Supported double membranes
Introduction
Biological membranes are characterized by their high level of complexity. Scaling down the complex real membranes to simpler model membrane systems has become a popular approach to achieve insights into the functions and interactions of their components. In recent years, supported bilayers (Tamm and McConnell, 1985) have received increasingly more attention due to their relative ease of preparation and their accessibility to sophisticated fluorescence techniques. Single particle techniques in particular are promising tools to gain detailed insight into biological mechanisms that are not accessible by ensemble measurements. Starting with the tracking of single lipids (Schmidt et al., 1995), supported membranes have since been employed in studies of more complex reactions such as, single vesicle fusion (Bowen et al., 2004, Fix et al., 2004, Liu et al., 2005) and the fusion of single viral particles (Floyd et al., 2008). For a recent review on preparation and characterization methods of supported bilayers and their applications see (Kiessling et al., 2009).
In parallel to modeling membrane processes, an additional application of supported bilayers has emerged in recent years. Supported membranes can be utilized to anchor the tethers to vesicles that encapsulate molecules of interest (Boukobza et al., 2001) or that themselves act as the model membrane of choice (Yoshina-Ishii and Boxer, 2003). Like supported bilayers, surface-tethered vesicles have been used to gain insight into SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor)-mediated neuronal vesicle fusion (Yoon et al., 2006). Tethering vesicles to a supported membrane has the advantage that nonspecific membrane surface interactions are reduced and, as a result, the observed reactions may more directly address the pertinent biological questions.
Our original motivation for this work was to develop a fusion assay with proteoliposomes that contain single copies of the fluorescently labeled transmembrane SNARE syntaxin-1A tethered to a supported bilayer. We utilized a biotin–streptavidin tether and found that most vesicles docked to supported bilayers that were prepared by a combined Langmuir–Blodgett/vesicle fusion (LB/VF) technique (Crane et al., 2005, Kalb et al., 1992) are laterally mobile above the supporting membrane. When vesicles were added at high concentrations, we found, to our surprise, that a second bilayer was formed on top of the first bilayer. These double membranes are comparable to systems that have been prepared by attaching much larger vesicles, giant unilamellar vesicles (GUV), to supported membranes as models for cell adhesion (Albersdorfer et al., 1997, Bruinsma et al., 2000, Kloboucek et al., 1999), or after rupturing as models for inter-membrane junctions (Kaizuka and Groves, 2004, Wong and Groves, 2001). Since it is relatively easy to reconstitute membrane proteins into smaller proteoliposomes, we introduce our system as a new model to study biological processes distributed between or located in the proximity of two membranes such as the double membranes of Gram-negative bacteria or mitochondria, the synaptic cleft between neurons, or cell–cell adhesion contacts.
In the following, we first characterize the biotin–streptavidin-tethered vesicles by single particle tracking (SPT) experiments. We then demonstrate the formation of a second bilayer by measuring the lateral diffusion of fluorescently labeled lipids by FRAP (fluorescence recovery after photobleaching) and the inter-membrane distance by FLIC (fluorescence interference contrast) microscopy. To test the feasibility of the double membranes as model systems we have reconstituted the transmembrane protein syntaxin-1A and characterized its mobility by SPT.
Section snippets
Materials and methods
The following materials were purchased and used without further purification: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[7-nitro-2-1,3-benzoxadiazol-4-yl] (NBD-DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[lissamine rhodamine B] (Rh-DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl (polyethylene glycol) 2000] (biotin-peg-DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[cap biotinyl)
Supported membrane
In order to prepare a surface with well-defined surface properties, we prepared biotinylated supported bilayers by the Langmuir–Blodgett/vesicle-fusion technique. Large unilamellar POPC vesicles that contained 1 mol% of biotin-peg-DSPE were added to supported monolayers composed of only POPC. The quality of these membranes was controlled by FRAP experiments with 0.5 mol% NBD-DOPE that was included in the bilayers. The diffusion coefficient was ∼1 μm2/s with a mobile fraction of >90% (data not
Discussion
We have shown that single vesicles can be tethered efficiently to biotinylated supported membranes that have been treated with streptavidin. By reconstituting the transmembrane protein syntaxin-1A at single molecule concentrations into biotinylated vesicles, we utilized a concept introduced by Boukobza et al. (2001), who encapsulated single soluble proteins in a similar tethered vesicle system. However, in contrast to the earlier work, most of the tethered vesicles in our system are laterally
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