title: Spatial localization of PtdInsP2 in phase-separated giant unilamellar vesicles with a fluorescent PLC-delta 1 PH domain.
abstract: This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate that are larger than 20 microm in size. The phospholipids composition of the vesicular membrane is such that fluid lamellar and liquid-ordered or gel phases are formed and separate within the confines of one vesicle. It outlines the preparation of a protein fluorescent label, pleckstrin homology domain from phospholipase C-delta 1, that binds specifically to phosphatidylinositol 4,5-bisphosphate. Using fluorescence microscopy, the presence and spatial position of this phosphorylated phosphatidylinositol lipid on the lipid membrane have been located with the pleckstrin homology domain. We show that phosphatidylinositol 4,5-bisphosphate and the phospholipase C-delta 1 pleckstrin homology domain are located to the fluid phase of the vesicle membrane. This approach can therefore show how membrane physical properties can affect enzyme binding to phosphatidylinositol 4,5-bisphosphate and thus further the understanding of important membrane processes such as endocytosis.
authors.affiliation: Chemical Biology Centre, Imperial College, London, London, UK. xavier.mulet@imperial.ac.uk
authors.forename: Xavier
authors.initials: X
authors.lastname: Mulet
authors.forename: Erika
authors.initials: E
authors.lastname: Rosivatz
authors.forename: Ka Kei
authors.initials: KK
authors.lastname: Ho
authors.forename: Béatrice L L E
authors.initials: BL
authors.lastname: Gauthé
authors.forename: Oscar
authors.initials: O
authors.lastname: Ces
authors.forename: Richard H
authors.initials: RH
authors.lastname: Templer
authors.forename: Rudiger
authors.initials: R
authors.lastname: Woscholski
mesh_terms: D000818:Animals; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry*; D008846:Micromanipulation; D008856:Microscopy, Fluorescence; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D054803:Phospholipase C delta / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D005456:Fluorescent Dyes; D019269:Phosphatidylinositol 4,5-Diphosphate; D053835:Unilamellar Liposomes; D054803:Phospholipase C delta