Francis X. Hart 1 , Adian S. Cook 1 and John R. Palisano 2

1. A Transition in Transduction Mechanisms For Amoeba Galvanotaxis From Electromechanical To Voltage-Gated Channels Francis X. Hart1, Adian S. Cook1 and John R. Palisano2 1: The Department of Physics; The University of the South; Sewanee, TN 37383; U.S.A. 2: The Department of Biology; The University of the South; Sewanee, TN 37383; U.S.A.

2. Galvanotaxis • Directed motion of cells under the application of a DC Electric Field. • Relatively easy to detect and measure compared to other field effects, such as calcium uptake. • Serves as a useful effect to compare mechanisms for electric field effects.

3. Amoeba Proteus

4. Experimental Chamber

5. Control; t = 0 min

6. Control; t = 10 min

7. Control; t = 20 min

8. E = 60 V/m; t = 30 min

9. E = 60 V/m; t = 40 min

10. E = 60 V/m; t = 50 min

11. E = 60 V/m; t = 60 min

14. Control; t = 0 min

15. Control; t = 10 min

16. Control; t = 20 min

17. E = 225 V/m; t = 30 min

18. E = 225 V/m; t = 40 min

19. E = 225 V/m; t = 50 min

20. E = 225 V/m; t = 60 min

23. What is the Primary Cellular Transduction Mechanism for Electric Fields ? • How does a cell know that an electric field is present? • 1. Electrodiffusion/osmosis • 2. Voltage-gated channels • 3. Electromechanical torques on transmembrane glycoproteins

24. E Electro-diffusion/osmosis Forces exerted on cell-surface-receptors (-) [CSR]and mobile counterions (+) redistribute them.

25. 0.5 mV 5 nm Eo = 100 V/m 1 mV E cyt = 0 10 µ

27. 2q Extracellular Fluid 2a Eappl L Membrane k h Cytosol

28. Keratinocyte Results{F.XHart et al. Bioelectromagnetics 34: 85-94 (2013)}

29. Why Amoeba? • Relatively easy to work with. • Intrinsically independent. • Much larger than tissue cells. • Both mechanisms may be studied depending on the applied field strength. • Low fields -> electromechanical transduction. • High fields -> voltage-gated channels.

30. Amoeba Protocols • Purchased from Carolina Biological Company. • Cells left at room temperature for up to 3 days to allow them to accumulate and divide. • Several aliqouts of 25 to 30 amoeba were placed in the center trough of the apparatus. • Cells were allowed to sit in the trough for 15 to 20 min before cover-slipping. • Trough was flooded with culture medium and cover slipped.

31. Experimental Protocols • No field applied for 20 minutes (control). • Field applied from 20 to 60 minutes. • During this time a movie records the moving amoeba. • The full movie is converted to a time-lapse movie with 1 minute intervals. • The position of each amoeba is digitized from that movie. • EACH AMOEBA SERVES AS ITS OWN CONTROL.

32. Directionality of Migration

33. Speed of Migration

34. Calculation • Assume an elongated amoeba is 0.4 mm long and 0.1 mm wide. • Suppose E = 200 V/m. • An amoeba oriented parallel to the field experiences a maximum change of transmembrane potential of 40 mV. • An amoeba oriented perpendicular to the field experiences a maximum change of transmembrane potential of 10 mV. • A change of 40 mV is likely to open voltage-gated ion channels, but a 10 mV change would not.

35. Could heating affect the results?

36. Conclusions • At low fields (~ 50 V/m) the results for amoeba agree with those reported previously for keratinocytes and support an electromechanical transduction model. • At high fields (> 200 V/m) changes in transmembrane potential may be sufficiently high to open voltage-gated channels. • In the first ten minutes directionality increases with field up to about 200 V/m and is essentially constant thereafter. • In the first ten minutes the relative speed increases steadily beyond about 350 V/m, but does appear to show a plateauing thereafter. • Thermal effects play no role is these results. • The opening of voltage-gated channels at the higher fields may modify the transduction mechanism.