Agonist-induced PIP(2) hydrolysis inhibits cortical actin dynamics: regulation at a global but not at a micrometer scale.
Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) 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 PIP(2) 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 PIP(2)-enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized at approximately 15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP(2) breakdown, and it resumes as soon as PIP(2) levels are back to normal. Thus, our data support a role for PIP(2) in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP(2) regulation of the cytoskeleton exist at a micrometer scale.
van Rheenen
Jacco
J
Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands.
Jalink
Kees
K
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D003599:Cytoskeleton / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D015233:Models, Statistical; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000819:agonists / Q000378:metabolism*; D038761:Photobleaching; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D000199:Actins; D008164:Luminescent Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D049452:Green Fluorescent Proteins