For over a century it has been known that motile cells exposed to external information from an applied direct current electric field will migrate along the orientation of the electrical potential (galvanotax/electrotax) [1
]. Cells respond to currents that are similar in magnitude to those that exist under normal physiological conditions, including during the development of embryos of some animals [2
] and wound formation [3
] due to a short-circuit of the trans-epithelial potential [4
]. In addition, exogenous electric fields applied in-vivo
are sufficient to disrupt development [5
] or produce directed migration [6
]. At this time the mechanisms that cells use to sense an external electrical field, transduce this signal to the cell migration apparatus, and then appropriately change the direction of migration remain controversial.
Galvanotactic behavior has been demonstrated thus far in over thirty metazoan-derived cell types including neurons [7
], lung cancer cells [8
], and leukocytes [9
] as well as in crawling single celled organisms including Dictyostelium discoideum
] and many swimming (ciliated) protozoa [11
]. It is far less common to see reports of animal cells that fail to galvanotax and this usually correlates with poorly motile behavior [6
]. Electric fields that produce galvanotaxis are typically in the range of 0.1 to 10 V/cm [3
]. It has been established that galvanotaxis operates independently of sensing an external chemical gradient [12
], therefore we can limit our discussion of a cellular sensor of an external electric field to the electrical dimensions of the cell.
These electrical properties of the cell are primarily dictated by the cell’s plasma membrane. External to the plasma membrane, the cell adheres to a charged substrate and is bathed by a conductive ionic media. Due to the high resistance of the cellular plasma membrane compared to the external media as well as the small size of the cell, most (
99.999%) of the current flow created by an external electric field will pass around the cell and will therefore have limited effect on intracellular components [13
]. The shielding effect of the plasma membrane is bridged primarily by a set of membrane channels with selective permeability to ions. In addition, the plasma membrane itself is embedded with a large set of charged macromolecules and lipids, which will be directly acted on by an external electric field through Coulombic interactions. These extracellular charged components and the charged substrate will also induce electro-osmotic flow in the presence of an external electric field.
Given these physical constraints we can limit our exploration of the galvanotactic sensing mechanism to the following set of four plausible physical hypotheses (). (A) Cells will be asymmetrically excited due to hyperpolarization of the anodal side and depolarization of the cathodal side of the cell, changing the opening probability of voltage gated ion channels as well as creating an asymmetric electro-motive force for ionic flow once ion channels are open [10
]. (B) Electro-osmotic flow created at the substrate will re-orient cells through hydrodynamic shear as is seen with laminar fluid flow [14
]. (C) Electrostatic and electro-osmotic forces at the plasma membrane will apply mechanical force on the cell or on tension sensitive cell surface components. (D) These same electrostatic and electro-osmotic forces at the plasma membrane will also redistribute the charged components of the membrane establishing a cathodal/anodal axis of polarity [15
]. These non-exclusive mechanisms are summarized in .
Models for directional sensing of a keratocyte in an electric field
Each of these putative sensors of an external electric field would require signal transduction pathways to relay the directional information to the cytoskeletal players that produce cell migration. Most cell types respond to an electric field by migrating towards the cathode, although some (often similar) cell types respond by migrating to the anode [16
]. The mechanism underlying these anti-parallel responses is unclear. Separate reports on the same cell type (human polymorphonuclear cells) have found opposing anodal versus cathodal galvanotactic responses [6
], which has been attributed to differences in extracellular calcium [19
]. In addition, a mutant strain of Dictyostelium
has been identified with a reversed (anodal) electrotactic response. This mutant phenotype could be replicated by inhibiting both cGMP and PI3K signaling activity [20
], supporting the hypothesis that there is a separation between the physical mechanism of sensing an electric field and the eventual directional response.
Downstream of the unknown sensing mechanism, the current literature supports a hypothesis where intracellular signaling pathways canonical to chemotaxis are used to transduce the galvanotactic signal. It is commonly noted that inhibition of PI3K disrupts the galvanotactic response of cells [21
]. Galvanotaxis can also be blocked by inhibition of alternative signaling pathways such as VEGF, ERK and Rho/ROCK [16
]. In addition cells in electric fields have asymmetric distributions of common polarity factors including phosphatidylinositol (3,4,5)-trisphosphate (PIP3), PTEN, and growth factor receptors [9
]. However, the signal transduction pathways of chemotaxis and galvanotaxis do not completely overlap, as unlike chemotaxis PTEN inhibition improves the strength of a cell’s galvanotactic response [9
], and in general the signaling pathways of galvanotaxis remain poorly understood.
The final step in the directional response to an electric field is the actual change in organization of the cytoskeleton of the motile cell to produce a change in direction. Little is known about the mechanical requirements for this process other than a described independence from the microtubule system and a general requirement for actin polymerization [25
In this work, we seek to identify the cellular sensor of an external electric field by investigating the validity of each of the hypothetical physical mechanisms that could produce a galvanotactic response using the motile fish epithelial keratocyte model system. Keratocytes move at high speeds, with a simple shape, and, unlike cultures of mammalian cells, are robust to extreme physical perturbations, making them useful for understanding mechanical effectors of motility. In addition, keratocytes operate largely without requirements for external stimuli and are not known to be chemotactic. We find that the most likely sensing mechanism for galvanotaxis occurs due to electrophoretic redistribution of membrane components to the anode of the cell defining the rear. This polarity of membrane components is transduced by canonical intracellular signalling pathways that then dictate the cell’s directionality.