Agonist-induced PIP2 hydrolysis actin dynamics:: Regulation at a global but not at a micrometer scale

Phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane has been proposed to locally regulate the actin cytoskeleton. Indeed, recent studies that use GFP-tagged pleckstrin homology domains (GFP-PH) as fluorescent PIP2 sensors suggest that this lipid is enriched in membrane microdomains. Here we report that this concept needs revision. Using three distinct fluorescent GFP-tagged pleckstrin homology domains, we show that highly mobile GFP-PH patches colocalize perfectly with various lipophilic membrane dyes and, hence, represent increased lipid content rather than PIP2-enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized at similar to15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP2 breakdown, and it resumes as soon as PIP2 levels are back to normal. Thus, our data support a role for PIP2 in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP2 regulation of the cytoskeleton exist at a micrometer scale.