A single-vesicle content mixing assay for SNARE-mediated membrane fusion

Abstract

The in vitro studies of membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have primarily been performed by following the mixing of the lipids. However, the formation a of fusion pore and its expansion has been difficult to detect directly due to the leakiness of proteoliposomes, vesicle aggregation and rupture that often complicate the interpretation of ensemble fusion experiments. Fusion pore expansion is an essential step for full collapse fusion and recycling of the fusion machineries. Here, we demonstrate a method to detect the inter-vesicular mixing of large cargoes at the single molecule and vesicle level. The change in FRET signal when a DNA hairpin encapsulated in a surface-tethered vesicle encounters a complementary DNA strand from another vesicle indicates content mixing. We found that that the yeast SNARE complex alone without any accessory proteins can expand the fusion pore large enough to transmit ~ 11 kD cargoes.

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

“…Previous work probing single SUV-SUV fusion [18][19][20]28 These properties make dequenching a good tool to obtain a spike in the signal to detect fusion events, but complicate quantitative kinetic analysis of lipid release using high time resolution. In contrast, changes in intensity due to dipole reorientation and evanescent field effects as a fluorophore transfers from a SUV into the SBL are linearly related to the fraction of dye still remaining in the SUV.…”

“…As the bulk assay is easy to set up and analyze, it has been widely used to study mechanisms of SNARE-mediated fusion . Ha and colleagues used v-SUVs tethered onto a surface and monitored their fusion with free t-SUVs 18,19 . Lipid mixing was monitored using FRET between a pair of lipid-bound fluorophores embedded in the v-and t-SUVs, respectively, using total internal reflection fluorescence (TIRF) microscopy 18 .…”

In vitro system capable of differentiating fast Ca 2+ -triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release

“…2E, Left). Using a different contentmixing assay, spontaneous complete fusion activity of neuronal SNAREs alone in the absence of Ca 2þ was also found to be slow (23); in fact, endogenous fusion activity appears to be slower for neuronal SNAREs than for yeast SNAREs (54,55). Thus, one can speculate that neuronal SNAREs have evolved to generate less power than yeast SNAREs to overcome the transition barriers for complete fusion between membranes.…”

Fusion pore formation and expansion induced by Ca 2+ and synaptotagmin 1

“…To monitor fusion pore expansion, we developed a singlemolecule/vesicle content-mixing assay based on vesicle-encapsulated DNA molecules (11,12). This assay can detect expansion of the fusion pore that is large enough to pass ∼11-kDa DNA probes between two apposed proteoliposomes.…”

“…This assay can detect expansion of the fusion pore that is large enough to pass ∼11-kDa DNA probes between two apposed proteoliposomes. With this method, we showed that yeast SNAREs alone can efficiently drive expansion of the fusion pore (12). In this work, we systematically dissect lipid mixing, fusion pore opening, and fusion pore expansion steps in neuronal SNARE-dependent membrane fusion.…”