Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

Abstract

Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods.

Keywords: SNARE; TIRF; fluorescence microscopy; supported membrane; synaptic vesicle fusion.

Figure 1

Sample preparation and requirements for supported membrane—SNARE applications. (A) Reconstitution of trans-membrane proteins into supported membranes is accomplished by a two-step technique. In step i, a lipid monolayer is transferred from the water-air interface of a Langmuir-Blodgett trough onto an appropriate hydrophilic substrate. In step ii, protein-containing liposomes are fused with the monolayer in a flow-through chamber to assemble the second leaflet of the supported bilayer and to incorporate membrane proteins. (B) Syntaxin-1a (Syx1a), reconstituted by the technique pictured in (A) is oriented with its SNARE motif facing away from the substrate. Incubation of labeled Syx1a (Alexa 546 at residue 192) with Co²⁺ results in ~90% fluorescence quenching while the same application with a symmetrically distributed rhodamine labeled lipid results in ~50% fluorescence quenching (Liang et al., 2013). (C,D) Formation of 1:1 Syx1a:SNAP-25 complex in DPC as demonstrated by ion-exchange chromatography and SDS-PAGE. Equal molar amounts of Syx1a and SNAP25 are mixed and incubated overnight in DPC before ion-exchange purification. (C) MonoQ elution profile: the blue trace shows UV absorption (left axis) and the red trace shows the eluted buffer conductivity (right axis). The red vertical lines at the bottom denote collected fractions, and fractions run on SDS-PAGE gels are labeled with corresponding capital letters. (D) SDS-PAGE of protein samples purified by MonoQ column chromatography. Since SNAP-25 is about twice the molecular mass of syntaxin (residues 183–288), the SNAP-25 band is twice as strong as the Syx1a band when they are in a molar ratio of 1:1 (Kreutzberger et al., 2016). (A) is reprinted from Kiessling et al. (2015a) with permission from Elsevier. (C,D) are reprinted from Kreutzberger et al. (2016) with permission from Elsevier.

Figure 2

Supported membrane—fluorescence microscopy assays. (A) Fluorescence recovery after photobleaching (FRAP) records the recovery of fluorescence due to lateral diffusion in a region of interest in the membrane after application of a strong laser pulse. The total intensity of an area is recorded before and after the bleach pulse has been applied (Smith and McConnell, 1978). The example graph on the right shows the recovery of Alexa488 labeled t-SNAREs in supported membranes (Wagner and Tamm, 2001). (B) Single particle tracking (SPT). The movement of membrane components labeled with a single fluorophore are tracked within the x/y plane of the lipid bilayer. The statistically analysis of the trajectories can quantify the lateral mobility as well as reveal different modes of diffusion (Schmidt et al., ; Kiessling et al., ; Vasquez et al., 2014). The example image on the right shows single Alexa647 labeled t-SNAREs in inverse contrast. Movies of the moving protein can be seen on the original publisher’s website (Domanska et al., 2009). (C) Binding assay using total internal reflection (TIRF) microscopy. A totally reflected laser beam produces an exponentially decaying electric field at the glass/water interface. Fluorescent molecules or organelles that bind at the membrane surface increase the observable fluorescent intensity I over time (Kalb et al., 1989). Binding isotherms can be determined with various ligand concentrations and acceptor densities. Data on the right shows SNARE-specific binding of Alexa546 labeled Syb2(1-96) to a supported membrane (Domanska et al., 2009). (D) Single vesicle fusion TIRF assay. Fluorescence originating from the membrane (red, I_M) or the content (green, I_C) of single vesicles can be imaged when they enter the evanescent field of a TIRF microscope (Fix et al., ; Domanska et al., 2009). Characteristic intensity traces from docking vesicles can be analyzed to determine docking and fusion efficiencies as well as fusion kinetics (Kiessling et al., 2015b). Data on the right shows fluorescence originating from the membrane and content during a single vesicle fusion event (Kiessling et al., 2015a). (E) Distance measurements by FLIC microscopy. A Si/SiO₂ substrate with different steps is used to probe the interference pattern originating from reflected excitation and emission light (Braun and Fromherz, 1997). Fitting the optical theory to the measured intensities I from different SiO₂ layers allows the determination of the distance of specifically labeled protein residues from the lipid bilayer surface (Lambacher and Fromherz, ; Kiessling and Tamm, 2003). Data on the right shows a FLIC curve obtained from Alexa546 labeled Syx obtained under the same conditions as published in Liang et al. (2013). Data in (A) is reprinted from Wagner and Tamm (2001) with permission from Elsevier. Data in (B,C) was originally published in Domanska et al. (2009), © the American Society for Biochemistry and Molecular Biology. Data in (D) is reprinted from Kiessling et al. (2013) with permission from Elsevier.