations to potassium channel gating. Together, these results demonstrate a function for ion channels in bacterial biofilms, and provide a prokaryotic paradigm for active, long-range electrical signalling in cellular communities.

Metabolic co-dependence gives rise to collective oscillations within biofilms.

Cells that reside within a community can cooperate and also compete with each other for resources. It remains unclear how these opposing interactions are resolved at the population level. Here we investigate such an internal conflict within a microbial (Bacillus subtilis) biofilm community: cells in the biofilm periphery not only protect interior cells from external attack but also starve them through nutrient consumption. We discover that this conflict between protection and starvation is resolved through emergence of long-range metabolic co-dependence between peripheral and interior cells. As a result, biofilm growth halts periodically, increasing nutrient availability for the sheltered interior cells. We show that this collective oscillation in biofilm growth benefits the community in the event of a chemical attack. These findings indicate that oscillations support population-level conflict resolution by coordinating competing metabolic demands in space and time, suggesting new strategies to control biofilm growth.

Scanning Ion Conductance Microscopy Reveals Differences in the Ionic Environments of Gram-Positive and Negative Bacteria.

This paper reports on the use of scanning ion conductance microscopy (SICM) to locally map the ionic properties and charge environment of two live bacterial strains: the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. SICM results find heterogeneities across the bacterial surface and significant differences among the Gram-positive and Gram-negative bacteria. The bioelectrical environment of the B. subtilis was found to be considerably more negatively charged compared to E. coli. SICM measurements, fitted to a simplified finite element method (FEM) model, revealed surface charge values of -80 to -140 mC m-2 for the Gram-negative E. coli. The Gram-positive B. subtilis show a much higher conductivity around the cell wall, and surface charge values between -350 and -450 mC m-2 were found using the same simplified model. SICM was also able to detect regions of high negative charge near B. subtilis, not detected in the topographical SICM response and attributed to the extracellular polymeric substance. To further explore how the B. subtilis cell wall structure can influence the SICM current response, a more comprehensive FEM model, accounting for the physical properties of the Gram-positive cell wall, was developed. The new model provides a more realistic description of the cell wall and allows investigation of the relation between its key properties and SICM currents, building foundations to further investigate and improve understanding of the Gram-positive cellular microenvironment.

Biofilm and swarming emergent behaviours controlled through the aid of biophysical understanding and tools.

Bacteria can organise themselves into communities in the forms of biofilms and swarms. Through chemical and physical interactions between cells, these communities exhibit emergent properties that individual cells alone do not have. While bacterial communities have been mainly studied in the context of biochemistry and molecular biology, recent years have seen rapid advancements in the biophysical understanding of emergent phenomena through physical interactions in biofilms and swarms. Moreover, new technologies to control bacterial emergent behaviours by physical means are emerging in synthetic biology. Such technologies are particularly promising for developing engineered living materials (ELM) and devices and controlling contamination and biofouling. In this minireview, we overview recent studies unveiling physical and mechanical cues that trigger and affect swarming and biofilm development. In particular, we focus on cell shape, motion and density as the key parameters for mechanical cell-cell interactions within a community. We then showcase recent studies that use physical stimuli for patterning bacterial communities, altering collective behaviours and preventing biofilm formation. Finally, we discuss the future potential extension of biophysical and bioengineering research on microbial communities through computational modelling and deeper investigation of mechano-electrophysiological coupling.

Nuclear retention of importin α coordinates cell fate through changes in gene expression.

Various cellular stresses including oxidative stress induce a collapse of the Ran gradient, which causes accumulation of importin α in the nucleus and a subsequent block of nuclear protein import. However, it is unknown whether accumulated importin α performs roles in the nucleus after its migration in response to stress. In this study, we found that nuclear-retained importin α2 binds with DNase I-sensitive nuclear component(s) and exhibits selective upregulation of mRNA encoding Serine/threonine kinase 35 (STK35) by microarray analysis. Chromatin immunoprecipitation and promoter analysis demonstrated that importin α2 can access to the promoter region of STK35 and accelerate its transcription in response to hydrogen peroxide exposure. Furthermore, constitutive overexpression of STK35 proteins enhances caspase-independent cell death under oxidative stress conditions. These results collectively reveal that nuclear-localized importin α2 influences gene expression and contributes directly to cell fate outcomes including non-apoptotic cell death.

Localized cell death focuses mechanical forces during 3D patterning in a biofilm.

