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PMID: 23222454 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

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

Nature protocols ·Vol. 8 ·No. 1 ·2013-01-00 ·Pages 1-16

Kyoung M, Zhang Y, Diao J, Chu S, Brunger AT

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.

MeSH Terms
Calcium/metabolism Image Processing, Computer-Assisted Lipid Metabolism Membrane Fusion Microscopy, Fluorescence/methods R-SNARE Proteins/metabolism Synaptic Vesicles/metabolism Synaptosomal-Associated Protein 25/metabolism Synaptotagmin I/metabolism Synaptotagmins/metabolism
Chemicals
R-SNARE Proteins Synaptosomal-Associated Protein 25 Synaptotagmin I Synaptotagmins Calcium
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Kyoung Minjoung
Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.
Zhang Yunxiang
Diao Jiajie
Chu Steven
Brunger Axel T
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Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2012.134
Published
2013-01-00
Epub
2012-00-06
Pages
1-16
Language
English
Region
England
NLM ID
101284307
PMCID
PMC3566647
Subset
IM
Grants
Howard Hughes Medical Institute · United States
NIMH NIH HHS · R37 MH063105 · United States
NIMH NIH HHS · R37-MH63105 · United States
External Links
PubMed source DOI: 10.1038/nprot.2012.134 PMC Full Text View journal details
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