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Spatial Localization of PtdInsP2 in Phase-Separated Giant Unilamellar Vesicles with a Fluorescent PLC-delta 1 PH Domain

  • Xavier MuletEmail author
  • Erika Rosivatz
  • Ka Kei Ho
  • Béatrice L.L.E. Gauthé
  • Oscar Ces
  • Richard H. Templer
  • Rudiger Woscholski
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 462)

Abstract

This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate that are larger than 20 μm 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.

Keywords

Giant unilamellar vesicles phosphatidylinositol 4,5-bisphosphate phospholipase C-delta 1 PH domain 

Abbreviations

BSA:

bovine serum albumin;

DOPtdCho:

dioleylphosphatdylcholine;

DOPtdEth:

dioleyl-phosphatdylethanolamine;

GUV:

giant unilamellar vesicle;

NBD:

[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl;

PH:

pleckstrin homology;

PtdInsP2:

phosphatidylinositol 4,5-bisphosphate;

PLC:

phospholipase C.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Xavier Mulet
    • 1
    Email author
  • Erika Rosivatz
  • Ka Kei Ho
  • Béatrice L.L.E. Gauthé
  • Oscar Ces
  • Richard H. Templer
  • Rudiger Woscholski
  1. 1.Chemical Biology CentreImperial CollegeLondonUK

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