Single vesicle fusion assays

Vesicle-vesicle assays

Single vesicle-vesicle fusion assays were developed to circumvent the disadvantages of ensemble fusion assays. One major approach to achieve this is to tether one vesicle population on a surface and add the second population to the immobilized vesicles.

Yoon et al. developed a system in which they tethered t-SNARE containing vesicles labeled with the acceptor fluorophore DiD via biotin-neutravidin interaction to a biotin-polyethyleneglycol coated glass surface and added v-SNARE containing vesicles labeled with the donor fluorophor DiI to the immobilized vesicles [45]. Lipid mixing was subsequently observed by total internal reflection (TIRF) microscopy considering the FRET efficiencies. With this approach they could distinguish between docked and fused vesicles. However, the assay did not allow to distinguish between hemifusion and full fusion.

In order to discriminate between hemifusion and full fusion, a single vesicle-vesicle content mixing assay was developed using an indicator for content- and lipid-mixing,

simultaneously [46, 47]. This assay allowed to distinguish between a variety of different fusion events including hemifusion, fast content mixing and delayed content mixing.

Vesicle-planar bilayer assays

Although single vesicle-vesicle assays allow to investigate lipid and content mixing simultaneously to obtain information about fusion intermediates, they suffer from the fact that the target-membrane is highly curved and not flat as observed in synaptic boutons. To consider this aspect but to maintain the possibility of single vesicle fusion detection, assays utilizing a supported planar target membrane to which freely diffusing vesicles are added were developed by several different groups [48–50].

Bowen et al. published an assay using calcein filled mobile vesicles containing dye-labeled synaptobrevin, which were added to a planar glass supported membrane containing syntaxin (Fig. 1.4) [48]. By simultaneously monitoring the content and membrane fluorescence, they were able to discriminate between docking and fusion of the vesicles with the planar membrane. However, SNARE fusion was rare, e.g.

independent of SNAP 25, and apparently triggered by laser light. Wanget al., who used a similar assay, mainly detected vesicle bursting rather than controlled fusion with the target-membrane [51]. A similar approach was established by Fix et al. also relying on glass supported target-membranes (Fig. 1.4) [49]. In contrast to the assay published by Bowen, they used the fluorescently labeled lipids NBD-PE and Rh-PE, which resemble a FRET pair also initially used in the first ensemble vesicle-vesicle assays (see Chapter 1.3.1). They could monitor docking, undocking as well as fusion.

Diffusion of the fluorophores in the planar membrane indicated lipid mixing.

Besides the rather inconsistent finding of these assays, most of the studies showed SNAP 25 independent fusion, indicating that fusion is most probably not triggered by SNAREs. A possible explanation for this might be the limited mobility of the proteins in the planar membrane due to the direct condensation of small liposomes on the glass surface [39].

To overcome the rather poor mobilities in the glass supported membranes, planar membranes were prepared on polyethylene glycol (PEG) brushes on glass, often referred to as polymer-cushions, to decouple the membrane from the support [52, 53]. In one of the approaches, small vesicles with reconstituted t-SNAREs were directly condensed on the PEG coated glass surface to obtain a supported bilayer [52]. In the second approach, a lipid monolayer was first formed on the PEG-support by a Langmuir-Blodgett transfer technique to which small t-SNARE containing vesicles were added.

Figure 1.4. Single-vesicle bilayer fusion assay. (A) Fusion assay developed by Fixet al. [49]. using lipid labeled vesicles, which fuse with an unlabeled lipid bilayer formed on a glass surface. (B) Fusion assay published by Bowen et al. [48] using content labeled vesicles, which fuse with an unlabeled lipid bilayer. In both assays, fusion was monitored by time dependent fluorescence microscopy. Lipid mixing was indicated by a sudden increase in fluorescence intensity at the bilayer surface subsequently followed by a decay as the molecules diffuse away. Reconstituted proteins are synaptobrevin 2 (blue), syntaxin 1A (red) and SNAP 25 (green).

The vesicles fused with the monolayer, resulting in an entire lipid bilayer [53]. Both assays showed SNAP 25 dependent fusion, indicating that fusion in these system is driven by SNAREs. A combination of a lipid and a content mixing indicator was also recently used in these systems to discriminate not only between docking and fusion but also between hemifusion and full fusion [54].

Inspired by the various fusion assays based on entirely supported planar target-membranes, Höfer and Schwenen et al. recently developed planar pore-spanning membranes (PSMs) as a suitable system to investigate SNARE-mediated membrane fusion [55, 56]. PSMs are composed of a lipid bilayer patch which is spanned over a functionalized porous microsieve structure harboring cylindrical holes resulting in supported membranes on the pore rim and freestanding membranes spanning the pores (Fig. 1.5). The areas between the pores, called pore rims were functionalized with a hydrophilic self-assembled monolayer (SAM) to decouple the membrane from the support. The PSMs contained the t-SNARE ∆N-complex (see Chapter 3.1.4) and were fluorescently labeled with OregonGreen-DPPE (OG). Large unilamellar vesicles reconstituted with the v-SNARE synaptobrevin 2 and fluorescently labeled with TexasRed-DPPE (TR) were added to the PSMs and docking as well as fusion was monitored with an upright confocal laser scanning microscope. For the experiments,

OG and TR were excited with a single laser line (488 nm) and separately detected.

This allowed to analyze docking and undocking of vesicles, as well as lipid mixing due to FRET between the donor OG and the acceptor TR on supported and freestanding membranes.

Figure 1.5. Single-vesicle PSM fusion assay. Fusion of TR labeled vesicles containing the v-SNARE synaptobrevin 2 with an OG labeled PSM containing the t-SNARE ∆N-complex is monitored by CLSM. Lipid mixing was observable by a FRET between the donor OG and the acceptor TR.