Single-molecule FRET study of SNARE-mediated membrane fusion

Abstract

Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.

Figure 1

Schematics of the bulk fusion assay (A) and the single-vesicle lipid mixing assay (B) for the mixture of protein free vesicles (35% PS & 65% PC, DiD or DiI labeled), C2AB (cytoplasmic domain of synaptotagmin) and 1 mM calcium. The single-vesicle FRET histogram is plotted by compiling FRET signals from over one thousand vesicles. Y-axis is normalized population, where we divided the distribution by the total number of vesicles measured and X-axis is FRET efficiency value.