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First published online 31 October 2006
doi: 10.1242/jcs.03248

Journal of Cell Science 119, 4741-4748 (2006)
Published by The Company of Biologists 2006
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Influx of extracellular Ca2+ is necessary for electrotaxis in Dictyostelium

Lynne J. Shanley1, Petr Walczysko1, Mary Bain1, David J. MacEwan1,2 and Min Zhao1,*

1 School of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK
2 School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK


Figure 1
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  		Fig. 1. Dictyostelium cells migrate directionally in an electric field. (A,B,C) Trajectories plotting the movement of the cell centres of AX2 cells in (A) the absence of an electric field, (B) the presence of an electric field and (C) following reversal of field polarity. (B',C') Consecutive micrographs show migration of cells in an electric field of 15 V/cm. (supplementary material Movie 1). Numbers in A-C' indicate individual cells.

 

Figure 2
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  		Fig. 2. Quantification of the directional migration of Dictyostelium cells in an electric field. (A) Direction of cell migration without and with an EF (EF1, cathode on the right) and reversal of polarity (EF2, cathode to the left). (B) Trajectory and displacement speeds; average of 33-43 cells from three independent experiments.

 

Figure 3
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  		Fig. 3. A direct-current EF induces [Ca2+]i elevation compared with the control. (A) F:F0 ratio of control cells and iplA mutants exposed to an EF or cAMP. (B-D) Relative fluorescence intensity (F:F0 is the average F:F0 ratio of control cells) obtained for (B) individual wild-type cells in, (C) an EF under control conditions and (D) following stimulation with cAMP. Arrows indicate start of EF exposure (in B) or addition of cAMP (in D).

 

Figure 4
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  		Fig. 4. A direct-current EF induces [Ca2+]i elevation from the control and later-appearing [Ca2+]i spikes. (A-J) Fluorescent images of Fluo-3-AM-loaded cells. (A,B) Entire field at time 0 and 240 seconds. The EF was switched on immediately after the frame in A was taken and the cells moved to the right (towards the cathode). (C-J) Selected field showing details of migrating cells and the [Ca2+]i spikes (E,I). (K) Tracks of the three cells shown in A-J. (L) EF exposure significantly increases [Ca2+]i (P=8–9), which is prevented in the presence of 5 mM EGTA (n=29-30 cells; supplementary material Movie 2). Numbers in A and C indicate individual cells.

 

Figure 5
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  		Fig. 5. A direct-current EF induces 45Ca2+ influx. EF stimulation increases the amount of radioactive 45Ca2+ (dpm) detected within both wild-type and iplA-null mutant cells (average of four independent experiments).

 

Figure 6
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  		Fig. 6. iplA gene is not required for electrotaxis. (A-C) Trajectories plotting the movement of cell centres under control conditions (A), in the presence of an electric field with the cathode to the right (B) and on reversal of field polarity (C). (B',C') Consecutive micrographs show directional migration of cells in an electric field of 15 V/cm at 0, 7.5 and 15 minutes (supplementary material, Movie 3). Numbers in A-C' indicate individual cells.

 

Figure 7
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  		Fig. 7. The iplA gene is not required for electrotaxis. (A) iplA-null cells and AX2 cells migrate towards the cathode with similar direction of cell migration. (B) The deletion of iplA does not significantly affect the trajectory speeds. (C) Displacement speeds increased significantly in both types of cells following field stimulation, but the increase of that of iplA-null cells was significantly less than that of AX2 cells (P=0.004; data are the average of 21-33 cells from three independent experiments).

 

Figure 8
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  		Fig. 8. Depletion of [Ca2+]e abolishes electrotactic response. (A,C) Varying [Ca2+]e concentrations did not affect cell migration in control conditions. (B) Increasing [Ca2+]e to 0.6 mM did not affect the electrotactic response. (D) depletion of [Ca2+]e (by using Ca2+-free buffer or addition of 5 mM EGTA) completely abolished the electrotactic response, while the cells still migrate actively. (supplementary material, Movie 4). Numbers in A-D indicate individual cells.

 

Figure 9
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  		Fig. 9. Requirement of [Ca2+]e for electrotactic migration. (A-C) Effects of varying [Ca2+]e concentrations on (A) direction of cell migration, (B) trajectory speed and (C) displacement speed. Data are the average of 30-72 cells from at least three independent experiments.

 

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© The Company of Biologists Ltd 2006