From microbial biofilm communities to multicellular organisms, 3D macroscopic structures develop through poorly understood interplay between cellular processes and mechanical forces. Investigating wrinkled biofilms of Bacillus subtilis, we discovered a pattern of localized cell death that spatially focuses mechanical forces, and thereby initiates wrinkle formation. Deletion of genes implicated in biofilm development, together with mathematical modeling, revealed that ECM production underlies the localization of cell death. Simultaneously with cell death, we quantitatively measured mechanical stiffness and movement in WT and mutant biofilms. Results suggest that localized cell death provides an outlet for lateral compressive forces, thereby promoting vertical mechanical buckling, which subsequently leads to wrinkle formation. Guided by these findings, we were able to generate artificial wrinkle patterns within biofilms. Formation of 3D structures facilitated by cell death may underlie self-organization in other developmental systems, and could enable engineering of macroscopic structures from cell populations.

Differential role for transcription factor Oct4 nucleocytoplasmic dynamics in somatic cell reprogramming and self-renewal of embryonic stem cells.

Oct4 is a member of the POU family of transcription factors and plays a critical role in both maintenance of the undifferentiated state of embryonic stem (ES) cells and in the reprogramming of somatic cells to induced pluripotent stem cells. Oct4 is imported into the nucleus where it functions as a transcription factor; however, the spatiotemporal dynamic behavior of Oct4 remains largely unknown. In the present study we show that Oct4 is a nucleocytoplasmic shuttling protein. Furthermore, although Oct4 mutants with altered nuclear import/export activity were able to maintain the self-renewal of ES cells, they displayed limited potential for cellular reprogramming. These results indicate that the intracellular localization of Oct4, which is dependent on nucleocytoplasmic shuttling, must be more strictly regulated for cellular reprogramming, suggesting that Oct4 plays differential roles in the self-renewal of ES cells and in somatic cell reprogramming.

Triggering neural differentiation of ES cells by subtype switching of importin-alpha.

Nuclear proteins are selectively imported into the nucleus by transport factors such as importin-alpha and importin-beta. Here, we show that the expression of importin-alpha subtypes is strictly regulated during neural differentiation of mouse embryonic stem (ES) cells, and that the switching of importin-alpha subtype expression is critical for neural differentiation. Moreover, reproducing the switching of importin-alpha subtype expression in undifferentiated ES cells induced neural differentiation in the presence of leukaemia inhibitory factor (LIF) and serum, coordinated with the regulated expression of Oct3/4, Brn2 and SOX2, which are involved in ES-neural identity determination. These transcription factors were selectively imported into the nucleus by specific subtypes of importin-alpha. Thus, importin-alpha subtype switching has a major impact on cell differentiation through the regulated nuclear import of a specific set of transcription factors. This is the first study to propose that transport factors should be considered as major players in cell-fate determination.

Electrotaxis behavior of droplets composed of aqueous Belousov-Zhabotinsky solutions suspended in oil phase.

Taxis is ubiquitous in biological and physical chemistry systems as a response to various external stimulations. We prepared aqueous droplets containing Belousov-Zhabotinsky (BZ) solutions suspended on an oleic acid oil phase subject to DC electric field and found that these BZ droplets undergo chemically driven translational motion towards the negative electrode under DC electric field. This electrotaxis phenomenon originates from the field-induced inhomogeneous distribution of reactants, in particular Br[Formula: see text] ions, and consequently the biased location of the leading centers towards the positive electrode. We define the 'leading center' (LC) as a specific location within the droplet where the BZ chemical wave (target pattern) is initiated. The chemical wave generated from the LC propagates passing the droplet center of mass and creates a gradient of interfacial tension when reaching the droplet-oil interface on the other side, resulting in a momentum exchange between the droplet and oil phases which drives the droplet motion in the direction of the electric field. A greater electric field strength renders a more substantial electrotaxis effect.

Thioflavin T indicates mitochondrial membrane potential in mammalian cells.

The fluorescent benzothiazole dye thioflavin T (ThT) is widely used as a marker for protein aggregates, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilization of ThT in research and diagnosis. Here, we show that ThT localizes into the mitochondria of HeLa cells in a membrane-potential-dependent manner. Specifically, ThT colocalized in cells with the mitochondrial membrane potential indicator tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ = 405 nm), but neither factor alone, caused depolarization of mitochondrial membrane potential. This additive effect of the concentration and blue light was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its effect on mitochondrial membrane potential through photosensitization. These results show that ThT can act as a mitochondrial membrane potential indicator in mammalian cells, when used at low concentrations and with low blue light exposure. However, it causes dissipation of the mitochondrial membrane potential depending additively on its concentrations and blue light exposure. This conclusion motivates a re-evaluation of ThT's use at micromolar range in live-cell analyses and indicates that this dye can enable future studies on the potential connections between mitochondrial membrane potential dynamics and protein aggregation.

