Article

Synaptotagmin-1 drives synchronous Ca2+-triggered fusion by C2B-domain-mediated synaptic-vesicle-membrane attachment

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Abstract

The synaptic vesicle (SV) protein synaptotagmin-1 (Syt1) is the Ca2+ sensor for fast synchronous release. Biochemical and structural data suggest that Syt1 interacts with phospholipids and SNARE complex, but the manner in which these interactions translate into SV fusion remains poorly understood. Using flash-and-freeze electron microscopy, which triggers action potentials with light and coordinately arrests synaptic structures with rapid freezing, we found that synchronous-release-impairing mutations in the Syt1 C2B domain (K325, 327; R398, 399) also disrupt SV-active-zone plasma-membrane attachment. Single action potential induction rescued membrane attachment in these mutants within less than 10 ms through activation of the Syt1 Ca2+-binding site. The rapid SV membrane translocation temporarily correlates with resynchronization of release and paired pulse facilitation. On the basis of these findings, we redefine the role of Syt1 as part of the Ca2+-dependent vesicle translocation machinery and propose that Syt1 enables fast neurotransmitter release by means of its dynamic membrane attachment activities.

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References

  1. 1.

    Imig, C. et al. The morphological and molecular nature of synaptic vesicle priming at presynaptic active zones. Neuron 84, 416–431 (2014).

  2. 2.

    Jahn, R. & Fasshauer, D. Molecular machines governing exocytosis of synaptic vesicles. Nature 490, 201–207 (2012).

  3. 3.

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

  4. 4.

    Südhof, T. C. Neurotransmitter release: the last millisecond in the life of a synaptic vesicle. Neuron 80, 675–690 (2013).

  5. 5.

    Brose, N., Petrenko, A. G., Südhof, T. C. & Jahn, R. Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science 256, 1021–1025 (1992).

  6. 6.

    Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).

  7. 7.

    Fernández-Chacón, R. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001).

  8. 8.

    Mackler, J. M., Drummond, J. A., Loewen, C. A., Robinson, I. M. & Reist, N. E. The C(2)B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature 418, 340–344 (2002).

  9. 9.

    Sutton, R. B., Davletov, B. A., Berghuis, A. M., Südhof, T. C. & Sprang, S. R. Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold. Cell 80, 929–938 (1995).

  10. 10.

    Chapman, E. R. & Davis, A. F. Direct interaction of a Ca2+-binding loop of synaptotagmin with lipid bilayers. J. Biol. Chem. 273, 13995–14001 (1998).

  11. 11.

    Fernandez, I. et al. Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine. Neuron 32, 1057–1069 (2001).

  12. 12.

    Bacaj, T. et al. Synaptotagmin-1 and -7 are redundantly essential for maintaining the capacity of the readily-releasable pool of synaptic vesicles. PLoS Biol. 13, e1002267 (2015).

  13. 13.

    de Wit, H. et al. Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes. Cell 138, 935–946 (2009).

  14. 14.

    Kedar, G. H. et al. A post-docking role of Synaptotagmin 1-C2B domain bottom residues R398/399 in mouse chromaffin cells. J. Neurosci. 35, 14172–14182 (2015).

  15. 15.

    Jorgensen, E. M. et al. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature 378, 196–199 (1995).

  16. 16.

    Reist, N. E. et al. Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila. J. Neurosci. 18, 7662–7673 (1998).

  17. 17.

    Siksou, L. et al. A common molecular basis for membrane docking and functional priming of synaptic vesicles. Eur. J. Neurosci. 30, 49–56 (2009).

  18. 18.

    Liu, H., Dean, C., Arthur, C. P., Dong, M. & Chapman, E. R. Autapses and networks of hippocampal neurons exhibit distinct synaptic transmission phenotypes in the absence of synaptotagmin I. J. Neurosci. 29, 7395–7403 (2009).

  19. 19.

    Poskanzer, K. E., Marek, K. W., Sweeney, S. T. & Davis, G. W. Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo. Nature 426, 559–563 (2003).

  20. 20.

    van den Bogaart, G. et al. Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation. Nat. Struct. Mol. Biol. 18, 805–812 (2011).

  21. 21.

    Honigmann, A. et al. Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nat. Struct. Mol. Biol. 20, 679–686 (2013).

  22. 22.

    Bai, J., Tucker, W. C. & Chapman, E. R. PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nat. Struct. Mol. Biol. 11, 36–44 (2004).

  23. 23.

    Li, L. et al. Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin 1. J. Biol. Chem. 281, 15845–15852 (2006).

  24. 24.

    Xue, M., Ma, C., Craig, T. K., Rosenmund, C. & Rizo, J. The Janus-faced nature of the C(2)B domain is fundamental for synaptotagmin-1 function. Nat. Struct. Mol. Biol. 15, 1160–1168 (2008).

  25. 25.

