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A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

Abstract

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.

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Figure 1: Experimental scheme.
Figure 2: The schematic illustration of a prism-type TIR fluorescence (TIRF) microscopy with both excitation and emission pathways as well as the TIR occurring at the interface between a quartz slide and water.
Figure 3: Standard versus direct methods of preparing SNARE-reconstituted vesicles preparation.
Figure 4: Sample chambers are made by putting a quartz slide (1) and a glass coverslip (3) together with pieces of double-sided tape (2) and sealing with epoxy.
Figure 5: Experimental results.
Figure 6: Real-time lipid mixing for lipophilic dyes.
Figure 7: Real-time content mixing for content labeling only.

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Acknowledgements

This work was supported by the US National Institutes of Health Grants (R21 GM074526 to T.H. and R01 GM51290 to Y.-K.S.) and by the National Research Foundation of Korea grants funded by the Korean government (2009-0069857 and 2009-0090781 to T.-Y.Y.). T.H. is an investigator of the Howard Hughes Medical Institute.

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Authors

Contributions

J.D. and Y.I. performed experiments. J.D., Y.I. and C.J. drew figures. H.L. and T.-Y.Y. contributed the neuronal SNARE vesicle preparation protocols based on the standard method. Z.S. and Y.-K.S. developed the yeast SNARE reconstitution protocol based on the direct method. S.S. wrote the PEG slide preparation part. J.D., Y.I. and T.H. wrote the paper.

Corresponding author

Correspondence to Taekjip Ha.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Photo of our homemade TIR fluorescence microscope setup (DOC 361 kb)

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Diao, J., Ishitsuka, Y., Lee, H. et al. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins. Nat Protoc 7, 921–934 (2012). https://doi.org/10.1038/nprot.2012.020

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