P. J. M. Van Haastert

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

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)P3]. Uniform cyclic AMP (cAMP) concentrations induce persistent translocation of PHCrac-GFP from the cytosol to multiple patches, which are similar to the single patch of PHCrac-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 PHCrac-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 PHCrac-GFP. Inhibition of phosphoinositide-3-kinase (PI3K) activity with LY294002 inhibits PHCrac-GFP patches and inhibits chemotaxis towards nanomolar cAMP but has no effect at higher cAMP concentrations. Thus, very low cAMP concentrations induce self-organizing PHCrac-GFP patches that serve as a spatial cue for pseudopod formation, which enhances the sensitivity and amplitude of chemotactic movement.

Distinction Between the Rate Theory and the Occupation Theory of Signal Transduction By Receptor Activation

Two ways to describe the response after an interaction between a ligand and a receptor are the rate theory and the occupation theory. The response of rate receptors is proportional to the rate of association while the response of occupation receptors is proportional to the fraction of receptors occupied. Ligands are bound to receptors by dipole bonds and ionic and hydrophobic interactions. Ligand derivatives in which one or more atomic interactions with the receptor cannot take place will dissociate faster than the natural ligand. A faster dissociation from a rate receptor allows more associations between the derivative and the receptor resulting in a higher maximal response than with the natural ligand. The maximal response of an occupation receptor to the natural ligand and to a ligand derivative are the same. Analysis of dose-response curves allows discrimination between these two receptors even if the response can only be recorded a long period after the activation of the receptor.