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 (PLCgamma), hydrolysis of phosphoinsotide 4,5-bisphosphate (PIP(2)) and subsequent release of gelsolin. We hypothesized this led to the mobilization of PIP(2)-binding proteins which modify the actin cytoskeleton and thus sought to determine whether the leading edge was a site of active PIP(2) 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 PIP(2) and a green fluorescence protein (GFP)-tagged pleckstrin homology (PH) domain of PLCdelta (GFP-PH) that recognizes both PIP(2) and inositol 1,4,5-trisphosphate (IP(3)). PIP(2) was absent from leading lamellipodia despite an increase in IP(3) generation, suggesting an increase in PIP(2) 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 PIP(2) hydrolysis at the leading edge causing a localized release of PIP(2)-binding proteins-particularly gelsolin-that drives cytoskeletal rearrangement and protrusion.
Chou
Jeffrey
J
Department of Pathology, University of Pittsburgh, 713 Scaife Street, Pittsburgh, PA 15261, USA.
Stolz
Donna B
DB
Burke
Nancy A
NA
Watkins
Simon C
SC
Wells
Alan
A
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology*; D003285:Contractile Proteins; D003599:Cytoskeleton / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D018260:Gelsolin / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D051966:Phospholipase C gamma; D051304:Profilins; D011554:Pseudopodia / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D010738:Type C Phospholipases / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D000199:Actins; D003285:Contractile Proteins; D018260:Gelsolin; D007527:Isoenzymes; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D051304:Profilins; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma