The Directional Control Of Electric Field Guided Cell Migration And Its Molecular Mechanisms

Precise control of cell movements is critical in development, angiogenesis, immune response, wound healing and cancer metastasis. During these processes, cells often migrate directionally towards targets by sensing chemical concentration gradients as well as electric fields (EFs). To do this, cells must not only become motile, but also migrate in the correct direction by integrating information from these two cues. The importance of chemotaxis is well accepted. That of electrotaxis remains controversial, despite the widespread existence of endogenous EFs and the evidence that disrupting these also disrupts development and wound healing.EF guided cell migration, termed electrotaxis galvanotaxis/galvanotaxis, has been reported for many cell types including corneal epithelial cells, keratinocytes, endothelial cells, lymphocytes, stem cells and cancer cells, by showing preference migrating towards either the cathode or anode. Recent research shows that the EFs activate multiple signaling pathways. Electrotaxis appears to be a consequence of EF-induced polarized signaling of epidermal growth factor receptors (EGFR), integrins, and phosphatidylinositol-3-kinase (PI3K), and other kinases, such as extracellular signal regulated kinase (ERK1/2), mitogen activated protein kinase (MAPK), protein kinase C and cAMP-or cGMP-dependent kinases, have also been implicated in electrotaxisWhile the mechanism of how cells respond to electrical gradient and promote directional migration has been largely determined, the directional control of electrotaxis and its molecular mechanisms are much less understood. Our previous study showed that keratocyte fragments respond to a direct-current electric field, by migrating towards the anode, unlike their mother cells, that migrate toward the cathode. By using the unique model, in the first chapter, we aimed to elucidate the roles of cyclic nucleotides (e.g., cAMP and cGMP) in regulation galvanotaxis and demonstrate the difference in signal transduction in migration of cell fragments and their parental cells. cAMP and cGMP are second messengers often exert opposing effects on cellular responses to extracellular factors. Here I found that cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre-incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells.on the other hand, as a cluster of specialized transmembrane proteins, integrins have long been proposed to be one of the most important modulator in electrotaxis, since they can mediate signal transduction bi-directionally trough the membrane and play an essential role in the regulation of dynamic interaction between extracellular microenvironment and migration related molecules. Therefore, in the second chapter, we set to determine the roles of integrins in directional control of electrotaxis. Previous studies have documented the role of integrin a5 and β4 in electrotaxis of fibroblasts and keratinocytes. Here we established cell lines that stably expressing specific integrins, and determined their responses to applied EFs with high throughput screen. Quantitative analysis revealed three types of effects on basal motility - enhancement, no significant effects and slowdown. Most striking effects were on the directional migration. Using electric field as a guidance cue, we found profound effects of expression of integrins on the directional migration. Expression of specific integrins rendered cells to migrate to the cathode (cathodal migration), to lose migration direction, and to migrate to the anode (anodal migration). Cells expressing αM, β1, αⅡbβ3,α2,α5 migrated to the cathode, whereas cells expressing β3, α6 and α9 migrated to the opposite direction to the anode. Cells expressing α4, αv, α6β4 lost directional galvanotaxis. Antibody neutralization confirmed the role of α9. Manipulation of a9 molecules suggested that the intracellular domain is critical for the directional reversal.In summary, this doctoral dissertation is organized in two main sections. The first section is devoted to the different roles of cyclic nucleotides in electrotaxis of fish epidermal keratocytes and cell fragments. I found that cAMP and cGMP are essential for galvanotaxis of cell fragments, but not for of their parental cells, suggesting the striking different signal transduction mechanisms in galvanotaxis of cells and cell fragments. The second section is devoted to the role of integrins in the directional control of electrotaxis. And we found that expression profile of integrins had extensive regulatory and even opposing roles in directional cell migration in electric fields.