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Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells

Nature Protocols volume 7, pages 903–920 (2012)Cite this article

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Abstract

Many biological processes rely on membrane fusion, and therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, and even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) liposomes and target-membrane-associated t-SNARE–reconstituted planar, supported bilayers (t-SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorescence microscopy. In this assay, fusion is dependent on SNAP-25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of: (i) bilayers covered with a thin layer of poly(ethylene glycol) (PEG) to control bilayer-bilayer and bilayer-substrate interactions, and (ii) microfluidic flow channels that present many advantages, such as the removal of nonspecifically bound liposomes by flow. The protocol takes 6–8 d to complete. Analysis can take up to 2 weeks.

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Figure 1: The experimental setup.
Figure 2: Verifying the fluidity of an SBL.
Figure 3: SUV-SBL fusion.
Figure 4: Microfabrication of the SU-8 template for the PDMS flow cell.
Figure 5: Making of the PDMS block.
Figure 6: Coverslip cleaning.
Figure 7: Analysis of data using PointPicker and MATLAB.
Figure 8: Analysis of TIRFM data.

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Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique (CNRS) and by a US National Institutes of Health grant to J.E.R. E.K. is indebted to M. Seagar and members of his laboratory (Institut National de la Santé et de la Recherche Médicale (INSERM) UMR641) for introducing him to proteoliposomes, and J. Coleman (Yale University) for teaching him how to express and purify SNARE proteins. We thank J.-P. Henry, F. Darchen and B. Gasnier (Laboratory of Membrane Dynamics and Neurological Diseases, CNRS/Université Paris Descartes UMR 8192, formerly CNRS UPR 1929); T. Melia and A. Gohlke (Department of Cell Biology, Yale University); M. Power (School of Engineering and Applied Science clean room, Yale University); B. O'Shaughnessy and J. Warner at Columbia University for many useful discussions and suggestions; the CNRS for granting a leave of absence to E.K.; and A. Gohlke for help with some of the photos. We thank A. Gohlke, W. Xu, B. Antonny, G. Melikyan, B. O'Shaughnessy, J. Warner and J. Diao for carefully reading and commenting on the manuscript.

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  1. Erdem Karatekin

    Present address: Present address: Department of Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, Connecticut, USA.,

Authors and Affiliations

  1. Department of Cell Biology, School of Medicine, Yale University, New Haven, Connecticut, USA

    Erdem Karatekin & James E Rothman

  2. Centre National de la Recherche Scientifique and Université Paris Descartes, UMR 8192, Centre Universitaire des Saints-Pères, Paris, France

    Erdem Karatekin

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Contributions

E.K. developed the assay first in the Laboratory of Membrane Dynamics and Neurological Diseases, CNRS/Université Paris Descartes UMR 8192 (formerly CNRS UPR 1929), then in the laboratory of J.E.R. at Yale University. J.E.R. oversaw the project and provided all the material support required since E.K. joined his lab. E.K. wrote the MATLAB analysis programs that are supplied. E.K. wrote the manuscript, which was read and approved by J.E.R.

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Correspondence to Erdem Karatekin.

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Supplementary information

Supplementary Fig. 1

Snapshots from a typical movie acquired using TIRFM (256 pixels by 402 pixels, 57 frames/s). (PDF 88 kb)

Supplementary Methods

The supplementary files below are packaged into a single zip file. The files are separated into three folders: (ZIP 67046 kb)

\FRAP\ Three sample FRAP recordings (in Nikon .nd2 file format), Matlab programs, instructions, and auxiliary files.

\Fusion\Epi\ Three sample fusion events cropped from larger movies (10 Hz acquisition rate, far-field epifluorescence mode), sample PointPicker files for analysis of docking and fusion events, Matlab programs, instructions, and auxiliary files. The movies are 16 bit tif stacks that can be opened using e.g. ImageJ.

\Fusion\TIRFM\ Three sample fusion events cropped from larger movies (57 Hz acquisition rate, TIRFM mode), sample SpeckleTrackerJ track files for analysis of docking and fusion events, Matlab programs, instructions, and auxiliary files. The movies are 16 bit tif stacks that can be opened using e.g. ImageJ.

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Karatekin, E., Rothman, J. Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells. Nat Protoc 7, 903–920 (2012). https://doi.org/10.1038/nprot.2012.019

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