Distribution of gelsolin and phosphoinositol 4,5-bisphosphate in lamellipodia during EGF-induced motility

During induced cell motility the actin cytoskeleton at the leading edge must undergo constant reorganization. Recently, phosphoinositides have been shown to be central to cytoskeleton-membrane linkages and actin organization and turnover. Epidermal growth factor (EGF) receptor (EGFR)-mediated cell motility requires phospholipase C-gamma (PLC-y), hydrolysis of phosphoinsotide 4,5-bisphosphate (PIP2) and subsequent release of gelsolin. We hypothesized this led to the mobilization of PIP2-binding proteins which modify the actin cytoskeleton and thus sought to determine whether the leading edge was a site of active PIP2 hydrolysis and gelsolin redistribution to cytoskeleton. Herein, we report that during EGF-induced motility, the leading edge's submembranous region constitutes a distinct subcellular locale. The relevant phosphoinositide composition of this space was determined by probing with an antibody to PIP2 and a green fluorescence protein (GFP)-tagged pleckstrin homology (PH) domain of PLCdelta (GFP-PH) that recognizes both PIP2 and inositol 1,4,5-trisphosphate (IP3)- PIP2 was absent from leading lamellipodia despite an increase in IP3 generation, suggesting an increase in PIP2 hydrolysis at the leading edge. Visualized with immunofluorescence, gelsolin preferentially concentrated near the leading edge in a punctate fashion. Examining the Triton X-insoluble actin cytoskeleton fractions, we observe a PLCgamma-dependent increase of gelsolin incorporation upon EGF stimulation. At a molecular level, field emission scanning electron microscopy (FE-SEM) shows that gelsolin incorporates preferentially into the submembranous actin arcs at the leading edge of the lamellipodia. Together these data suggest a model of PIP2-hydrolysis at the leading edge causing a localized release of PIP2-binding proteins-particularly gelsolin-that drives cytoskeletal rearrangement and protrusion. (C) 2002 Elsevier Science Ltd. All rights reserved.