Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia

Abstract

The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1microM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 s after stimulation and disappears after approximately 20 s. A second translocation of PHCrac-GFP starts after approximately 30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of approximately 4-5 microm, each covering approximately 10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at approximately 10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.

Figure 1.

cAMP-mediated translocation of PH_Crac-GFP. Aggregation competent cells were stimulated in a perfusion chamber with a homogeneous cAMP concentration of 1 μM at t = 0 s. Confocal images were taken at the times indicated. The figure shows GFP in the cytosol before cAMP stimulation, a transient translocation of GFP to the entire boundary of the cell at 8 s after addition of cAMP, and patches of GFP at the boundary after 40 s.

Figure 2.

Quantification of fluorescence intensity in the cytosol and along the cell boundary. A representative cell before and 8 s after stimulation with 1 μM cAMP is shown in panels A and B, respectively. An area of the cytosol was selected that is devoid of the nucleus and large vesicles. The mean fluorescence intensity in the selected area was determined and is presented in C as relative fluorescence at different times after stimulation with cAMP. The results shown are the means and SD (large bars) or SE of the mean (small bars) of 20 cells. (D) The boundary contour of the cAMP-stimulated cell. On this contour thousand short perpendicular lines were positioned (inset in D); the fluorescence intensity along one line segment is presented in E. The fluorescence intensity along the membrane was determined for the 1000 boundary points as described in MATERIALS AND METHODS and is presented in F. The thin gray line represent the original data, which are smoothed by adjacent averaging in the black line. Î_C + σ̂_C refers to the fluorescence intensity of the cytosol Î_C and the SD σ̂_C. The 0-position and direction are indicated by the arrow in D.

Figure 3.

Fluorescence intensity in the cytosol and at the at the cell boundary. (A) Cell at different times after stimulation with 1 μM cAMP. (B) PH_Crac-GFP at the boundary, presented as the difference of fluorescence intensity between the boundary and the cytosol, color-coded as shown below panel A. The ordinate refers to the position along the cell with the 0-position and direction indicated by the arrow in the cell image. (C) Fluorescence intensity in the cytosol of this cell and along its entire boundary obtained from seven cells. The data show two phases of depletion in the cytosol that coincide with increases of fluorescence at the boundary.

Figure 4.

Fraction of boundary with significant translocation (FoT). The FoT value is defined as the fraction of the boundary that shows a significantly higher fluorescence intensity than in the cytosol. The FoT values were determined for 7 cells at 8 and 58 s after stimulation. The FoT value at 8 s after stimulation is very high, indicating PH_Crac-GFP associates to nearly the entire boundary, whereas at 58 s after stimulation PH_Crac-GFP is found at only ∼50% of the boundary in patches.

Figure 5.

Three-dimensional reconstruction of patches. Cells expressing PH_Crac-GFP were stimulated with 1 μM cAMP for 2-3 min. From a series of confocal Z-scans, the fluorescence intensity at the cell boundary was determined. The figure reveals one patch with a diameter of ∼8 μm.

Figure 6.

Correlation between PH_Crac-GFP patches and pseudopodia. The life cycles of two patches are presented that appear ∼35 s after stimulation with 1 μM cAMP. Phase I, onset of patch formation. The first appearance of increased levels of PH_Crac-GFP at the boundary is indicated by the arrow at t = 0 s; the patch grows during the next 10 s. Phase II, protrusions and pseudopod extension occurs at the location of the PH_Crac-GFP patch (arrow; the asterisk at 24 s indicates the start of a new patch). Phase II termination of the patch and retraction of the pseudopod. At about 60 s the patch reduces in size and disappears, and the pseudopod is retracted (double arrow).

Figure 7.

Fluorescent intensity at the boundary of a locally stimulated cell. Data obtained from Parent et al. (1998). Cells were stimulated with a pipette filled with 1 μM cAMP. (A) The fluorescence intensity of a typical cell (the position of the pipette is indicated; note that this is not a confocal image). The fluorescence intensity at the boundary is presented for this cell in B; the position of the pipette is at 0 μm. The results show a nearly uniform response at t = 0 s, followed by a patch of ∼6 μm that is formed in the direction of the pipette and is present from 28 to 104 s. A new patch is formed around 108 s, not at the position of the old patch but immediately to the right side. These patches of PH_Crac-GFP are associated with the formation of pseudopodia at those positions.