1 / 36

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

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

ivor-hale
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

  12. Control; t = 0 min

  13. Control; t = 10 min

  14. Control; t = 20 min

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

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

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

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

  19. 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

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

  21. 0.5 mV 5 nm Eo = 100 V/m 1 mV E cyt = 0 10 µ

  22. 2q Extracellular Fluid 2a Eappl L Membrane k h Cytosol

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

  24. 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.

  25. 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.

  26. 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.

  27. Directionality of Migration

  28. Speed of Migration

  29. 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.

  30. Could heating affect the results?

  31. 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.

More Related