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)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.
Postma
Marten
M
Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Roelofs
Jeroen
J
Goedhart
Joachim
J
Loovers
Harriët M
HM
Visser
Antonie J W G
AJ
Van Haastert
Peter J M
PJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism / Q000502:physiology*
D000199:Actins; D002867:Chromones; D000242:Cyclic AMP; D049452:Green Fluorescent Proteins; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015800:Protozoan Proteins; C089420:dagA protein, Dictyostelium; C060974:phosphatidylinositol 3,4,5-triphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one