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A more recent version of this article appeared on December 1, 2003
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Submitted on August 7, 2003
Revised on September 23, 2003
Accepted on October 9, 2003
1 Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands
2 Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands, Department of Cell Biology, Harvard
Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115-5730, U.S.A
3 Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, 6703 HA Wageningen, The Netherlands., Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands
4 MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, The Netherlands
* Corresponding author. E-mail address: P.J.M.van.Haastert{at}chem.rug.nl.
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 1 µM 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.
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