    Brewer, K. D. et al. Dynamic binding mode of a Synaptotagmin-1-SNARE complex in solution. Nat. Struct. Mol. Biol. 22, 555–564 (2015).

  26. 26.

    Seven, A. B., Brewer, K. D., Shi, L., Jiang, Q. X. & Rizo, J. Prevalent mechanism of membrane bridging by synaptotagmin-1. Proc. Natl. Acad. Sci. USA 110, E3243–E3252 (2013).

  27. 27.

    Zhou, Q. et al. Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis. Nature 525, 62–67 (2015).

  28. 28.

    Zhou, A., Brewer, K. D. & Rizo, J. Analysis of SNARE complex/synaptotagmin-1 interactions by one-dimensional NMR spectroscopy. Biochemistry 52, 3446–3456 (2013).

  29. 29.

    Martens, S., Kozlov, M. M. & McMahon, H. T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007).

  30. 30.

    Lynch, K. L. et al. Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion. Mol. Biol. Cell 19, 5093–5103 (2008).

  31. 31.

    Hui, E., Johnson, C. P., Yao, J., Dunning, F. M. & Chapman, E. R. Synaptotagmin-mediated bending of the target membrane is a critical step in Ca2+-regulated fusion. Cell 138, 709–721 (2009).

  32. 32.

    Schaub, J. R., Lu, X., Doneske, B., Shin, Y. K. & McNew, J. A. Hemifusion arrest by complexin is relieved by Ca2+-synaptotagmin I. Nat. Struct. Mol. Biol. 13, 748–750 (2006).

  33. 33.

    Heuser, J. E. & Reese, T. S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344 (1973).

  34. 34.

    Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013).

  35. 35.

    Young, S. M. Jr. & Neher, E. Synaptotagmin has an essential function in synaptic vesicle positioning for synchronous release in addition to its role as a calcium sensor. Neuron 63, 482–496 (2009).

  36. 36.

    Nishiki, T. & Augustine, G. J. Dual roles of the C2B domain of synaptotagmin I in synchronizing Ca2+-dependent neurotransmitter release. J. Neurosci. 24, 8542–8550 (2004).

  37. 37.

    Gerber, S. H., Rizo, J. & Südhof, T. C. The top loops of the C(2) domains from synaptotagmin and phospholipase A(2) control functional specificity. J. Biol. Chem. 276, 32288–32292 (2001).

  38. 38.

    Schneggenburger, R. & Neher, E. Presynaptic calcium and control of vesicle fusion. Curr. Opin. Neurobiol. 15, 266–274 (2005).

  39. 39.

    Binz, T. et al. Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. J. Biol. Chem. 269, 1617–1620 (1994).

  40. 40.

    Schiavo, G. et al. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359, 832–835 (1992).

  41. 41.

    Chen, Y. A., Scales, S. J. & Scheller, R. H. Sequential SNARE assembly underlies priming and triggering of exocytosis. Neuron 30, 161–170 (2001).

  42. 42.

    Chernomordik, L. V. & Kozlov, M. M. Protein-lipid interplay in fusion and fission of biological membranes. Annu. Rev. Biochem. 72, 175–207 (2003).

  43. 43.

    Xu, J., Pang, Z. P., Shin, O. H. & Südhof, T. C. Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release. Nat. Neurosci. 12, 759–766 (2009).

  44. 44.

    Rosenmund, C. & Stevens, C. F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 (1996).

  45. 45.

    Min, D. et al. Mechanical unzipping and rezipping of a single SNARE complex reveals hysteresis as a force-generating mechanism. Nat. Commun. 4, 1705 (2013).

  46. 46.

    Jackman, S. L. & Regehr, W. G. The mechanisms and functions of synaptic facilitation. Neuron 94, 447–464 (2017).

  47. 47.

    Lois, C., Hong, E. J., Pease, S., Brown, E. J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

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Acknowledgements

We thank A. Plested, M. Herman, J. Rizo, C. Garner and T. Südhof for discussions and comments on the manuscript, S. Watanabe and E. Jorgensen for technical support, the Charité viral core facility for virus production, and B. Söhl-Kielszinski for sample preparation. This work was supported by ERC grant SynVGLUT, Berlin Institute of Health, Stiftung Charite, German Research Council grants SFB958, Ro1296/7-1 and TRR186.

Author information

Affiliations

  1. Institut für Neurophysiologie, Charité - Universitätsmedizin, Berlin, Germany

    • Shuwen Chang
    • , Thorsten Trimbuch
    •  & Christian Rosenmund
  2. NeuroCure Cluster of Excellence Cluster, Berlin, Germany

    • Shuwen Chang
    • , Thorsten Trimbuch
    •  & Christian Rosenmund

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Contributions

S.C. performed experiments and analyzed data. T.T. produced molecular reagents. S.C. and C.R. designed the experiments and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Christian Rosenmund.

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Supplementary information