The molecular mechanism by which cells detect and migrate directionally in response to an EF remains vague. The early theory that the EF redistributed molecules in the extracellular medium, thus creating a chemical gradient which guides cell response, is not entirely true. This is because cells retain their characteristic electrotropic response to EFs with perfusion of fresh medium orthogonal to the vector of the EFs (Patel and Poo, 1982; Patel and Poo, 1984). Another proposal was that a direct voltage difference across a cell might produce a cytoplasmic voltage gradient which means the cell has positively and negatively charged ends. However, because of the very high resistance of the plasma membrane, the vast majority of the voltage drop remains outside the cells, with almost no internal gradient arising (Betz et al., 1980). The focus then changed to examining which molecular elements of the cells might be asymmetrically affected on the exposure to an EF.
1.9.1 Nerve Cells
It was very controversial finding that showed that growing nerve cell processes (neurites) respond to an electric field; Jaffe and Poo found that neurites grew faster toward the cathode than toward the anode by using embryonic chicken dorsal root ganglia (Jaffe and Poo, 1979). But continued research revealed that it was indeed an electrotactic response, and neurites even turned 180 degree in order to move to the cathode. Further study discovered that EFs also increased total neurite production, and what’s more, there were more neurites initiating on the cathode-facing side of the cell body than the anodal side.
Rajnicek and Mccaig first tried to explain the molecular mechanism behind the EF- induced neurite migration (Rajnicek et al., 2006a, b). They found that dynamic microfilaments and microtubules are very important for EF-induced growth cone migration. Further, growth cone turning can be divided into two stages: filopodia reorientation is in advance of turning and lamellipodia reorientation is coincident with the turning. Finally they suggested that Rac/Cdc42-mediated dynamics of microfilaments and microtubules act cooperatively to generate cathodal steering.
1.9.2 Muscle Cells
Myoblast cells from frog embryos were aligned perpendicularly to the vector of an applied EF. The threshold to induce the alignment of myoblasts was as low as 0.3 mV/mm. Mccaig and Dover (McCaig and Dover, 1991) found that calcium potentially contributed to the EF-induced orientation change, because a calcium inhibitor blocked the perpendicular orientation of myoblasts. Calcium entry is essential for EF-induced perpendicular orientation of mouse embryonic fibroblasts as well (McCaig and Dover, 1991). Further experiments indicated that L-type calcium channels might not be as important as T and N type calcium channels in this effect. Another factor which affected the EF-induced orientation change was recognized to be cytoskeleton proteins, and pharmacological inhibitor studies implied that the limited amount of F-actin present played a more important role than microtubules in the morphology transformation.
1.9.3 Neural Crest Cells
Neural crest cells are a transient, multipotent migratory cell population that first accumulates on the dorsal side of the vertebrate neural tube, and then disperses as these cells follow a characteristic pathway to form a remarkable number of derivatives, including melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia. A number of studies were carried out mainly on amphibian and quail material, and the results were consistent, which were that the cells responded directionally to a transcellular voltage difference of 0.7 mV. In agreement with muscle cells, the neural crest cells have similar morphology transformations in applied EFs, which is difficult to explain because any asymmetries induced by the
explained that the perpendicular alignment minimizes the perturbing effect of the field on the membrane potential and suggest that cells respond in such a way as to achieve this minimization.
1.9.4 Epithelial Cells and Fibroblasts
There are a quite a number studies discussing how epithelial cells and fibroblasts respond to an applied EF. The threshold of the field able to induce directional migration in these two cell types is around 100 mV/mm. Cornea epithelia cells in an EF of this magnitude have a migration speed of 10 μm/hour with directedness of 0.6, and fibroblasts appear to have a similar response. However, most of the fibroblasts studied have shown migration towards the anode, which is contrary to the response of the majority of other types of cells (Guo et al., 2010; McCaig et al., 2005). The mechanism of the difference is largely unknown so far.
