In vitro single vesicle fusion assays based on pore-spanning membranes: merits and drawbacks

Abstract

Neuronal fusion mediated by soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) is a fundamental cellular process by which two initially distinct membranes merge resulting in one interconnected structure to release neurotransmitters into the presynaptic cleft. To get access to the different stages of the fusion process, several in vitro assays have been developed. In this review, we provide a short overview of the current in vitro single vesicle fusion assays. Among those assays, we developed a single vesicle assay based on pore-spanning membranes (PSMs) on micrometre-sized pores in silicon, which might overcome some of the drawbacks associated with the other membrane architectures used for investigating fusion processes. Prepared by spreading of giant unilamellar vesicles with reconstituted t-SNAREs, PSMs provide an alternative tool to supported lipid bilayers to measure single vesicle fusion events by means of fluorescence microscopy. Here, we discuss the diffusive behaviour of the reconstituted membrane components as well as that of the fusing synthetic vesicles with reconstituted synaptobrevin 2 (v-SNARE). We compare our results with those obtained if the synthetic vesicles are replaced by natural chromaffin granules under otherwise identical conditions. The fusion efficiency as well as the different fusion states observable in this assay by means of both lipid mixing and content release are illuminated.

Forces, Kinetics, and Fusion Efficiency Altered by the Full-Length Synaptotagmin-1 -PI(4,5)P2 Interaction in Constrained Geometries

“…To analyze how full-length syt-1, PI­(4,5)­P 2 , and Ca 2+ influence the fusion process, a single vesicle fusion assay based on pore-spanning membranes (PSMs) was employed (Figure A). − PSMs have the advantage to unequivocally discern different states of fusion such as stalled docking, intermediate fusion, and full fusion unlike bulk fusion assays.…”

The role of PI(4,5)P2 and synaptotagmin in membrane fusion - an in vitro study

“…In order to facilitate uptake of additional lipid material during full fusion, we employed single vesicle experiments on pore-spanning membranes (PSMs) that have been shown previously to be well-suited to measure fusion events with high time resolution by means of fluorescence microscopy. [24][25][26][27]40 We first investigated the fusion efficiency of the system in the absence of syt-1 as a function of different PI(4,5)P 2 concentrations. PSMs composed of DOPC/POPE/POPS/Chol/PI(4,5)P 2 were prepared on porous silicon substrates with pore diameters of 1.2 µm.…”

Special issue: Multicomponent lipid membranes—how molecular organisation leads to function

“…A large part of the CRC 803 was dedicated to elucidate the process of membrane fusion. Here, five articles contribute to our understanding of how the process of neuronal fusion takes place, identifying prominent intermediate states of fusion and quantifying the energetics of fusion (Witt et al 2021;Mühlenbrock et al 2020;Smirnova et al 2021;Scheu et al2021;Risselada et al 2020). In the last article of this special issue, protein-dependent membrane remodelling is discussed (Tarasenko and Meinecke 2021).…”