Agonist-induced PIP2Hydrolysis Inhibits Cortical Actin Dynamics: Regulation at a Global but not at a Micrometer Scale

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Abstract
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 PIP2sensors 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 ∼15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP2breakdown, and it resumes as soon as PIP2levels are back to normal. Thus, our data support a role for PIP2in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP2regulation of the cytoskeleton exist at a micrometer scale.

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