Fig 1.
System for Electrotaxis Experiments.
(A) Schematic drawing of the electric field application. (B) The galvanotaxis chamber was constructed with PDMS in a Petri dish, shown from above or (C) as a transverse section. (D) The total voltage applied to the system and the voltage on the galvanotaxis chamber changed with the current.
Fig 2.
Cell Migration in Pulsed Directional Current Fields, Constant Electric Fields of Decreasing Intensity or an Anion-exchange Membrane Between an Agar Bridge and a Platinum Electrode.
Mouse fibroblasts attached to the galvanotaxis chamber were exposed to a direct current electric field for 7 h, and cells’ migration was imaged every 6 min during the experiment. (A) and (B) are pulse direction waveforms of 50% and 75% duty cycles in a 5 V/cm field. (C) Mean speed, vector speed and average cosine of the cellular translocation under different pulse EFs. (D) Mean speed, vector speed and average cosine of the cellular translocation with decreasing EF intensity. The linear relationship between vector speed and electric field intensity is y = 1.865x+0.5913. * (p < 0.05) and ** (p < 0.01). Error bars indicate standard error.
Fig 3.
Function of Calcium Ions in Cell Migration in Response to an EF.
(A) EGTA (2.5 mM) in only the anode culture medium significantly inhibited mouse fibroblasts’ migration toward the cathode, but EGTA (2.5 mM) in the cathode medium did not inhibit migration. Amplifying the current from 0.115 mA to 2.3 mA abolished the migration in a larger chamber with a cross-sectional area 20 times larger than the original chamber. (B) The concentration of calcium in the anode culture media decreased in the larger chamber over time. (C) Migration speed in the vector direction decreased with time. * (p < 0.05) and ** (p < 0.01) vs EF 5 V/cm control. Error bars indicate standard error.
Fig 4.
Calcium Channel Blockers Inhibit Cathode-Directed Migration.
Several inorganic calcium channel blockers, including La3+ (200 μM), Gd2+ (100 μM), and Ni2+ (300 μM), and store-operated calcium channel (SOC) blockers, including 2-APB (100 μM) and SKF96365 (10 μM), inhibit cathode-directed cell migration, but Sr2+ (1 mM), a substitute for calcium, nifedipine (20 μM), a voltage-dependent channel inhibitor, and BAPTA-AM (25 μM), an intracellular calcium chelator, failed to inhibit cellular responses to EF. * (p < 0.05) and ** (p < 0.01) vs EF 5 V/cm control. Error bars indicate standard error.
Fig 5.
Calcium Distributions and Concentrations In Control Cells and Cells Exposed to EFs.
(A) Calcium distributions in mouse fibroblasts without EFs as imaged using Fluo-4FF AM for 10 minutes. (B) Electric fields do not induce calcium gradients in cell bodies for 10 minutes or (C) 150 min. (D) EFs do not induce increased [Ca2+]i compared with controls, presented as the ratio (F:F0) for 10 minutes or (E) 150 minutes. Scale bar, 20μm.
Fig 6.
The PI3K Pathway Was Involved in Galvanotaxis.
Treatment with the RTK inhibitor AG1478 (10 μM), the PI3K inhibitor LY294002 (30 μM), the actin polymerization inhibitor CB (1 μg/ml) or the myosin-inhibitor BB (25 μM) abolished cathode-oriented cell migration, but treatment with Y27632 (10 μM) or U0126 (10 μM) did not. * (p < 0.05) and ** (p < 0.01) vs EF 5 V/cm control. Error bars indicate standard error.
Fig 7.
PC3 cells Utilized the Same Galvanotactic Mechanism as Mouse Fibroblasts.
(A) Ni2+ (300 μM), SKF96365 (10 μM), AG1478 (10 μM), LY294002 (30 μM), Y27632 (10 μM), U0126 (10 μM), BB (25 μM), CB (1 μg/ml) and knocking down Orai1 expression restricted the galvanotaxis of PC3 cells as well as mouse fibroblasts. (B) The efficiency of the siRNA-mediated knockdown was evaluated using western blotting. n = 3. * (p < 0.05) and ** (p < 0.01). Error bars indicate standard error.
Fig 8.
Schematic Illustration of Fibroblast Directional Sensing in EF.
In fields of direct current, cations move toward the cathode, and anions move toward the anode. Calcium ions permeate the cell through SOCs at the drift velocity, and this was the primary physical mechanism for directional cell motility and sensation of the electrical field. A positively charged RTK moves to the cathode-facing membrane in an electrophoretic process and recruits the PI3K to that surface; then, the PI3K pathway is responsible for cytoskeletal polymerization and cell migration toward the cathode.