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First published online 25 May 2004
doi: 10.1242/jcs.01143
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Research Article |
1 Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
2 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-5730, USA
3 Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, 6703 HA Wageningen, The Netherlands
4 Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands
5 MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands
* Author for correspondence (e-mail: haastert{at}chem.rug.nl)
Accepted 3 February 2004
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.
Key words: Chemotaxis, PtdIns(3,4,5)P3, Self-organization, Patches, Pseudopod
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