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

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.

References

  1. Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

    CAS  PubMed  Article  Google Scholar 

  2. Sudhof, T.C. & Rothman, J.E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  3. Yoon, T.Y., Okumus, B., Zhang, F., Shin, Y.K. & Ha, T. Multiple intermediates in SNARE-induced membrane fusion. Proc. Natl. Acad. Sci. USA 103, 19731–19736 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Kyoung, M. et al. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc. Natl. Acad. Sci. USA 108, E304–E313 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Diao, J. et al. A single-vesicle content mixing assay for SNARE-mediated membrane fusion. Nat. Commun. 1, 54 (2010).

    PubMed  Article  CAS  Google Scholar 

  6. Smith, E.A. & Weisshaar, J.C. Docking, not fusion, as the rate-limiting step in a SNARE-driven vesicle fusion assay. Biophys. J. 100, 2141–2150 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Diao, J. et al. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins. Nat. Protoc. 7, 921–934 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Karatekin, E. et al. A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proc. Natl. Acad. Sci. USA 107, 3517–3521 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Smith, M.B. et al. Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion. Biophys. J. 101, 1794–1804 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Brian, A.A. & McConnell, H.M. Allogeneic stimulation of cytotoxic T cells by supported planar membranes. Proc. Natl. Acad. Sci. USA 81, 6159–6163 (1984).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Tamm, L.K. & McConnell, H.M. Supported phospholipid bilayers. Biophys. J. 47, 105–113 (1985).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Fix, M. et al. Imaging single membrane fusion events mediated by SNARE proteins. Proc. Natl. Acad. Sci. USA 101, 7311–7316 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Bowen, M.E., Weninger, K., Brunger, A.T. & Chu, S. Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs). Biophys. J. 87, 3569–3584 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Liu, T., Tucker, W.C., Bhalla, A., Chapman, E.R. & Weisshaar, J.C. SNARE-driven, 25-millisecond vesicle fusion in vitro. Biophys. J. 89, 2458–2472 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. de Gennes, P.G. Polymers at an interface; a simplified view. Adv. Colloid Interface Sci. 27, 189–209 (1987).

    CAS  Article  Google Scholar 

  16. Kenworthy, A.K., Hristova, K., Needham, D. & McIntosh, T.J. Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1921–1936 (1995).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Milner, S.T. Polymer brushes. Science 251, 905–914 (1991).

    CAS  PubMed  Article  Google Scholar 

  18. Israelachvili, J. The different faces of poly(ethylene glycol). Proc. Natl. Acad. Sci. USA 94, 8378–8379 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Oesterhelt, F., Rief, M. & Gaub, H.E. Single molecule force spectroscopy by AFM indicates helical structure of poly(ethylene-glycol) in water. N. J. Phys. 1, 6.1–6.11 (1999).

    Article  Google Scholar 

  20. Perrret, E., Leung, A., Morel, A., Feracci, H. & Nassoy, P. Versatile decoration of glass surfaces to probe individual protein-protein interactions and cellular adhesion. Langmuir 18, 846–854 (2002).

    Article  CAS  Google Scholar 

  21. Knoll, W. et al. Solid supported lipid membranes: new concepts for the biomimetic functionalization of solid surfaces. Biointerphases 3, Fa125–Fa135 (2008).

    CAS  PubMed  Article  Google Scholar 

  22. Hiergeist, C. & Lipowsky, R. Elastic properties of polymer-decorated membranes. J. De Physique II 6, 1465–1481 (1996).

    CAS  Article  Google Scholar 

  23. Kenworthy, A.K., Simon, S.A. & McIntosh, T.J. Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1903–1920 (1995).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Lasic, D.D. & Needham, D. The ''Stealth'' liposome: a prototypical biomaterial. Chem. Rev. 95, 2601–2628 (1995).

    CAS  Article  Google Scholar 

  25. Albertorio, F. et al. Fluid and air-stable lipopolymer membranes for biosensor applications. Langmuir 21, 7476–7482 (2005).

    CAS  PubMed  Article  Google Scholar 

  26. Tanaka, M. & Sackmann, E. Polymer-supported membranes as models of the cell surface. Nature 437, 656–663 (2005).

    CAS  PubMed  Article  Google Scholar 

  27. Deng, Y. et al. Fluidic and air-stable supported lipid bilayer and cell-mimicking microarrays. J. Am. Chem. Soc. 130, 6267–6271 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Lin, J., Szymanski, J., Searson, P.C. & Hristova, K. Effect of a polymer cushion on the electrical properties and stability of surface-supported lipid bilayers. Langmuir 26, 3544–3548 (2010).

    CAS  PubMed  Article  Google Scholar 

  29. Wong, J.Y. et al. Polymer-cushioned bilayers. I. A structural study of various preparation methods using neutron reflectometry. Biophys. J. 77, 1445–1457 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Cornell, B.A. et al. A biosensor that uses ion-channel switches. Nature 387, 580–583 (1997).

