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SNAREs pp 335-344 | Cite as

Single-Molecule Fluorescence Measurement of SNARE-Mediated Vesicle Fusion

  • Yachong Hu
  • Zhiqi Tian
  • Jiajie Diao
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

This chapter expounds the single vesicle-vesicle fluorescence resonance energy transfer (FRET) measurement to study the membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. The formation of a four-α-helix bundle of SNARE proteins can drive two membranes to a close proximity for fusion. Through single-molecule FRET-based microscopy, the lipid-mixing process at the single-vesicle level can be tracked in real time. This reconstitution system is applicable to study the molecular mechanism of SNAREs during different membrane fusion stages, such as docking, hemifusion, and full fusion. Four main parts are described in this chapter, including SNARE reconstitution, imaging preparation, data collection, and analysis.

Key words

FRET SNARE Vesicle Membrane fusion Lipid mixing Reconstitution 

Notes

Acknowledgments

This work was supported by the 973-program (2015CB856304) and National Institutes of Health (R35GM128837).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Cancer BiologyUniversity of Cincinnati College of MedicineCincinnatiUSA
  2. 2.School of Life Science and TechnologyXi’an Jiaotong UniversityXi’anChina

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