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

Nature Protocols volume 8, pages 1–16 (2013)Cite this article

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Abstract

This protocol describes a single vesicle-vesicle microscopy system to study Ca2+-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-Ca2+ 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 Ca2+ 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 Ca2+ injection.

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Figure 1: Schematic of the single vesicle-vesicle microscopy system.
Figure 2: Schematic diagram of the objective-based TIR setup.
Figure 3: Sample chamber and outlet ports.
Figure 4: Representative time traces of content- and lipid-mixing events for a fluorescent spot that corresponds to a single interacting vesicle-vesicle pair.
Figure 5: Testing nonspecific binding to the surface.
Figure 6
Figure 7: Display of an example of a fluorescence intensity time trace.
Figure 8: Display of time points of content- and lipid-mixing events.
Figure 9: Bead mapping.
Figure 10: Examples of content- and lipid-mixing histograms (the fraction of vesicles that show a jump in fluorescence intensity ('occurrence') in a particular time bin) vs. time.

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References

  1. Katz, B. & Miledi, R. Spontaneous and evoked activity of motor nerve endings in calcium Ringer. J. Physiol. 203, 689–706 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sudhof, T.C. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547 (2004).

    Article  PubMed  Google Scholar 

  3. Bean, A.J., Zhang, X. & Hokfelt, T. Peptide secretion: what do we know? FASEB J. 8, 630–638 (1994).

    Article  CAS  PubMed  Google Scholar 

  4. Voets, T., Neher, E. & Moser, T. Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices. Neuron 23, 607–615 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Lindau, M. & Gomperts, B.D. Techniques and concepts in exocytosis: focus on mast cells. Biochim. Biophys. Acta. 1071, 429–471 (1991).

    Article  CAS  PubMed  Google Scholar 

  6. Sollner, T. et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324 (1993).

    Article  CAS  PubMed  Google Scholar 

  7. Sutton, R.B., Fasshauer, D., Jahn, R. & Brunger, A.T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347–353 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Jackson, M.B. & Chapman, E.R. Fusion pores and fusion machines in Ca2+-triggered exocytosis. Ann. Rev. Biophys. Biomol. Struct. 35, 135–160 (2006).

    Article  CAS  Google Scholar 

  9. Tang, J. et al. A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  11. Ma, C., Li, W., Xu, Y. & Rizo, J. Munc13 mediates the transition from the closed syntaxin-Munc18 complex to the SNARE complex. Nat. Struct. Mol. Biol. 18, 542–549 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Jun, Y. & Wickner, W. Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing. Proc. Natl. Acad. Sci. USA 104, 13010–13015 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chan, Y.H.M., van Lengerich, B. & Boxer, S.G. Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides. Proc. Natl. Acad. Sci. USA 106, 979–984 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Diao, J. et al. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. eLife 1, e00109 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Cypionka, A. et al. Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS. Proc. Natl. Acad. Sci. USA 106, 18575–18580 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lee, H.K. et al. Dynamic Ca2+-dependent stimulation of vesicle fusion by membrane-anchored synaptotagmin 1. Science 328, 760–763 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Karatekin, E. & Rothman, J.E. Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells. Nat. Protoc. 7, 903–920 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  26. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Schneggenburger, R. & Neher, E. Intracellular calcium dependence of transmitter release rates at a fast central synapse. Nature 406, 889–893 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Sun, J. et al. A dual-Ca2+-sensor model for neurotransmitter release in a central synapse. Nature 450, 676–U674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Vennekate, W. et al. Cis- and trans-membrane interactions of synaptotagmin-1. Proc. Natl. Acad. Sci. USA 109, 11037–11042 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gao, Y. et al. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science (2012).

  31. Weninger, K., Bowen, M.E., Chu, S. & Brunger, A.T. Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations. Proc. Natl. Acad. Sci. USA 100, 14800–14805 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ghosh, S.K. et al. Measuring Ca2+-induced structural changes in lipid monolayers: implications for synaptic vesicle exocytosis. Biophys. J. 102, 1394–1402 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Heidelberger, R., Heinemann, C., Neher, E. & Matthews, G. Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature 371, 513–515 (1994).

    Article  CAS  PubMed  Google Scholar 

  34. Wang, Z., Liu, H., Gu, Y. & Chapman, E.R. Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion. J. Cell. Biol. 195, 1159–1170 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hernandez, J.M. et al. Membrane fusion intermediates via directional and full assembly of the SNARE complex. Science 336, 1581–1584 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. van den Bogaart, G. et al. Membrane protein sequestering by ionic protein-lipid interactions. Nature 479, 552–555 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhang, Y., Sivasankar, S., Nelson, W.J. & Chu, S. Resolving cadherin interactions and binding cooperativity at the single-molecule level. Proc. Natl. Acad. Sci. USA 106, 109–114 (2009).

    Article  CAS  PubMed  Google Scholar 

  40. Pertsinidis, A., Zhang, Y. & Chu, S. Subnanometre single-molecule localization, registration and distance measurements. Nature 466, 647–651 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Bates, M., Dempsey, G.T., Chen, K.H. & Zhuang, X. Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection. Chemphyschem. 13, 99–107 (2012).

    Article  CAS  PubMed  Google Scholar 

  42. Kiessling, V., Domanska, M.K. & Tamm, L.K. Single SNARE-mediated vesicle fusion observed in vitro by polarized TIRFM. Biophys. J. 99, 4047–4055 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health grant no. R37-MH63105 to A.T.B. We thank M. Padolina for making the video of the acceptor and donor vesicle preparation; and D. Cipriano, R. Garland and M. Padolina for critical reading of the manuscript.

Author information

Author notes

  1. Minjoung Kyoung

    Present address: Present address: Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland, USA.,

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA

    Minjoung Kyoung, Yunxiang Zhang, Jiajie Diao & Axel T Brunger

  2. Department of Neurology and Neurological Science, Stanford University, Stanford, California, USA

    Minjoung Kyoung, Yunxiang Zhang, Jiajie Diao & Axel T Brunger

  3. Department of Structural Biology, Stanford University, Stanford, California, USA

    Minjoung Kyoung, Yunxiang Zhang, Jiajie Diao & Axel T Brunger

  4. Department of Photon Science, Stanford University, Stanford, California, USA

    Minjoung Kyoung, Yunxiang Zhang, Jiajie Diao & Axel T Brunger

  5. Howard Hughes Medical Institute, Stanford University, Stanford, California, USA

    Minjoung Kyoung, Jiajie Diao & Axel T Brunger

  6. Formerly of Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Yunxiang Zhang & Steven Chu

  7. Formerly of Department of Physics, University of California at Berkeley, Berkeley, California, USA

    Yunxiang Zhang & Steven Chu

  8. Formerly of Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA

    Yunxiang Zhang & Steven Chu

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  1. Minjoung Kyoung
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Contributions

M.K. designed the single-vesicle content and lipid-mixing system described in this protocol with input from S.C. and A.T.B. Y.Z. developed the injection system and programs for data analysis. J.D. made recent improvements to the vesicle content and lipid-mixing system. M.K. and A.T.B. wrote the paper.

Corresponding author

Correspondence to Axel T Brunger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Donor and acceptor vesicle preparation (MP4 16487 kb)

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Kyoung, M., Zhang, Y., Diao, J. et al. Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system. Nat Protoc 8, 1–16 (2013). https://doi.org/10.1038/nprot.2012.134

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