    CAS  PubMed  Article  Google Scholar 

  31. Deverall, M.A. et al. Transbilayer coupling of obstructed lipid diffusion in polymer-tethered phospholipid bilayers. Soft Matter 4, 1899–1908 (2008).

    CAS  Article  Google Scholar 

  32. Floyd, D.L., Ragains, J.R., Skehel, J.J., Harrison, S.C. & van Oijen, A.M. Single-particle kinetics of influenza virus membrane fusion. Proc. Natl. Acad. Sci. USA 105, 15382–15387 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Kataoka-Hamai, C., Higuchi, M., Iwai, H. & Miyahara, Y. Detergent-mediated formation of polymer-supported phospholipid bilayers. Langmuir 26, 14600–14605 (2010).

    CAS  PubMed  Article  Google Scholar 

  34. Daniel, S., Albertorio, F. & Cremer, P.S. Making lipid membranes rough, tough, and ready to hit the road. MRS Bull. 31, 536–540 (2006).

    CAS  Article  Google Scholar 

  35. Diaz, A.J., Albertorio, F., Daniel, S. & Cremer, P.S. Double cushions preserve transmembrane protein mobility in supported bilayer systems. Langmuir 24, 6820–6826 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Fasshauer, D., Otto, H., Eliason, W.K., Jahn, R. & Brunger, A.T. Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation. J. Biol. Chem. 272, 28036–28041 (1997).

    CAS  PubMed  Article  Google Scholar 

  37. Hazzard, J., Sudhof, T.C. & Rizo, J. NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin. J. Biomol. NMR 14, 203–207 (1999).

    CAS  PubMed  Article  Google Scholar 

  38. Bright, J.N., Woolf, T.B. & Hoh, J.H. Predicting properties of intrinsically unstructured proteins. Prog. Biophys. Mol. Biol. 76, 131–173 (2001).

    CAS  PubMed  Article  Google Scholar 

  39. Quinn, P., Griffiths, G. & Warren, G. Den sity of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies. J. Cell Biol. 98, 2142–2147 (1984).

    CAS  PubMed  Article  Google Scholar 

  40. Wessels, L., Elting, M.W., Scimeca, D. & Weninger, K. Rapid membrane fusion of individual virus particles with supported lipid bilayers. Biophys. J. 93, 526–538 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Brunger, A.T., Weninger, K., Bowen, M. & Chu, S. Single-molecule studies of the neuronal SNARE fusion machinery. Annu. Rev. Biochem. 78, 903–928 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Domanska, M.K., Kiessling, V., Stein, A., Fasshauer, D. & Tamm, L.K. Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion. J. Biol. Chem. 284, 32158–32166 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Pobbati, A.V., Stein, A. & Fasshauer, D. N- to C-terminal SNARE complex assembly promotes rapid membrane fusion. Science 313, 673–676 (2006).

    CAS  PubMed  Article  Google Scholar 

  44. Needham, D. & Nunn, R.S. Elastic deformation and failure of lipid bilayer membranes containing cholesterol. Biophys. J. 58, 997–1009 (1990).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Rawicz, W., Smith, B.A., McIntosh, T.J., Simon, S.A. & Evans, E. Elasticity, strength, and water permeability of bilayers that contain raft microdomain-forming lipids. Biophys. J. 94, 4725–4736 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Nikolaus, J., Stockl, M., Langosch, D., Volkmer, R. & Herrmann, A. Direct visualization of large and protein-free hemifusion diaphragms. Biophys. J. 98, 1192–1199 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. Ohki, S. A mechanism of divalent ion-induced phosphatidylserine membrane fusion. Biochim. Biophys. Acta 689, 1–11 (1982).

    CAS  PubMed  Article  Google Scholar 

  48. Berquand, A. et al. Two-step formation of streptavidin-supported lipid bilayers by PEG-triggered vesicle fusion. Fluorescence and atomic force microscopy characterization. Langmuir 19, 1700–1707 (2003).

    CAS  Article  Google Scholar 

  49. Israelachvili, J.N. Intermolecular and Surface Forces (Academic Press, 1991).

  50. Finkelstein, A. Bilayers: formation, measurements, and incorporation of components. Methods Enzymol. 32, 489–501 (1974).

    CAS  PubMed  Article  Google Scholar 

  51. Scott, B.L. et al. Liposome fusion assay to monitor intracellular membrane fusion machines. Methods Enzymol. 372, 274–300 (2003).

    CAS  PubMed  Article  Google Scholar 

  52. Wang, T., Smith, E.A., Chapman, E.R. & Weisshaar, J.C. Lipid mixing and content release in single-vesicle, SNARE-driven fusion assay with 1–5 ms resolution. Biophys. J. 96, 4122–4131 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Soumpasis, D.M. Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys. J. 41, 95–97 (1983).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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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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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Erdem Karatekin.

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The authors declare no competing financial interests.

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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