Letter | Published:

Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties

Nature volume 554, pages 260263 (08 February 2018) | Download Citation

Abstract

The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures1. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans-SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, which mimics the action of botulinum neurotoxin A, markedly reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determines fundamental properties of individual pores.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. SNAREs can promote complete fusion and hemifusion as alternative outcomes. J. Cell Biol. 170, 249–260 (2005)

  2. 2.

    , & Activity-dependent differential transmitter release in mouse adrenal chromaffin cells. J. Neurosci. 25, 7324–7332 (2005)

  3. 3.

    Vesicular release mode shapes the postsynaptic response at hippocampal synapses. J. Physiol. (Lond.) 587, 5073–5080 (2009)

  4. 4.

    , & Fusion pore modulation as a presynaptic mechanism contributing to expression of long-term potentiation. Phil. Trans. R. Soc. Lond. B 358, 695–705 (2003)

  5. 5.

    & Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu. Rev. Physiol. 75, 393–422 (2013)

  6. 6.

    , & A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation. Neuron 29, 469–484 (2001)

  7. 7.

    et al. SNARE proteins: one to fuse and three to keep the nascent fusion pore open. Science 335, 1355–1359 (2012)

  8. 8.

    , & Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices. Neuron 22, 395–409 (1999)

  9. 9.

    et al. Exocytotic fusion pores are composed of both lipids and proteins. Nat. Struct. Mol. Biol. 23, 67–73 (2016)

  10. 10.

    , , & Reconstitution of cell membrane structure in vitro and its transformation into an excitable system. Nature 194, 979–980 (1962)

  11. 11.

    et al. Nanodisc–cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains. Sci. Rep. 6, 27287 (2016)

  12. 12.

    , , & Ca2+-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nat. Struct. Mol. Biol. 13, 323–330 (2006)

  13. 13.

    et al. A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex. J. Biol. Chem. 285, 21549–21559 (2010)

  14. 14.

    & Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009)

  15. 15.

    et al. The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389, 509–512 (1997)

  16. 16.

    & Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals. Nature 418, 89–92 (2002)

  17. 17.

    , , , & Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells. EMBO J. 19, 84–93 (2000)

  18. 18.

    , & Dopamine neurons release transmitter via a flickering fusion pore. Nat. Neurosci. 7, 341–346 (2004)

  19. 19.

    et al. Dilation of fusion pores by crowding of SNARE proteins. Elife 6, e22964 (2017)

  20. 20.

    , , & Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses. Proc. Natl Acad. Sci. USA 108, 14318–14323 (2011)

  21. 21.

    et al. Fusion pore formation and expansion induced by Ca2+ and synaptotagmin 1. Proc. Natl Acad. Sci. USA 110, 1333–1338 (2013)

  22. 22.

    & Membrane fusion. Nat. Struct. Mol. Biol. 15, 658–664 (2008)

  23. 23.

    et al. Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin. Nature 460, 978–983 (2009)

  24. 24.

    et al. MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion. Nature 542, 372–376 (2017)

  25. 25.

    Virus and cell fusion mechanisms. Annu. Rev. Cell Dev. Biol. 30, 111–139 (2014)

  26. 26.

    et al. SV2 mediates entry of tetanus neurotoxin into central neurons. PLOS Pathogens 6, e1001207 (2010)

  27. 27.

    , & Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protocols 4, 495–505 (2009)

  28. 28.

    et al. Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat. Methods 14, 49–52 (2017)

  29. 29.

    , , & A genetically encoded, high-signal-to-noise maltose sensor. Proteins 79, 3025–3036 (2011)

  30. 30.

    , & Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs. Science 304, 435–438 (2004)

  31. 31.

    & Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2. PLoS ONE 7, e34836 (2012)

  32. 32.

    et al. CHARMM: the biomolecular simulation program. J. Comput. Chem. 30, 1545–1614 (2009)

  33. 33.

    et al. OpenMM 4: a reusable, extensible, hardware independent library for high performance molecular simulation. J. Chem. Theory Comput. 9, 461–469 (2013)

  34. 34.

    et al. Additive empirical force field for hexopyranose monosaccharides. J. Comput. Chem. 29, 2543–2564 (2008)

  35. 35.

    , , , & CHARMM additive all-atom force field for glycosidic linkages between hexopyranoses. J. Chem. Theory Comput. 5, 2353–2370 (2009)

  36. 36.

    , , , & Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983)

  37. 37.

    , & SWEET—WWW-based rapid 3D construction of oligo- and polysaccharides. Bioinformatics 15, 767–768 (1999)

  38. 38.

    , , & CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008)

  39. 39.

    , , , & Glycan Reader: automated sugar identification and simulation preparation for carbohydrates and glycoproteins. J. Comput. Chem. 32, 3135–3141 (2011)

  40. 40.

    , , , & Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system. Nat. Protocols 8, 1–16 (2013)

  41. 41.

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

  42. 42.

    Ion Channels of Excitable Membranes 3rd edn (Sinauer, 2001)

Download references

Acknowledgements

This study was supported by grants from the NIH (MH061876 and NS097362 to E.R.C.; NS081293 to B.C.). H.B. was supported by a postdoctoral fellowship from the Human Frontier Science Program. D.R. was supported by an NIH fellowship (F32GM112371). E.R.C. is an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

    • Huan Bao
    •  & Debasis Das

    These authors contributed equally to this work.

Affiliations

  1. Department of Neuroscience, University of Wisconsin–Madison, 1111 Highland Avenue, Madison, Wisconsin 53705, USA

    • Huan Bao
    • , Debasis Das
    • , Nicholas A. Courtney
    • , Yihao Jiang
    • , Joseph S. Briguglio
    • , Xiaochu Lou
    • , Baron Chanda
    •  & Edwin R. Chapman
  2. Howard Hughes Medical Institute, 1111 Highland Avenue, Madison, Wisconsin 53705, USA

    • Huan Bao
    • , Debasis Das
    • , Nicholas A. Courtney
    • , Joseph S. Briguglio
    • , Xiaochu Lou
    •  & Edwin R. Chapman
  3. Department of Chemistry and Theoretical Chemistry Institute, University of Wisconsin, Madison, Wisconsin 53706, USA

    • Daniel Roston
    •  & Qiang Cui
  4. Department of Biomolecular Chemistry, University of Wisconsin, Madison, 420 Henry Mall, Madison, Wisconsin 53706, USA

    • Baron Chanda

Authors

  1. Search for Huan Bao in:

  2. Search for Debasis Das in:

  3. Search for Nicholas A. Courtney in:

  4. Search for Yihao Jiang in:

  5. Search for Joseph S. Briguglio in:

  6. Search for Xiaochu Lou in:

  7. Search for Daniel Roston in:

  8. Search for Qiang Cui in:

  9. Search for Baron Chanda in:

  10. Search for Edwin R. Chapman in:

Contributions

H.B. and E.R.C. conceived of the project and designed the biochemistry experiments. H.B. performed nanodisc reconstitution and fusion assays. H.B. and D.D. performed the planar lipid bilayer recordings. N.A.C. designed and conducted the experiments using neurons. Y.J. and B.C. aided in the initial planar lipid bilayer recordings. J.S.B. contributed neurons. X.L. and H.B. contributed to the single vesicle fusion assays. D.R. and Q.C. conducted molecular dynamics simulations. H.B., D.D., N.A.C. and E.R.C. wrote the paper, and all other authors edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Edwin R. Chapman.

Reviewer Information Nature thanks J. Dittman, R. Heidelberger, J. Sørensen and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains full scans of the blots shown in Figure 1c.

  2. 2.

    Life Sciences Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature25481

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.