SNAREs pp 263-275 | Cite as

A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity and Sub-millisecond Time Resolution

  • Natasha R. Dudzinski
  • Zhenyong WuEmail author
  • Erdem KaratekinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)


During exocytosis, vesicles fuse with the plasma membrane and release their contents. The fusion pore is the initial, nanometer-sized connection between the plasma membrane and the cargo-laden vesicle. A growing body of evidence points toward the fusion pore being a regulator of exocytosis, but the shortcomings of current experimental techniques to investigate single-fusion pores make it difficult to study factors governing pore behavior. Here we describe an assay that fuses v-SNARE-reconstituted nanodiscs with cells ectopically expressing “flipped” t-SNAREs to monitor dynamics of single fusion pores in a biochemically defined system using electrical recordings. We also describe a fluorescence microscopy-based approach to monitor nanodisc-cell fusion that is much simpler to employ, but cannot resolve single pores.

Key words

Membrane fusion Exocytosis Nanodisc SNAREs Fusion pore Electrophysiology 



We thank all members of the Karatekin laboratory for stimulating discussions, D. Zenisek and F. Sigworth (Cellular and Molecular Physiology, Yale University) for expert advice and discussions, and James E. Rothman, Oscar Bello, Shyam Krishnakumar, and other members of the Rothman laboratory (Cell Biology, Yale University) for critical advice and introducing us to the use of nanodiscs. This work was supported by the National Institute of General Medical Sciences (grant R01GM108954), and a Kavli Foundation Neuroscience Scholar Award (to EK). NRD was supported by NIH Training Grant T32 NS41228 funded by the Jointly Sponsored NIH Predoctoral Training Program in the Neurosciences.


  1. 1.
    Chernomordik LV, Kozlov MM (2008) Mechanics of membrane fusion. Nat Struct Mol Biol 15:675–683CrossRefGoogle Scholar
  2. 2.
    Jahn R, Fasshauer D (2012) Molecular machines governing exocytosis of synaptic vesicles. Nature 490:201–207. Scholar
  3. 3.
    Sudhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477. Scholar
  4. 4.
    Lindau M, de Toledo GA (2003) The fusion pore. BBA-Mol Cell Res 1641:167–173Google Scholar
  5. 5.
    Jackson MB, Chapman ER (2008) The fusion pores of Ca2+ −triggered exocytosis. Nat Struct Mol Biol 15:684–689. Scholar
  6. 6.
    Weber T et al (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92:759–772CrossRefGoogle Scholar
  7. 7.
    Gao Y et al (2012) Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337:1340–1343. Scholar
  8. 8.
    Lindau M (2012) High resolution electrophysiological techniques for the study of calcium-activated exocytosis. BBA-Gen Subjects 1820:1234–1242CrossRefGoogle Scholar
  9. 9.
    Fulop T, Radabaugh S, Smith C (2005) Activity-dependent differential transmitter release in mouse adrenal chromaffin cells. J Neurosci 25:7324–7332CrossRefGoogle Scholar
  10. 10.
    Hastoy B, Clark A, Rorsman P, Lang J (2017) Fusion pore in exocytosis: more than an exit gate? A beta-cell perspective. Cell Calcium 68:45–61. Scholar
  11. 11.
    Collins SC et al (2016) Increased expression of the diabetes gene SOX4 reduces insulin secretion by impaired fusion pore expansion. Diabetes 65:1952–1961. Scholar
  12. 12.
    Staal RGW, Mosharov EV, Sulzer D (2004) Dopamine neurons release transmitter via a flickering fusion pore. Nat Neurosci 7:341–346CrossRefGoogle Scholar
  13. 13.
    Pawlu C, DiAntonio A, Heckmann M (2004) Postfusional control of quantal current shape. Neuron 42:607–618CrossRefGoogle Scholar
  14. 14.
    Chapochnikov NM et al (2014) Uniquantal release through a dynamic fusion pore is a candidate mechanism of hair cell exocytosis. Neuron 83:1389–1403. Scholar
  15. 15.
    He LM, Wu XS, Mohan R, Wu LG (2006) Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature 444:102–105CrossRefGoogle Scholar
  16. 16.
    Alabi AA, Tsien RW (2013) Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu Rev Physiol 75:393–422. Scholar
  17. 17.
    Travis ER, Wightman RM (1998) Spatio-temporal resolution of exocytosis from individual cells. Annu Rev Biophys Biomol Struct 27:77–103. Scholar
  18. 18.
    Kyoung M, Zhang Y, Diao J, Chu S, Brunger AT (2013) Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system. Nat Protoc 8:1–16. Scholar
  19. 19.
    Yoon TY, Okumus B, Zhang F, Shin YK, Ha T (2006) Multiple intermediates in SNARE-induced membrane fusion. Proc Natl Acad Sci U S A 103:19731–19736CrossRefGoogle Scholar
  20. 20.
    Lai Y et al (2013) Fusion pore formation and expansion induced by Ca2+ and synaptotagmin 1. Proc Natl Acad Sci U S A 110:1333–1338. Scholar
  21. 21.
    Kiessling V, Liang B, Kreutzberger AJ, Tamm LK (2017) Planar supported membranes with mobile SNARE proteins and quantitative fluorescence microscopy assays to study synaptic vesicle fusion. Front Mol Neurosci 10:72. Scholar
  22. 22.
    Karatekin E et al (2010) A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proc Natl Acad Sci U S A 107:3517–3521. Scholar
  23. 23.
    Karatekin E, Rothman JE (2012) Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells. Nat Protoc 7:903–920. Scholar
  24. 24.
    Smith MB et al (2011) Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion. Biophys J 101:1794–1804. Scholar
  25. 25.
    Stratton BS et al (2016) Cholesterol increases the openness of SNARE-mediated flickering fusion pores. Biophys J 110:1538–1550. Scholar
  26. 26.
    Wu Z et al (2016) Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains. Sci Rep 6:27287. Scholar
  27. 27.
    Wu Z et al (2017) Dilation of fusion pores by crowding of SNARE proteins. elife 6:e22964. Scholar
  28. 28.
    Shi L et al (2012) SNARE proteins: one to fuse and three to keep the nascent fusion pore open. Science 335:1355–1359CrossRefGoogle Scholar
  29. 29.
    Hu C et al (2003) Fusion of cells by flipped SNAREs. Science 300:1745–1749CrossRefGoogle Scholar
  30. 30.
    Sakmann B, Neher E (2009) Single-channel recording, 2nd edn. Springer, New YorkGoogle Scholar
  31. 31.
    Bello OD, Auclair SM, Rothman JE, Krishnakumar SS (2016) Using ApoE Nanolipoprotein particles to analyze SNARE-induced fusion pores. Langmuir 32:3015–3023. Scholar
  32. 32.
    Stroeva E, Krishnakumar SS (2018) Using nanodiscs to probe Ca2+-dependent membrane interaction of Synaptotagmin-1. In: Fratti R (ed) SNAREs, Methods and protocols. Springer, New YorkGoogle Scholar
  33. 33.
    Breckenridge LJ, Almers W (1987) Currents through the fusion pore that forms during exocytosis of a secretory vesicle. Nature 328:814–817CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Interdepartmental Neuroscience ProgramYale UniversityNew HavenUSA
  2. 2.Nanobiology InstituteYale UniversityWest HavenUSA
  3. 3.Department of Cellular and Molecular PhysiologyYale University School of MedicineNew HavenUSA
  4. 4.Department of Molecular Biophysics and BiochemistryYale UniversityNew HavenUSA
  5. 5.Centre National de la Recherche Scientifique (CNRS)ParisFrance

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