Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches.

Abstract

The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate [PtdIns(3,4,5)P(3)]. Uniform cyclic AMP (cAMP) concentrations induce persistent translocation of PH(Crac)-GFP from the cytosol to multiple patches, which are similar to the single patch of PH(Crac)-GFP at the leading edge in a cAMP gradient. We show that cAMP determines the probability of patch formation (half-maximal effect at 0.5 nM cAMP) but not the size, lifetime or intensity of patches, indicating that patches are self-organizing structures. A pseudopod is extended from the area of the cell with a PH(Crac)-GFP patch at about 10 seconds after patch formation. Cells treated with the F-actin inhibitor latrunculin A are round without pseudopodia; uniform cAMP still induces localized patches of PH(Crac)-GFP. Inhibition of phosphoinositide-3-kinase (PI3K) activity with LY294002 inhibits PH(Crac)-GFP patches and inhibits chemotaxis towards nanomolar cAMP but has no effect at higher cAMP concentrations. Thus, very low cAMP concentrations induce self-organizing PH(Crac)-GFP patches that serve as a spatial cue for pseudopod formation, which enhances the sensitivity and amplitude of chemotactic movement.