Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures

AbstractPhosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is known to regulate a wide range of molecular targets and cellular processes, from ion channels to actin polymerization [1–6]. Recent studies have used the phospholipase C-δ1 (PLC-δ1) pleckstrin-homology (PH) domain fused to green fluorescent protein (GFP) as a detector for PI(4,5)P2in vivo[7–10]. Although these studies demonstrated that PI(4,5)P2 is concentrated in the plasma membrane, its association with actin-containing structures was not reported. In the present study, fluorescence imaging of living NIH-3T3 fibroblasts expressing the PLC-δ1 PH domain linked to enhanced green fluorescent protein (PH–EGFP) reveals intense, non-uniform fluorescence in distinct structures at the cell periphery. Corresponding fluorescence and phase-contrast imaging over time shows that these fluorescent structures correlate with dynamic, phase-dense features identified as ruffles and with microvillus-like protrusions from the cell’s dorsal surface. Imaging of fixed and permeabilized cells shows co-localization of PH–EGFP with F-actin in ruffles, but not with vinculin in focal adhesions. The selective concentration of the PH–EGFP fusion protein in highly dynamic regions of the plasma membrane that are rich in F-actin supports the hypothesis that localized synthesis and lateral segregation of PI(4,5)P2 spatially restricts actin polymerization and thereby affects cell spreading and retraction