Endogenous voltage gradients as mediators of cell-cell communication: strategies for investigating bioelectrical signals during pattern formation

Abstract

Alongside the well-known chemical modes of cell-cell communication, we find an important and powerful system of bioelectrical signaling: changes in the resting voltage potential (Vmem) of the plasma membrane driven by ion channels, pumps and gap junctions. Slow Vmem changes in all cells serve as a highly conserved, information-bearing pathway that regulates cell proliferation, migration and differentiation. In embryonic and regenerative pattern formation and in the disorganization of neoplasia, bioelectrical cues serve as mediators of large-scale anatomical polarity, organ identity and positional information. Recent developments have resulted in tools that enable a high-resolution analysis of these biophysical signals and their linkage with upstream and downstream canonical genetic pathways. Here, we provide an overview for the study of bioelectric signaling, focusing on state-of-the-art approaches that use molecular physiology and developmental genetics to probe the roles of bioelectric events functionally. We highlight the logic, strategies and well-developed technologies that any group of researchers can employ to identify and dissect ionic signaling components in their own work and thus to help crack the bioelectric code. The dissection of bioelectric events as instructive signals enabling the orchestration of cell behaviors into large-scale coherent patterning programs will enrich on-going work in diverse areas of biology, as biophysical factors become incorporated into our systems-level understanding of cell interactions.

Fig. 1

Overview of bioelectric signaling in cell regulation. a Transcriptional events (up- or down-regulation of specific ion channel/pump genes) establish the complement of ion translocators expressed in any cell. Their activity results in changes of ion content and transmembrane potential, which is transduced by a number of mechanisms that couple biophysical events to changes of the expression of target genes. These in turn also control key cell behaviors and large-scale anatomical properties such as axial patterning and organ specification/morphogenesis. b Interplay between physiological properties (red) and specific proteins (black). An important aspect of physiological signaling is the multiple feedback loops that occur because of the physiological (post-translational) gating of channels and pumps. For example, the level of gap-junctional connectivity determines the way that voltage spreads between adjacent cells but gap junctions are themselves regulated by voltage gradient. Thus, cells and cell groups can implement self-organizing spatially distributed stable patterns (e.g., auto-catalytic Turing-Child dynamics) even in the absence of differential transcription or genetic prepattern. Cell fields can thus be described as complex dynamical systems with multiple stable attractors, supporting symmetry breaking, hysteresis (memory) and the amplification of stochastic heterogeneity (V_mem resting voltage potential)

Fig. 2

Strategy for investigating bioelectric signaling. One often begins by detecting an interesting bioelectrical gradient during some process and hypothesizing that this voltage or ion flow pattern is functionally important. In this case, the next step is to develop a robust assay recapitulating this process and to conduct a loss-of-function hierarchical drug screen to narrow down a list of candidate transporters that might underlie the observed gradient. In contrast, a specific transporter candidate might be suggested by the results of a genetic screen, network analysis, or microarray comparison. Because V_mem is determined by ion flux, part of characterizing bioelectric signals is determining the endogenous translocators involved. Thus, the next step would involve the use of fluorescent voltage- or ion-reporting dyes (with and without a blocker of the channel/pump) to characterize the spatiotemporal properties of whatever gradient pattern might be produced by the transporter in question (Adams and Levin 2012a, 2012b; Oviedo et al. 2008). Molecular-genetic validation of the pharmacological data follows; a loss-of-function experiment with the knockout or misexpression of a dominant negative mutant should be used to establish that this transporter is indeed important for the phenotype under study. Next, to determine the physical process carrying instructive information, the transporter is blocked and a rescue of the phenotype is attempted with other transporters that distinguish among non-ionic, ion-specific and voltage-specific functions. Subsequently, this biophysical signal has to be connected to canonical pathways by (1) identifying the second-messenger mechanism(s) by which a biophysical signal is transduced into changes in cell behaviors, (2) identifying its downstream effects (which genes does it turn on/off, how does it affect cell behavior) and (3) modeling the physiology (quantitative spatial simulation of the ion flows that result in the relevant voltage gradient)

