ABSTRACT Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 +/- 1.0 nm. The cleft distance was increased to 3.9 +/- 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 +/- 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.
Abbreviations used: DiI, 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocya-- nineperchlorate; DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine DPS -DMPE-PEG-- triethoxysilane; FLIC, fluorescence interference-contrast; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; HEPES, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]); NBD-- eggPE -N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-egg phosphoethanolamine; beta-OG, beta-octylglucoside; PEG, poly(ethylene glycol); POPC, 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine; RB, reconstitution buffer (25 mM HEPES/KOH, 100 mM KCl, pH 7.4); SNAP, soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein; SNAP-25, synaptosome-- associated protein of 25 kDa; SNARE, SNAP receptor; t-SNARE, target SNARE; v-SNARE, vesicle SNARE; VAMP, vesicle-associated membrane protein.
INTRODUCTION
Supported lipid bilayer membranes (Tamm and McConnell, 1985) have found wide application as models of cellular membranes in fundamental and applied biophysical research (see Sackmann and Tanaka, 2000; Boxer, 2000, for recent reviews). Interest in supported membranes ranges from studies of lipid-protein interactions and membrane selfassembly to the design of membrane-based biosensors and modeling interactions between cell surfaces in the immune system and elsewhere. We have recently been interested in reconstituting biological, i.e., protein-mediated membrane fusion in a supported bilayer membrane format (Hinterdorfer et al., 1994; Wagner and Tamm, 2001).
A common problem of membranes that are directly supported on hydrophilic glass or other hard substrates is that integral membrane proteins, such as cell surface receptors are not laterally mobile in these systems. To overcome this problem, several investigators have suggested supporting the membranes on soft polymer cushions that are intercalated between the hard surface and the lipid bilayer membrane. To detach the membrane proteins from the glass support in a physiological environment, the polymer must be well soluble in water (i.e., water must approximate a theta solvent for the polymer of choice) and must exhibit minimal interactions with the supporting glass and the lipids and proteins of the supported membrane. We have chosen polyethylene glycol as a polymer that fulfills these criteria. To achieve a stable attachment of the cushioned bilayer to the solid support, we have designed a tripartite molecule (DPS), which consists of a lipid, a 3400 molecular weight polyethylene glycol, and a reactive silane for covalent attachment to glass or quartz supports. Supported lipid bilayers containing 3 mol % of DPS in the leaflet facing the solid substrate could be stably attached to quartz or glass, were uniformly fluorescent, and allowed membrane lipids and proteins to diffuse relatively freely in the plane of the membrane (Wagner and Tamm, 2000; 2001). In this previous work, we estimated from simple polymer theory that the distance between the support and bilayer should be of the order of 4.8 nm, i.e., the Flory diameter of PEG3400. Although some membrane proteins diffused laterally, the bilayer-support separation distance has not been directly measured in this system.
Neutron reflectivity with deuterium-labeled components is a relatively established technique to measure distances of layers of different scattering density in supported membranes (Naumann et al., 1996; Wong et al., 1999). However, the technique requires relatively large amounts of material, expensive equipment, and a model-dependent data analysis. Neutron reflectivity measurements also have a poor lateral resolution. Ellipsometry unfortunately works only in samples exposed to air. Surface plasmon resonance and quartz crystal microbalance techniques simply detect refractive index and surface mass changes, respectively, and therefore are insufficient to resolve structural details of the supported layers.
A very promising new optical technique to probe details of stratified layers on reflective surfaces is fluorescence interference-contrast microscopy (Lambacher and Fromherz, 1996). In this technique, an interference pattern between the incoming and reflected light is generated on the surface of an oxidized silicon chip. The fluorescence intensity of a deposited layer varies depending on the distance of the layer from the reflective surface. To simultaneously probe several distances in the same sample and thereby provide for an internal standard, Fromherz and co-workers suggested using patterned silicon chips with oxide layers of different thickness. These workers showed that supported lipid bilayers (without an interstitial polymer layer) are supported on a 1-2 nm thin film of water (Fromherz et al., 1999), as had been suspected from lateral diffusion measurements (Tamm and McConnell, 1985) and later measured by neutron reflectivity (Johnson et al., 1991). In the present work, we are using FLIC microscopy to measure the distance between the silicon dioxide surface of an oxidized silicon chip and a DPS-polymer supported bilayer. We also demonstrate the first application of this method to measure the surface distance of a fluorescent labeled protein ligand (GFP-VAMP) after its specific binding to integral membrane receptors (t-SNAREs) that were functionally reconstituted into polymer-supported bilayers. Fig. 1 illustrates the general configuration of the two types of experiments.
MATERIALS AND METHODS
We thank Dr. P. Fromherz (Max-Planck-Institute for Biochemistry, Martinsried, Germany) for allowing us to manufacture FLIC chips in his laboratory and for providing the FLIC fitting software, and Drs. J. Rothman and T. Melia (Memorial Sloan-Kettering Cancer Center, New York) for the generous gift of the SNARE proteins used in this study. We also thank Drs. D. Braun and A. Lambacher for helpful discussions on the FLIC theory.
This work was supported by National Institutes of Health (AI30557). V.K. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft.
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[Author Affiliation]
Volker Kiessling and Lukas K. Tamm
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908-0736 USA
[Author Affiliation]
Submitted June 21, 2002, and accepted for publication September 25, 2002.
Address reprint requests to Lukas K. Tamm, Dept. of Molecular Physiology and Biological Physics, University of Virginia, P.O. Box 800736, Charlottesville, VA 22908-0736. Tel.: 434-982-3578; Fax: 434-982-1616; E-mail: lkt2e@virginia.edu.
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