Membrane Targeted Azobenzene Drives Optical Modulation of Bacterial Membrane Potential.

Recent studies have shown that bacterial membrane potential is dynamic and plays signaling roles. Yet, little is still known about the mechanisms of membrane potential dynamics regulation-owing to a scarcity of appropriate research tools. Optical modulation of bacterial membrane potential could fill this gap and provide a new approach for studying and controlling bacterial physiology and electrical signaling. Here, the authors show that a membrane-targeted azobenzene (Ziapin2) can be used to photo-modulate the membrane potential in cells of the Gram-positive bacterium Bacillus subtilis. It is found that upon exposure to blue-green light (λ = 470 nm), isomerization of Ziapin2 in the bacteria membrane induces hyperpolarization of the potential. To investigate the origin of this phenomenon, ion-channel-deletion strains and ion channel blockers are examined. The authors found that in presence of the chloride channel blocker idanyloxyacetic acid-94 (IAA-94) or in absence of KtrAB potassium transporter, the hyperpolarization response is attenuated. These results reveal that the Ziapin2 isomerization can induce ion channel opening in the bacterial membrane and suggest that Ziapin2 can be used for studying and controlling bacterial electrical signaling. This new optical tool could contribute to better understand various microbial phenomena, such as biofilm electric signaling and antimicrobial resistance.

Phase-separated nuclear bodies of nucleoporin fusions promote condensation of MLL1/CRM1 and rearrangement of 3D genome structure.

NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies containing CRM1, a nuclear export receptor. However, these nuclear bodies' function in controlling gene expression remains elusive. Here, we demonstrate that the nuclear bodies of NUP98::HOXA9 and SET::NUP214 promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase essential for the maintenance of HOX gene expression. These nuclear bodies are robustly associated with MLL1/CRM1 and co-localized on chromatin. Furthermore, whole-genome chromatin-conformation capture analysis reveals that NUP98::HOXA9 induces a drastic alteration in high-order genome structure at target regions concomitant with the generation of chromatin loops and/or rearrangement of topologically associating domains in a phase-separation-dependent manner. Collectively, these results show that the phase-separated nuclear bodies of nucleoporin fusion proteins can enhance the activation of target genes by promoting the condensation of MLL1/CRM1 and rearrangement of the 3D genome structure.

Seeking Insights into Aging Through Yeast Mitochondrial Electrophysiology.

During aging, mitochondrial membrane potential, a key indicator for bioenergetics of cells, depolarizes in a wide range of species-from yeasts, plants to animals. In humans, the decline of mitochondrial activities can impact the high-energy-consuming organs, such as the brain and heart, and increase the risks of age-linked diseases. Intriguingly, a mild depolarization of mitochondria has lifespan-extending effects, suggesting an important role played by bioelectricity during aging. However, the underpinning biophysical mechanism is not very well understood due in part to the difficulties associated with a multiscale process. Budding yeast Saccharomyces cerevisiae could provide a model system to bridge this knowledge gap and provide insights into aging. In this perspective, we overview recent studies on the yeast mitochondrial membrane electrophysiology and aging and call for more electrochemical and biophysical studies on aging.

Swarming bacteria undergo localized dynamic phase transition to form stress-induced biofilms.

Self-organized multicellular behaviors enable cells to adapt and tolerate stressors to a greater degree than isolated cells. However, whether and how cellular communities alter their collective behaviors adaptively upon exposure to stress is largely unclear. Here, we investigate this question using Bacillus subtilis, a model system for bacterial multicellularity. We discover that, upon exposure to a spatial gradient of kanamycin, swarming bacteria activate matrix genes and transit to biofilms. The initial stage of this transition is underpinned by a stress-induced multilayer formation, emerging from a biophysical mechanism reminiscent of motility-induced phase separation (MIPS). The physical nature of the process suggests that stressors which suppress the expansion of swarms would induce biofilm formation. Indeed, a simple physical barrier also induces a swarm-to-biofilm transition. Based on the gained insight, we propose a strategy of antibiotic treatment to inhibit the transition from swarms to biofilms by targeting the localized phase transition.

