Switching direction in electric-signal-induced cell migration by cyclic guanosine monophosphate and phosphatidylinositol signaling

Abstract

Switching between attractive and repulsive migration in cell movement in response to extracellular guidance cues has been found in various cell types and is an important cellular function for translocation during cellular and developmental processes. Here we show that the preferential direction of migration during electrotaxis in Dictyostelium cells can be reversed by genetically modulating both guanylyl cyclases (GCases) and the cyclic guanosine monophosphate (cGMP)-binding protein C (GbpC) in combination with the inhibition of phosphatidylinositol-3-OH kinases (PI3Ks). The PI3K-dependent pathway is involved in cathode-directed migration under a direct-current electric field. The catalytic domains of soluble GCase (sGC) and GbpC also mediate cathode-directed signaling via cGMP, whereas the N-terminal domain of sGC mediates anode-directed signaling in conjunction with both the inhibition of PI3Ks and cGMP production. These observations provide an identification of the genes required for directional switching in electrotaxis and suggest that a parallel processing of electric signals, in which multiple-signaling pathways act to bias cell movement toward the cathode or anode, is used to determine the direction of migration.

Fig. 1.

Reversal of directional preference during electrotaxis in KI-8, a particular chemotaxis-deficient mutant. (A–H) Migration of WT cells (A and B) and the mutants KI-5 (C and D), KI-8 (E and F), and KI-10 (G and H) under a dcEF (10 V·cm⁻¹) in which WT and the KI-5 mutant migrated toward the cathode. The KI-8 mutant moved toward the anode. The KI-10 mutant migrated in random directions. Blue lines and orange arrows represent the cell trajectory and its direction of migration, respectively. (B, D, F, and H) Cell trajectories in 10 V·cm⁻¹. The starting points for cell migration were at the origin. (I) Dependence of directedness on the dcEF strength. (J) Although migration velocity was specific for cell type, velocity had minimal dependence on electric field strength. Data (mean ± SEM) for each cell type were quantified from 7–9 independent experiments. (Scale bar, 100 μm.)

Fig. 2.

Switching migration direction during electrotaxis by simultaneous inhibition of GCase and PI3Ks activities. (A and B) Cell trajectories of gc-null (A) and gbpC-null (B) cells at 10 V·cm⁻¹. (C and D) Reversal of preferential direction was observed in gc-null (C) and gbpC-null (D) cells in the presence of 60 μM LY294002, a PI3K inhibitor, and 1 μM cAMP at 10 V·cm⁻¹. (E) Directedness at 10 V·cm⁻¹. Migration direction was reversed only when the activity of cGMP- and PI3K-dependent pathways was suppressed simultaneously. *, P < 0.01 compared with WT cells at 10 V·cm⁻¹; #, no statistical significance, unpaired Student's t test. (F) Time course of directedness (instantaneous directedness) at 10 V·cm⁻¹ in which directedness of cells was obtained in 1-min intervals. Data (mean ± SEM) for each cell type were quantified from 8–11 independent experiments.

Fig. 3.

Opposite function of GCase subdomains to determine migration direction during electrotaxis. (A and B) Control. (C and D) cAMP at 1 μM. (E and F) cAMP at 1 μM and LY294002, a PI3K inhibitor, at 60 μM. (A–F) Cell trajectories of gc-null cells expressing sGCΔΝ (gc-null/sGCΔN) (A, C, and E) or sGCΔCat (gc-null/sGCΔCat) (B, D, and F) at 10 V·cm⁻¹. Expression of sGCΔΝ or sGCΔCat into gc-null cells consistently caused the cathode-directed and anode-directed bias in electrotaxis, respectively, regardless of whether cAMP and the PI3K inhibitor were present or not. (G) A summary of directedness. *, P < 0.01 compared with WT cells at 10 V·cm⁻¹. **, P < 0.01 compared with gc-null/sGCΔN cells at 10 V·cm⁻¹. ***, P < 0.01 compared with gc-null/sGCΔCat cells treated with 1 μM cAMP at 10 V·cm⁻¹. #, no statistical significance, unpaired Student's t test. (H) Time course of directedness (instantaneous directedness) obtained in 1-min intervals at 10 V·cm⁻¹. Data (mean ± SEM) for each cell type was quantified from 8–11 independent experiments.

Fig. 4.

Intracellular localization of signaling molecules responsible for electrotaxis under a dcEF. (A–F) Confocal images of cells expressing sGC-GFP (A), GbpC-GFP (B), PI3K2-GFP (C), PH_Akt/PKB-GFP (D), sGCΔN-GFP (E), and sGCΔCat-GFP (F) at 10 V·cm⁻¹ in the absence of cAMP and LY294002, a PI3K inhibitor. In this condition, cells exhibited cathode-directed electrotaxis. (G and H) Confocal images of cells expressing sGCΔN-GFP (G) and sGCΔCat-GFP (H) at 10 V·cm⁻¹ in the presence of 1 μM cAMP and 60 μM LY294002. Migration directions are represented by white arrows. (A–D) No treatment Latrunculin A. (E–H) Presence of Latrunculin A at 5 μM. (Scale bar, 10 μm.)

Fig. 5.

Model for directional switching in electrotaxis. Multiple-signaling pathways mediate electrotactic signals to bias migration toward the cathode and anode. PI3Ks and the catalytic domain of sGC with GbpC are involved in cathode-directed migration, whereas the N-terminal domain of sGC and unidentified cAMP-activated pathways (X) mediate anode-directed migration. Migration direction is determined by a tug-of-war-like mechanism between the multiple-signaling pathways. The sGC-dependent pathways can be switched between cathode-directed and anode-directed signaling through intracellular cGMP levels.