Session H65: Controlling Cells with Electric Fields

Controlling the intracellular dynamics of neuronal model systems

Excitable tissues in the body have unique properties that allow for the transfer of information between individual cells and across larger scales. For example, recent work has investigated the spatiotemporal dynamics of electrical activity propagation in neural and cardiac tissues in both in vivo and in vitro settings. In the present work, we study how the intracellular behavior of in vitro cultures of primary rat cortical neurons can be tuned and controlled with electric fields. However, in addition to the effects on electrical signal propagation that are traditionally studied, we are also interested in the morphological responses of neurons to these electric fields with the goal of understanding the interaction between electrical activity propagation and cytoskeletal dynamics, particularly of actin. To accomplish this goal, we do the following: 1) apply electric field stimulation to the in vitro neuronal culture, 2) image changes in transmembrane potential or intracellular actin, and 3) quantify the intracellular response. We show that the dynamics of actin change significantly over time as the structural components of neurons (axons and dendrites) mature and become more stable. Moreover, we show that neurons can be electrically activated by direct current (DC) electric fields, but the degree and complexity of the response to the field strongly depends on the age of the cells with a more prevalent response observed as the neurons (and therefore, synapses) have matured. In sum, we have found that we can tune neuronal cell behavior with electric fields, and in future work, we hope to apply these same perturbations to other cells and make inferences about the intracellular behavior of other electrically excitable cell types.   

Precisely regulate ERK signaling pathway with local electric fields

Our main research interest is to understand how artificial electronics can interact with biological systems. Live cells rely on a big network of signaling pathways that sense and respond to biochemical, electrical and mechanical (BEM) stimuli. We want to explore if we can modulate this BEM network, particularly with external electric field, and investigate the mechanism at the molecular level.
In this presentation I will talk about our recent discovery of modulation of extracellular-signal-regulated kinase (ERK) pathway using alternative current (AC) electric fields (EFs). The amplitude, duration, and frequency of activation of the ERK pathway code diverse spectrum of information at cell, tissue and organism levels to instruct cells to migrate, proliferate, or differentiate. Synchronized control of ERK activation would provide a powerful approach to regulate cell behaviors. Here we show for the first time that AC EFs in a new frequency range can reproducibly activate ERK activities through patterned local microelectrodes with single-cell resolution. Both the amplitude and frequency of ERK activation can be precisely synchronized and modulated. We pinpointed a new mechanism of AC EF induced highly specific phosphorylation of epidermal growth factor (EGF) receptor (EGFR) to activate the EGFR-ERK pathway, which may serve as a powerful platform for control of cell behaviors with implications in wide range of biomedical applications.   

Control of immune cells via chemical, electrical and mechanical cues

Directed migration of immune cells is essential in facilitating the wound healing process. Coordination of collagen scaffolding, chemical gradients and endogenous electric fields guides cells to the site of inflammation. However, mimicking this system in vitrohas not been previously explored. In this work, we combine the above guidance cues artificially, and in a controlled manner, to understand better immune responses. Specifically, we are studying in the cells’ intracellular response via the actin cytoskeleton. By investigating and analyzing the actin cytoskeleton as an excitable system, a more complete understanding of contact guidance, chemotaxis and electrotaxis may be obtained.
  

Investigating wave morphodynamics in electrically active cell sheets

Biological systems exhibit many emergent phenomena, from schooling behavior in fish to electrical network dynamics in neurons and cardiomyocytes. On a tissue scale, these collective dynamics often arise in response to changes in the extracellular and intracellular environment. In this talk, we describe the collective signaling of HEK293T/17 cells modified to express sodium and potassium ion channels such that they form electrically-active monolayers. We investigate the collective firing dynamics across multiple spatio-temporal scales in these cell sheets in a highly-controlled fashion in order to understand the emergence of various, driven electrical wave morphologies and propagation dynamics. To accomplish this goal, we apply electrical and optical perturbations utilizing a novel optical system—a Multi-Scale Microscope—that allows for simultaneous imaging across two different spatial scales using epifluorescence and rescan confocal imaging techniques.   

Electric-Field Manipulation of a Cell-Free Gene Expression Reaction

Biological systems are regulated dynamically to respond to external environmental perturbations, and change their internal state as a result. Introducing controlled dynamical perturbations in minimal systems can provide insight into those processes. Currently, small biomolecular inducers are commonly used, yet applying these inducers locally and dynamically to compartmentalized micron-scaled reactions remains challenging. Here, we report on a DNA compartment fabricated in silicon and connected to thin electrodes, capable of external control of cell-free protein synthesis under steady-state conditions. We demonstrate manipulation of RNA polymerase, ribosomes and GFP in a nonuniform electrical field, using dielectrophoresis (DEP). We show local depletion of nutrients and machinery in an active gene expression reaction at physiologically relevant conditions. The response to the applied field is rapid at the scale of expression dynamics, and spatially confined thereby establishing spatiotemporal resolution of electric field control of gene expression.
  

Characterizing electrotaxis for control of cellular migration

It is well established that bioelectric fields arise during morphogenetic processes across many cell types, influencing development, metastasis, and wound healing. Many cell types use endogenous electric fields as a cue to guide cell migration in a process known as electrotaxis. While electrotaxis presents a tremendous opportunity for remote electronic control of cell migration, a formal physical approach exploring the limits and plasticity of this guided migration has not been conducted. In this work, we examine the input/output dynamics of electrotaxis in MDCK epithelial cell sheets, using rapid prototyping techniques to produce a versatile, reconfigurable platform capable of applying a programmable electric field to tissues. We modulate stimulation current density and duty cycle to probe the electrotaxis impulse and step responses. We also use live fluorescence imaging in labeled cell lines to characterize the biophysical response of the cytoskeleton during electrotaxis, which generates force during cell migration. Because collective cell migration is crucial to multicellular form and function, tools for reliable control would be invaluable for further studies of the biophysical processes underlying tissue development, wound healing, and other multicellular programs.   

Design considerations for high-performance dielectrophoretic devices

Dielectrophoresis (DEP) uses electric field gradients to trap and manipulate the position of particles in solution. The DEP force does not require direct contact with the particle nor the use of any form of labelling mechanism, making it an attractive candidate for manipulation of cells for biological applications. Performing dielectrophoresis in high-conductivity solutions, such as physiological samples, presents significant challenges for the force magnitude, power delivery, and device integrity. We present a coherent set of circuit-level design principles for optimizing electrode geometry for enhanced performance in high-conductivity environments. Investigations of the influence of various design parameters on the DEP force are performed by monitoring changes in the equilibrium velocity, as DEP competes with the Stokes force during fluid flow.   

Large-scale Actin Wave Patterns Perturbed by Electric and Mechanical Cues in Giant Dictyostelium discoideum

Dictyostelium discoideum(Dd) provides a good system to study actin dynamics guided by extracellular cues such as electric field, mechanical cues and chemical gradients. However, waves in normal Dd extinguish at the boundary and only show confined sections of wave patterns. Here we apply electrofusion to produce giant Dd, a polykaryotic cell which is up to ten times the size of normal Dd. In those cells F-actin waves travel freely across plasma membrane and show large-scale wave patterns independent of boundary effects. We use this system to explore how nanoridges and DC electric fields perturb actin waves on a scale as large as 50 microns.   

Propagation of electrical activity coupled to actin dynamics in electrically active neuron-like cells

Coupling between excitable systems is a well-known phenomenon. Recent work focused on the excitability of neural and cardiovascular systems has illuminated the dynamics behind electrical signal propagation in these tissues through the use of recording microelectrodes. Other studies have shown that dendritic spines are enriched with dynamic actin at the synapses of electrically-coupled neurons. Here we investigate both electrical activity propagation and actin dynamics in a simple, excitable system: in vitro cultures of human embryonic kidney (HEK-293) cells. This cell line (NK-HEKs) has been engineered to be electrically excitable through the expression of sodium (Na+) and potassium (K+) channels. We electrically stimulate the cells to induce changes in transmembrane potential monitored noninvasively through new, fast-acting- voltage sensitive dyes. We simultaneously image actin dynamics in the same cells using internal fluorophores with the goal of understanding how changes in actin dynamics may drive or be driven by changes in transmembrane potential. Our results suggest that actin fluctuates in response to stimulation in these electrically active cells.   

Escherichia coli's physiology can turn membrane voltage dyes into actuators

Bacteria tend to maintain an energy-costly electric potential across the biological membrane. The voltage thus stored can then be reinvested to fuel essential reactions, such as those required for feeding, movement and anabolism. Assays of Nernstian membrane voltage dyes accumulation are arguably the most widespread techniques to quantify such potential. However, interactions of such molecules with the complex cellular environment and physiology are often poorly understood. Here, we characterize the parametrical landscape in which these molecules behave like sensors and where they actually take the role of actuators. We recommend an experimental framework that can be used to characterize Nernstian dyes and we apply it to the characterization of the dye Thioflavin T in E. coli.   

The Tell-Tale Heart: Failures of Control and Descent into Cardiac Chaos

Proper contraction of cardiac muscle relies on the coordinated propagation of transmembrane voltage. Disturbances of this propagation can result in deadly cardiac arrhythmias such as fibrillation, the manifestation of chaos in the heart. Even in healthy tissue, high heart rates can drive the system to a dynamical instability known as alternans, a period doubling bifurcation in action potential duration (APD) which is strongly correlated with the onset of fibrillation and sudden cardiac death. Much theoretical effort based on the relationship between the APD and preceding diastolic interval (DI) has aimed to suppress the onset of alternans. Results from simulation and theory claim the suppression of alternans under stimulation at a constant DI; however, few experiments have addressed these predictions. In this talk, I will discuss comparative cardiac dynamics in the hearts of species including rabbit, dog, cat, pig, frog, zebrafish, snake, lizard, and alligator through the use of microelectrode recordings and high spatiotemporal resolution optical mapping of fluorescent voltage and calcium signals across the surfaces of hearts. Furthermore, I will discuss my closed-loop control system for performing constant DI stimulation and the highly unexpected results.