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  • Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system.

Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system.

Nature protocols (2012-12-12)
Minjoung Kyoung, Yunxiang Zhang, Jiajie Diao, Steven Chu, Axel T Brunger
ABSTRACT

This protocol describes a single vesicle-vesicle microscopy system to study Ca(2+)-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and synaptosomal-associated protein 25 (SNAP-25), and they are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at a zero-Ca(2+) concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca(2+) injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-100-millisecond time scale. Other factors such as complexin can be easily added. Our system is unique in that it monitors both content and lipid mixing and starts from a metastable state of interacting vesicle pairs before Ca(2+) injection.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
(3-Aminopropyl)triethoxysilane, 99%
Avanti
18:1 PS (DOPS), Avanti Research - A Croda Brand