1.9.5 Mechanism of interaction with electric fields
Although it is still unclear how cells sense and respond to an electric field as small as 0.1 mV, their responses to larger voltage gradients are partially due to the influence of the difference between calcium entry into the cathode-facing sides and anode- facing sides of the cells. The symmetric zygotes of the brown algae focus and pelvetia will polarize and develop their rhizoides on the anodal side in an electric field; the argument is that the imposed electric fields polarize the zygotes by driving in calcium on the anodal side, which mimics the normal physiological calcium drive process (Figure 1-7).
However, this straight-forward explanation with perturbation of calcium flux cannot explain all observed scenarios. An alternative mechanism whereby an applied electric field might induce asymmetric distribution of membrane proteins is by electrophoresis or electroosmosis.
In the case of ion channels on the cell membrane, it has been proposed that an electric field might redistribute and polarize charged and mobile components in the cell membrane. The degree of redistribution a molecule achieves in a given electric field has been shown to be dependent on the diffusion coefficient of the electrophoretic mobility and the voltage drop per cell required to produce asymmetry of 0.1 (0 representing uniform distribution and -1 or +1 representing completely
redistribution of the component to the one pole or the other). A variety of membrane receptors has been shown to be redistributed to the cathode side of the cell in an applied EF (McCaig et al., 2005; Robinson, 1985), including the Con A receptor, acetylcholine receptor, and the Fcε receptor.
Figure 1-7 Electric Fields interact with a cell by changing calcium uptake
(a) A spherical cell with a membrane potential of -70 mV. (b) The effect of a uniform applied electric field. The electric field is distorted by the highly resistive cell as shown. The potential outside the cell will vary sinusoidally and thus the transmembrane potential, will vary as well so that the anode-facing side is hyper polarized and cathode facing side depolarized. (c) The effects of a field on one voltage gated calcium channel. Because the anodal side is hyper- polarized meaning cytoplasm is more negative, the force driving calcium inwards is increased, while the calcium driving force on the other side is decreased. (d) The effects of the field on calcium flux through voltage-gated channels. The calcium channels on the cathodal side are open due to depolarization, while anodal side channels remain closed. (e) Electrophoretic redistribution of calcium channels. The other proteins studied have been shown to be redistributed by the field. Calcium channels here are only for illustrative purposes, similar consideration applies to other ion channels or protein (Robinson, 1985).
glycoproteins which have a net negative charge at physiological pH. This observation has led to the suggestion that the mechanism of electric field-induced receptor redistribution is due to electroosmotic water flow near the surface of the cell, produced by the immobile negative charge on the surface (Robinson, 1985). The evidence has come from Poo’s experiment that showed the direction of migration of the Con A receptor was reversed following the treatment of the cells with neuraminidase, which would be expected to remove much of fixed negative charge, allowing a direct electrophoretic response by the receptors (McLaughlin and Poo, 1981).
Another model is cornea epithelia cells as wounds in the cornea epithelium generate endogenous currents that control the process of reepithelialization. This small wound-induced electric field can affect both cell migration and division. During electrotaxis, several growth factors have also been found to act together, perhaps using parallel signalling pathways to transduce the effect of EF. Most electrotaxis work has concentrated on EGF, because during cornea wound healing EGF is upregulated and EGF receptor is activated at the leading edge, which experiences the strongest electric field. Flow cytometry results have confirmed that applying EF increases the expression of EGF receptors and resulted in redistribution of EGF receptors and F-actin to the cathodal side of the cells.
EF-induced asymmetry of EGFRs also induced asymmetric intracellular signalling through the mitogen-activated protein (MAP) kinase signalling cascade. Western blot results have shown increased activation of dual phosphorylated ERK1/2 distributed at the cathodal side. Moreover, activated ERK1/2 and F-actin have become colocalized at the leading lamellae in CECs migration.
In short, the mechanisms driving EF-induced cell directional migration in different kinds of cells share several elements in common. They all can be transduced by an induced asymmetry of membrane receptors which interact with the chemical gradient. They all involve signal transmission at the leading edge by second messenger pathways and eventually connect to cytoskeleton proteins. Figure Figure1-8 compares and contrasts the mechanisms that control electrically and chemically directed cell movement (McCaig et al., 2005).