title: Triggering actin polymerization in Xenopus egg extracts from phosphoinositide-containing lipid bilayers.
abstract: Xenopus egg extracts are a powerful tool to reconstitute complex cell biological processes using a cell-free strategy. When used in conjunction with liposomes and supported lipid bilayers, they can recapitulate the biochemical activities occurring at the cytosol/plasma membrane interface of the cell that underlie remodeling of the actin cytoskeleton. We use these in vitro systems to elucidate how membranes and proteins collaborate to make the appropriate actin structure at a given time and place. We have recently broadened the types of membrane substrate used, and also optimized protocols for preparation of Xenopus egg extracts for actin assembly assays from membranes. Tuning the lipid composition and curvature appropriately demands an appreciation of the native phospholipid and curvature environments that can form transiently in cells. Supported lipid bilayers on glass coverslips that contain phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) make actin bundles termed filopodia-like structures that contain fascin and have vasodilator-stimulated phosphoprotein (VASP) at their growing tips, indicating that these resemble filopodia growing from the plasma membrane. The combination of PI(4,5)P2 and phosphatidylinositol 3-phosphate in curved liposomes or supported bilayers on glass nanospheres uses Snx9, Cdc42, N-WASP (neuronal-Wiskott-Aldrich syndrome protein), and Arp2/3 complex for actin polymerization suggesting that this membrane may mimic the progression from plasma membrane to endosomes. Here we describe how to prepare high-speed supernatant frog egg extracts and phosphoinositide-containing liposomes and supported lipid bilayers that can assemble actin structures. We also describe the methods we use to assay actin polymerization using microscopy and spectrofluorometry and our protocol for immunodepleting specific proteins from extracts.
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Astrid
authors.initials: A
authors.lastname: Walrant
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Saxton
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Guilherme Pereira
authors.initials: GP
authors.lastname: Correia
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Gallop
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D005260:Female; D008051:Lipid Bilayers / Q000138:chemical synthesis* / Q000378:metabolism; D008081:Liposomes / Q000138:chemical synthesis* / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D054118:Nanospheres / Q000378:metabolism; D010063:Ovum / Q000166:cytology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D058105:Polymerization; D011554:Pseudopodia; D058305:Sorting Nexins / Q000378:metabolism; D014020:Tissue Extracts / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D002352:Carrier Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D008840:Microfilament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010718:Phosphatidylserines; C581908:SNX9 protein, Xenopus; D058305:Sorting Nexins; D014020:Tissue Extracts; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D029867:Xenopus Proteins; C055525:phosphatidylinositol 3-phosphate; C020324:fascin; C559910:Cdc42 protein, Xenopus; D020559:Monomeric GTP-Binding Proteins
keywords: Arp2/3 complex; Cdc42; Curvature; Cytoskeleton; Filopodia; Membrane; Microscopy; N-WASP; PI(4,5)P(2); Reconstitution