Fig. 3

Identification of a transduction mechanism. To identify which of several possible mechanisms is involved in transducing an epigenetic (biophysical) event into a biochemical signaling pathway, a suppression screen should be performed (Blackiston et al. 2011). Each candidate transduction mechanism can be probed in turn by inhibiting it in an assay in which an induced change in transmembrane potential leads to an observable phenotype (increase in proliferation, up-regulation of a target gene, cell migration, etc.). For example, if gene X is up-regulated upon depolarization but this up-regulation does not occur in the presence of a GdCl, then voltage-gated calcium channel (VGCC) opening is implicated. If on the other hand a blocker of gap-junctional communication (GJC) abolishes the effects of depolarization, this suggests that the voltage-gated control of GJC and small molecule movement mediate the effects. Similar experiments with inhibitory drugs, dominant negative constructs and genetic deletion can be used to implicate serotonergic (5HT) signaling, butyrate-driven chromatin modifications (SLC5A8), integrin signaling, voltage-sensitive phosphatases (VSP), or other transduction mechanisms

Fig. 4

Loss-of-function hierarchical drug screens. To determine whether a bioelectric process is likely to be involved in some process of interest and simultaneously to implicate specific channel/pump family(ies) as endogenous mediators of the biophysical signals, a loss-of-function pharmacological screen is performed. Known blockers of different types of channels/pumps are arranged (Adams and Levin 2006a, 2006b) in a hierarchical tree, from least specific to most specific (a). This chemical genetics approach is applied in an assay (b), such as the scoring of asymmetric positioning of internal organs following drug exposure at early embryonic stages (a screen to identify roles for ion flow in a left-right patterning during development; Adams et al. 2006; Levin et al. 2002). A negative result at any node in the tree means that the nodes of drugs (and their matching protein targets) below that point do not have to be pursued further, whereas a positive result (an observable change in the assay outcome) leads to the testing of the nodes below. This screen does not saturate (since not every transporter is blocked by a known drug) and in some (surprisingly rare) cases, pleiotropic functions of a given transporter lead to a toxicity phenotype that precludes a clean yes/no answer. Nevertheless, the existence (and continued development) of a plethora of pharmacological reagents and the demonstrated possibility of dissociating subtle patterning functions of ion flows from housekeeping roles combine to make this a powerful, rapid and inexpensive method to identify a manageable number of candidates for further molecular analysis and to motivate investigations of bioelectric components in a process of interest. Figure in panel a is taken from Adams and Levin 2006a, Fig. 1, with permission of John Wiley and Sons. Figure in panel b is taken from Levin et al. 2006, Fig. 3, with permission of S. Karger AG, Basel

Fig. 5

Pharmacological strategy for specific control of V_mem. To control V_mem experimentally, the best strategy is first to express a convenient ion channel in cell(s) of interest (or make sure it is expressed there natively); alternatively, an ionophore can be used if the targeting of specific cell subpopulations is not needed. Then, the channel is opened with a specific pharmacological activator and the extracellular concentration of that ion is varied in the medium. For example, in a field of embryonic cells, some express the glycine-gated chloride channel GlyR (a). When targeted by Ivermectin (IVM), a specific GlyR opener (b), these cells can be depolarized in medium of low chloride content (because negative chloride ions will tend to leave the cells via the open channel down their concentration gradient) and hyperpolarized in a medium of high chloride content (since the negative Cl⁻ ions will enter the cells). By varying the chloride level, one can observe, for example, the disappearance of a depolarization-induced phenotype, as the V_mem is driven toward hyperpolarization by the high chloride content (c typical result of a hypothetical experiment). To use this method quantitatively to measure or create absolute values of V_mem, it helps to know the internal and external concentrations of all the ions and their permeability coefficients. Then, the Goldman-Katz-Hodgkin equation can be used to calculate V_mem

