title: Solid supported membranes doped with PIP2: influence of ionic strength and pH on bilayer formation and membrane organization.
abstract: Phosphoinositides and in particular L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) are key lipids controlling many cellular events and serve as receptors for a large number of intracellular proteins. To quantitatively analyze protein-PIP2 interactions in vitro in a time-resolved manner, planar membranes on solid substrates are highly desirable. Here, we describe an optimized protocol to form PIP2 containing planar solid supported membranes on silicon surfaces by vesicle spreading. Supported lipid bilayers (SLBs) were obtained by spreading POPC/PIP2 (92:8) small unilamellar vesicles onto hydrophilic silicon substrates at a low pH of 4.8. These membranes were capable of binding ezrin, resulting in large protein coverage as concluded from reflectometric interference spectroscopy and fluorescence microscopy. As deduced from fluorescence microscopy, only under low pH conditions, a homogeneously appearing distribution of fluorescently labeled PIP2 molecules in the membrane was achieved. Fluorescence recovery after photobleaching experiments revealed that PIP2 is not mobile in the bottom layer of the SLBs, while PIP2 is fully mobile in the top layer with diffusion coefficients of about 3 μm(2)/s. This diffusion coefficient was considerably reduced by a factor of about 3 if ezrin has been bound to PIP2 in the membrane.
authors.affiliation: Institut für Organische und Biomolekulare Chemie, Georg-August Universität , Tammannstr. 2, 37077 Göttingen, Germany.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Braunger
authors.forename: Corinna
authors.initials: C
authors.lastname: Kramer
authors.forename: Daniela
authors.initials: D
authors.lastname: Morick
authors.forename: Claudia
authors.initials: C
authors.lastname: Steinem
mesh_terms: D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004058:Diffusion; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D009994:Osmolar Concentration; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D008051:Lipid Bilayers; D018129:Phosphatidylinositol Phosphates; D000099149:Ezrin