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First published online 25 May 2004
doi: 10.1242/jcs.01143

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

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

View larger version (29K): [in a new window]   Fig. 1. cAMP-mediated translocation of PHCrac-GFP. Dictyostelium cells were stimulated at t=0 seconds in a perfusion chamber with a homogeneous cAMP concentration of 1 µM or 1 nM cAMP. Confocal images were taken at the times indicated. PHCrac-GFP is in the cytosol before cAMP stimulation, and is translocated to the entire boundary (8 seconds) and to patches (30 seconds and later) after stimulation with 1 µM cAMP, whereas stimulation with 1 nM cAMP induces only patches of PHCrac-GFP (20 seconds and later).

View larger version (45K): [in a new window]   Fig. 2. Disappearance of patches after removal of cAMP. Cells were stimulated with 1 µM cAMP for 240 seconds (first 60 seconds shown), washed with buffer for 30 seconds (from 240-270 seconds), and then re-stimulated with 1 µM cAMP for 30 seconds. (A) The response at the boundary of a typical cell. (B) The cell was washed with buffer for only 10 seconds. (C) The means and standard deviations of the fluorescence intensity of the cytosol from 20 cells; the black line shows the intensity for the wash with buffer for 30 seconds, and the green line for 10 seconds. The dotted line represents a first-order decay of the fluorescence intensity with a latency time (tlat) and a half-life (t1/2).

View larger version (85K): [in a new window]   Fig. 3. cAMP dependency of PHCrac-GFP localization. Cells were incubated in buffer for 30 seconds, followed at t=0 seconds by perfusion with the indicated cAMP concentrations. At t=30 seconds, all cells were perfused with 1 µM cAMP. (A-E) The response at the boundary of a typical cell; the colours represent the differences between fluorescence intensity at the boundary and the cytosol. (F-J) The response in the cytosol, shown as the fluorescence intensity after stimulation relative to the fluorescence intensity before stimulation; the figure shows the means and standard deviations (large bars) and standard errors of the means (small bars) of about 20 cells. The experiment with 0.1 nM cAMP was slightly different: cells were stimulated for 45 seconds followed by the upshift. Because only about 15% of the cells responded to 0.1 nM cAMP with a patch, the fluorescence intensity in the cytosol (J) is presented for 18 cells without a patch (a) and two cells with a patch (b). (E) The response at the boundary in a cell with a patch. (D) The patch before cAMP stimulation is a bright cytosolic vesicle located next to the boundary.

View larger version (23K): [in a new window]   Fig. 4. Dose response curves of PHCrac-GFP translocation and patch formation. Cells were incubated in buffer for 30 seconds, followed at t=0 seconds by perfusion with the indicated cAMP concentrations. At t=30 seconds, all cells were perfused with 1 µM cAMP. The experiment with 0.1 nM cAMP was slightly different: cells were stimulated for 45 seconds followed by the upshift. Different aspects of these responses are presented as a percentage of the maximal response: the first depletion of the fluorescence intensity in the cytosol (maximum 53%); recovery of the fluorescence intensity in the cytosol (maximum 100%); the proportion of cells with one or more patches (maximum 100%); the number of patches per cell (maximum 2.2); the fluorescence intensity of the patches (maximum 2.4); and the size of the patches (maximum 9.85 µm). The results shown are the means and standard error of the means of about 20 cells (except the size and fluorescence intensity of the patches at 0.1 nM cAMP, which were for five patches).

View larger version (79K): [in a new window]   Fig. 5. Patch formation in latrunculin-A-treated cells. Cells in the perfusion chamber were incubated with 1 µM latrunculin A for 15 minutes, subsequently stimulated at t=0 seconds with 1 µM cAMP and washed with buffer at t=90 seconds. (A) The fluorescence intensity and the shape of a representative cell before and after stimulation with cAMP. (B) The fluorescence intensity at the boundary for the same cell. Before mounting the perfusion chamber, cells were overlaid with a thin layer of agarose to prevent elution of these spherical cells. The small indentations at 0 seconds, 90 seconds and 120 seconds are perfusion artefacts; at the onset of perfusion, the cover slip bends for a few seconds and cells move in the plane of focus.

View larger version (23K): [in a new window]   Fig. 6. Inhibition of chemotaxis by LY294002. Wild-type cells were starved for 5 hours on plates in phosphate buffer, resuspended in buffer and deposited as small droplets on hydrophobic agar containing different concentrations of LY294002. After about 15 minutes, droplets with different cAMP concentrations were deposited close to the amoebae and the chemotaxis response was observed during the following 30 minutes. The data are presented as a percentage of the populations (about 20 for each cAMP concentration) that showed a positive chemotactic response.