Fig. 6

Isolation of the information-bearing aspect of an ion-translocator function. In any given assay (process involving cell signaling), the function of a given ion transporter can be dissected by inhibiting the transporter and attempting to rescue the process with several distinct constructs that distinguish among ion-independent roles, ion-specific roles, or voltage-dependent roles (a). If a rescue can be made with an inactive (e.g., pore-mutant) form of the channel, then a non-ion-passing function (e.g., binding partner for some other protein) is implicated. If a rescue can be made with only the same channel family and no others, an ion-specific role is implicated. If any transporter of a different ion but with similar effects on transmembrane potential is sufficient to reproduce the same phenotype, then voltage alone is implicated. An example is shown in b: in the amputated tadpole tail, the native V-ATPase proton pump that is required for regeneration was blocked. Misexpression of a heterologous (yeast) P-type proton pump protein (with no sequence or structure homology to the V-ATPase) rescued regeneration, indicating that the proton pumping, not some ion-independent function, carried the signal necessary to initiate regeneration. In contrast, regeneration was not rescued by the electroneutral sodium/proton exchanger, ruling out the pH gradient as the causal factor in initiating regeneration (instead implicating V_mem regulation as the information-bearing aspect of this process). A V_mem role would be directly demonstrated by misxpressing a chloride channel and controlling intracellular chloride concentration in accordance with the Goldman equation, to reveal the voltage at which regeneration is initiated

Fig. 7

Known mechanisms by which transmembrane potential changes are transduced to downstream effector pathways. Changes of V_mem are physical processes that need to be converted into second messenger pathways and ultimately into transcriptional responses. Cell-autonomous mechanisms by which cells sense their depolarization or hyperpolarization include: electrophoretic transfer of small signaling molecules (Ca⁺⁺, 5-HT) through gap-junctional connections (GJC); voltage potential-mediated changes of integrin conformation (leading to activation of integrin pathways); regulation of transport of small molecules (e.g., the sodium/butyrate transporter) that impact chromatin modification; activation of voltage-gated calcium channels (thus impinging on the many pathways controlled by calcium); voltage-dependent activity of small signaling molecule transporters (such as SERT, the serotonin transporter) or of phosphatases (such as the phosphatase and tensin homolog; VSP voltage-sensitive phosphatases). Illustration is taken from Levin 2007b, Fig. 1a, with permission of Elsevier

Fig. 8

Multiple domains of membrane voltage within single cells in culture. a Monolayer of cultured COS M6 cells were imaged by using the membrane potential reporters DiBAC₄(3) and CC2-DMPE (red the most negatively charged membrane, blue the least negatively charged membrane). A domain was defined by coloring the original black and white image by using a red/green/blue lookup table in IPLabs, the software used to collect the images. The image was then opened in Photoshop and any stretch of color in which the pixels were pure red, pure green, or pure blue were counted as a region. Thus, this count reveals three possible values of membrane voltage. Inset Higher magnification of a single cell showing that this cell was determined to have four domains. b Domains were counted on 33 randomly chosen cells. These cells had 5±2 (mean±SD) domains per circumference; the median number of domains was four. Thus, even on using a conservative counting protocol, these cells were found to maintain four to five voltage domains around the circumference of the cell within the plane of focus

Fig. 9

Multiple domains of membrane voltage within frog embryonic cells in vivo. This 16- to 32-cell frog embryo has been stained with the voltage-sensitive dye DiSBAC₂(3). In this image, red areas are depolarized and blue are hyperpolarized. Regions near the cleavage furrows are depolarized relative to the rest of the cell. Time-lapse videos of cleavage reveal that areas of depolarization appear immediately before visible signs of the cleavage furrow, expand as the furrow moves and then become relatively hyperpolarized once cleavage is complete (white arrowhead pairs regions in which the V_mem of adjacent cells match, even though each individual cell has multiple domains)

Fig. 10

Multiple domains of membrane voltage within the single-celled ciliated protist Stentor coeruleus. An actively feeding Stentor was photographed at two different focal planes (a, b and c, d). In the differential interference contrast images left, the dark lines visible at the edges correspond to stripes of cilia that run along the cell from the oral to the aboral end. Right Images of the intricate and stable stripes of relatively hyperpolarized (lighter) and depolarized (darker) regions of the plasma membrane. The hyperpolarized stripes co-localize with the stripes of cilia. In d, the interior of the spiral feeding apparatus is visible as a spot of light surrounded by dark membrane. Despite the active phagocytosis of particles at this position on the membrane, this pattern is maintained. These fine scale differences in voltage are highly stable