Spatial localization of PtdInsP2 in phase-separated giant unilamellar vesicles with a fluorescent PLC-delta 1 PH domain.
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.
Mulet
Xavier
X
Chemical Biology Centre, Imperial College, London, London, UK. xavier.mulet@imperial.ac.uk
Rosivatz
Erika
E
Ho
Ka Kei
KK
Gauthé
Béatrice L L E
BL
Ces
Oscar
O
Templer
Richard H
RH
Woscholski
Rudiger
R
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*
D005456:Fluorescent Dyes; D019269:Phosphatidylinositol 4,5-Diphosphate; D053835:Unilamellar Liposomes; D054803:Phospholipase C delta