An agent-based model of the interrelation between the COVID-19 outbreak and economic activities.

As of July 2020, COVID-19 caused by SARS-COV-2 is spreading worldwide, causing severe economic damage. While minimizing human contact is effective in managing outbreaks, it causes severe economic losses. Strategies to solve this dilemma by considering the interrelation between the spread of the virus and economic activities are urgently needed to mitigate the health and economic damage. Here, we propose an abstract agent-based model of the COVID-19 outbreak that accounts for economic activities. The computational simulation of the model recapitulates the trade-off between the health and economic damage associated with voluntary restraint measures. Based on the simulation results, we discuss how the macroscopic dynamics of infection and economics emerge from individuals' behaviours. We believe our model can serve as a platform for discussing solutions to the above-mentioned dilemma.

The Microbiologist's Guide to Membrane Potential Dynamics.

All cellular membranes have the functionality of generating and maintaining the gradients of electrical and electrochemical potentials. Such potentials were generally thought to be an essential but homeostatic contributor to complex bacterial behaviors. Recent studies have revised this view, and we now know that bacterial membrane potential is dynamic and plays signaling roles in cell-cell interaction, adaptation to antibiotics, and sensation of cellular conditions and environments. These discoveries argue that bacterial membrane potential dynamics deserve more attention. Here, we review the recent studies revealing the signaling roles of bacterial membrane potential dynamics. We also introduce basic biophysical theories of the membrane potential to the microbiology community and discuss the needs to revise these theories for applications in bacterial electrophysiology.

Rapid Detection of Proliferative Bacteria by Electrical Stimulation.

Detecting live bacteria is an important task for antimicrobial susceptibility testing (AST) in the medical sector and for quality-monitoring in biological industries. Current methods for live-bacteria detection suffer limitations in speed or sensitivity. In a recent paper, we reported that electrical response dynamics in membrane potential enable single-cell rapid detection of live bacteria. The electrical response can be observed within a minute after electrical stimulation. Thus, it has potential in accelerating AST and the monitoring of biological samples. This method also enables experiments for biophysical and microbiological investigations into bacterial electrophysiology. With the hope that more researchers, scientists and engineers will use electrical stimulation for their assays, here we detail each step of the electrical stimulation experiment.

Pattern Engineering of Living Bacterial Colonies Using Meniscus-Driven Fluidic Channels.

Creating adaptive, sustainable, and dynamic biomaterials is a forthcoming mission of synthetic biology. Engineering spatially organized bacterial communities has a potential to develop such bio-metamaterials. However, generating living patterns with precision, robustness, and a low technical barrier remains as a challenge. Here we present an easily implementable technique for patterning live bacterial populations using a controlled meniscus-driven fluidics system, named as MeniFluidics. We demonstrate multiscale patterning of biofilm colonies and swarms with submillimeter resolution. Utilizing the faster bacterial spreading in liquid channels, MeniFluidics allows controlled bacterial colonies both in space and time to organize fluorescently labeled Bacillus subtilis strains into a converged pattern and to form dynamic vortex patterns in confined bacterial swarms. The robustness, accuracy, and low technical barrier of MeniFluidics offer a tool for advancing and inventing new living materials that can be combined with genetically engineered systems, and adding to fundamental research into ecological, evolutional, and physical interactions between microbes.

Bioelectrical understanding and engineering of cell biology.

The last five decades of molecular and systems biology research have provided unprecedented insights into the molecular and genetic basis of many cellular processes. Despite these insights, however, it is arguable that there is still only limited predictive understanding of cell behaviours. In particular, the basis of heterogeneity in single-cell behaviour and the initiation of many different metabolic, transcriptional or mechanical responses to environmental stimuli remain largely unexplained. To go beyond the status quo, the understanding of cell behaviours emerging from molecular genetics must be complemented with physical and physiological ones, focusing on the intracellular and extracellular conditions within and around cells. Here, we argue that such a combination of genetics, physics and physiology can be grounded on a bioelectrical conceptualization of cells. We motivate the reasoning behind such a proposal and describe examples where a bioelectrical view has been shown to, or can, provide predictive biological understanding. In addition, we discuss how this view opens up novel ways to control cell behaviours by electrical and electrochemical means, setting the stage for the emergence of bioelectrical engineering.