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First published online 7 October 2008
doi: 10.1242/jcs.031781

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PI3-kinase signaling contributes to orientation in shallow gradients and enhances speed in steep chemoattractant gradients

Department of Molecular Cell Biology, University of Groningen, Kerklaan 30, 9751NN Haren, The Netherlands

View larger version (26K): [in this window] [in a new window]   Fig. 1. Formation of PHCRAC-GFP patches in steep cAMP gradients. Cells expressing PHCRAC-GFP were stimulated with a micropipette filled with 10–4 M cAMP. (A) The confocal image reveals PHCRAC-GFP patch formation at the leading edge of cells close to the pipette, but more uniform cytosolic localization of PHCRAC-GFP in cells further away from the pipette. Two cells are shown at higher magnification. The asterisk indicates the position of the pipette. Scales bars: 50 µm (middle) and 10 µm (left and right). (B) The fraction of cells containing a PHCRAC-GFP patch at the leading edge and the chemotaxis index were determined at different distances from the pipette. Half-maximal PHCRAC-GFP patch formation occurs at a distance of about 50 µm from the pipette, and significant chemotaxis is observed up to 1000 µm from the pipette. (C) The chemotaxis index and speed of cells at 40-60 µm from the pipette was determined for 35 cells without and 22 cells with a PHCRAC-GFP patch. Values are averages ± s.d.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Inhibition of PI3K signaling inhibits chemotaxis in shallow gradients but not in steep gradients. (A) Chemotactic activity was measured for pi3k-null cells and wild-type cells in the absence and presence of 50 µM LY294002 using (A) the small-population assay, (B) a Zigmond chamber or (C) the pipette assay. The cAMP gradients to which the cells are exposed were calculated using the equations presented in the Materials and Methods. (D) The chemotaxis index as function of the absolute gradient is shown. The data are the means of three experiments involving about 20 populations or cells analyzed in each experiment.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Depletion of PHCRAC-GFP from the cytosol after uniform stimulation with very low cAMP concentrations. Cells expressing PHCRAC-GFP were stimulated in a perfusion chamber with the indicated concentration of cAMP. (A) Images before (top) and 14 seconds after (bottom) stimulation with 0.3 nM cAMP, showing one cell with PHCRAC-GFP patches (left) and two cells with a more uniform cytosolic distribution of PHCRAC-GFP (right). Scale bar: 10 µm. (B) Quantification of the decrease of fluorescence intensity of the cytosol after cAMP stimulation in cells with or without a PHCRAC-GFP patch. The results show the means ± s.d. of 10 to 22 cells per cAMP concentration from two independent experiments. (C) Frequency distribution of the number of cells with different amounts of depletion of fluorescence intensity in the cytosol after stimulation with 0.1, 0.3 or 1 nM cAMP for cells with or without a PHCRAC-GFP patch.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Accumulation of PHCRAC-GFP at the leading edge of cells stimulated with a gradient of cAMP. Cells expressing PHCRAC-GFP and cytosolic mRFP were stimulated with a micropipette filled with 100 µM cAMP (see supplementary material Movie 1). (A) A typical cell at a distance of about 100 µm from the pipette is shown. The cell in frame 41 exhibits a faint PHCRAC-GFP patch that is just visible in the confocal image of the GFP signal. The normalized mRFP signal of each pixel was subtracted from the GFP signal in that pixel to correct for the difference in cytosolic content of boundary pixel, yielding ccPHCRAC-GFP. Scale bar: 10 µm. (B) The fluorescence intensity of PHCRAC-GFP and ccPHCRAC-GFP at the boundary of the cell for all frames is presented. For the PHCRAC-GFP signal, a patch is visible around frames 3 and 40, but otherwise not many details can be recognized. In the panel showing ccPHCRAC-GFP, not only these patches can be seen but nearly all frames show an increased fluorescence at the leading edge. (C) The fluorescence intensity at the boundary of the cell is quantified for the PHCRAC-GFP and ccPHCRAC-GFP signal. Data for a visible PHCRAC-GFP patch are obtained from frames 3 or 4 and 39-42, and data for no visible PHCRAC-GFP patch are obtained from the remaining frames. The data show the means ± s.d. (D) The fluorescence intensity at the boundary in the front (f, black symbols) or back (b, white symbols) of the cell for the PHCRAC-GFP (triangles) and ccPHCRAC-GFP (circles) signal is shown. The left panel in D is the same cell as is shown in A-C; the gray area indicates the strong patches. The right panel in D was of the same batch of cells after incubation with 50 µM LY294002.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Cells lacking PI3K activity are out competed by wild-type cells. (A) Calculations. The chemotaxis index and speed of the cells at different distances from a pipette containing 100 µM cAMP were determined for AX3 cells in the presence and absence of the PI3K inhibitor LY294002 (see Fig. 1C; Fig. 2; supplementary material Table S1). Using these data, the displacement in the direction of the pipette due to chemotaxis and random movement was calculated (see Materials and Methods for equations). Presented is the average time needed to reach the pipette from different starting positions. The data predict that PI3K-inhibited cells always arrive later at the source than wild-type cells and perform relatively well when starting from a distance of about 100 µm, but perform increasingly worse at larger distances from the source. (B-D) Experiment. Wild-type cells were transformed with a plasmid expressing cytosolic GFP, whereas pi3k-null cells were transformed with a plasmid expressing RFP. Cells were starved, mixed (wild-type:pi3k-null ratio is 1:3) and exposed to a cAMP gradient from a pipette containing 100 µM cAMP. Confocal images were recorded before (C) and 50 minutes after (D) application of the cAMP gradient. The white dot in the centre in D indicates the position of the pipette; the field of observation is 725x725 µm, recorded as 5x5 tile scans. The cell density was measured at different distances from the pipette before and after stimulation (B). The data show the increase of cell density as means ± s.d. of four determinations.

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