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Vol. 14, Issue 12, 5019-5027, December 2003
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* Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands;
Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, 6703 HA Wageningen, The Netherlands; and
¶ MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, The Netherlands
Submitted May 22, 2003;
Revised September 23, 2003;
Accepted October 9, 2003
Monitoring Editor: Paul Matsudiara
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 1µM 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 
20 s. A second translocation of PHCrac-GFP starts after 30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of 4-5 µm, each covering 10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at 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.
Present address: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-5730
Present address: Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
|| Corresponding author. E-mail address: P.J.M.van.Haastert{at}chem.rug.nl.
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