A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

Abstract

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.

Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction

“…Nor do the present data formally prove the existence of a radial assembly of SNARE complexes only, as other proteins could be implicated in the interaction with R206 and D253 within the NMJ. However, the present data are strictly pertinent to the studies using cell-free membrane fusion model systems (Ellena et al, 2009;Gao et al, 2012;Hua and Scheller, 2001;Shi et al, 2012;Pobbati et al, 2006;Lu et al, 2008;Domanska et al, 2009;Giraudo et al, 2009;Karatekin et al, 2010;van den Bogaart et al, 2011;Diao et al, 2012).…”

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

“…4 B and C, unilamellar liposomes with the specified diameter were tethered on a surface through biotin-streptavidin coupling (65). For these experiments, Cpx was labeled at the eighth amino acid residue with Alexa 647 dye.…”

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

“…43). The details of TIRF microscope imaging and the data analysis of the single vesicle content mixing assay were described in detail elsewhere (46).…”