The involvement of Ca²⁺ and integrins in directional responses of zebrafish keratocytes to electric fields

Abstract

Many cells respond directionally to small DC electrical fields (EFs) by an unknown mechanism, but changes in intracellular Ca(2+) are widely assumed to be involved. We have used zebrafish (Danio rerio) keratocytes in an effort to understand the nature of the EF-cell interaction. We find that the adult zebrafish integument drives substantial currents outward through wounds produced by scale removal, establishing that keratocytes near the wound will experience endogenous EFs. Isolated keratocytes in culture turn toward the cathode in fields as small as 7 mV mm(-1), and the response is independent of cell size. Epidermal sheets are similarly sensitive. The frequency of intracellular Ca(2+) spikes and basal Ca(2+) levels were increased by EFs, but the spikes were not a necessary aspect of migration or EF response. Two-photon imaging failed to detect a pattern of gradients of Ca(2+) across the lamellipodia during normal or EF-induced turning but did detect a sharp, stable Ca(2+) gradient at the junction of the lamellipodium and the cell body. We conclude that gradients of Ca(2+) within the lamellipodium are not required for the EF response. Immunostaining revealed an anode to cathode gradient of integrin beta1 during EF-induced turning, and interference with integrin function attenuated the EF response. Neither electrophoretic redistribution of membrane proteins nor asymmetric perturbations of the membrane potential appear to be involved in the EF response, and we propose a new model in which hydrodynamic forces generated by electro-osmotic water flow mediate EF-cell interactions via effects on focal adhesions.

Epidermal keratinocyte polarity and motility require Ca2+ influx through TRPV1

“…In support of this idea, DC electric fields rescue polarized migration of zebrafish keratinocytes in the absence of extracellular Ca 2+ . Under these conditions, cells maintained an average speed that is only 55% lower than that of cells in normal [Ca 2+ ] (Huang et al, 2009). This speed is much higher than the speed reported here when cells migrate in the absence of both extracellular Ca 2+ and a polarizing applied electric field.…”

“…Time-accumulated Ca 2+ sparks reveal spatial Ca 2+ gradients in motile cells that correlate with turning. magnitude between the two sides of the lamellipodia of cells (Huang et al, 2009). Here, we tested whether DC electric fields act by generating an asymmetric distribution of the frequency of Ca 2+ sparks.…”

“…In the presence of TRPV1 inhibitors, cells lost their defined lamellipodia, an effect that prevented Ca 2+ spark analysis. As an alternative, we characterized the changes in whole-cell Ca 2+ -pulse activity, which is also due to Ca 2+ influx through plasma membrane mechanosensitive channels (Lee et al, 1999;Huang et al, 2009). Spontaneous whole-cell Ca 2+ pulses from cell sheets increased ,7-fold and 14-fold in response to 10 mM capsaicin (P,0.02) and 1 mM resiniferatoxin (P,0.03), respectively (Fig.…”

“…Transient Ca 2+ influx at the leading edge of migrating cells promotes lamellipodial extension during chemotaxis (Wei et al, 2009). Despite the close association of Ca 2+ with these processes, cell migration still occurs in the absence of extracellular Ca 2+ when an electrical polarizing signal is applied (Brown and Loew, 1994;Fang et al, 1998;Huang et al, 2009). These seemingly conflicting reports encourage further investigations into the importance of Ca 2+ signals during migration and in the presence of external polarizing cues.…”

“…It has been proposed that electric fields polarize cells by inducing Ca 2+ i gradients (Mycielska and Djamgoz, 2004;McCaig et al, 2005;Zhao, 2009 (Brown and Loew, 1994;Fang et al, 1998;Palmer et al, 2000;Huang et al, 2009). Interestingly, weak DC electric fields and low-frequency AC electric fields (1 Hz) increase Ca 2+ i , in vitro (Cho et al, 1999;Huang et al, 2009;Dubé et al, 2012). Careful analysis of Ca 2+ i changes during galvanotaxis are necessary to resolve these controversies and may provide insight toward the molecular and physical mechanisms by which cells sense and respond to weak DC electric fields.…”