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Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation

Abstract

Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting interactions between SNARE proteins on synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is unclear, and the massive increase in membrane fusion efficiency of Ca2+-bound synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator that tethers the liposomes too far from the plasma membrane for SNARE nucleation in the absence of Ca2+, but while bringing the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively weak electrostatic interactions between synaptotagmin-1 and membranes substantially accelerate fusion.

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Figure 1: Electrostatic repulsion blocks SNARE-mediated membrane fusion at low ionic strength.
Figure 2: At low ionic strength, Ca2+–synaptotagmin-1 triggers lipid mixing.
Figure 3: Ca2+–synaptotagmin-1 triggers full membrane fusion at low ionic strength.
Figure 4: Liposome clustering by synaptotagmin-1.
Figure 5: Preclustering of liposomes accelerates lipid mixing.
Figure 6: Models of distance regulation by synaptotagmin-1.

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References

  1. 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).

    Article  CAS  Google Scholar 

  2. Jahn, R. & Scheller, R.H. SNAREs—engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).

    Article  CAS  Google Scholar 

  3. Chapman, E.R. How does synaptotagmin trigger neurotransmitter release? Annu. Rev. Biochem. 77, 615–641 (2008).

    Article  CAS  Google Scholar 

  4. Martens, S. & McMahon, H.T. Mechanisms of membrane fusion: disparate players and common principles. Nat. Rev. Mol. Cell Biol. 9, 543–556 (2008).

    Article  CAS  Google Scholar 

  5. Radhakrishnan, A., Stein, A., Jahn, R. & Fasshauer, D. The Ca2+ affinity of synaptotagmin 1 is markedly increased by a specific interaction of its C2B domain with phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem. 284, 25749–25760 (2009).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Araç, D. et al. Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids. Nat. Struct. Mol. Biol. 13, 209–217 (2006).

    Article  Google Scholar 

  10. Gaffaney, J.D., Dunning, F.M., Wang, Z., Hui, E. & Chapman, E.R. Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis. J. Biol. Chem. 283, 31763–31775 (2008).

    Article  CAS  Google Scholar 

  11. Bhalla, A., Chicka, M.C., Tucker, W.C. & Chapman, E.R. Ca2+-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nat. Struct. Mol. Biol. 13, 323–330 (2006).

    Article  CAS  Google Scholar 

  12. Herrick, D.Z., Sterbling, S., Rasch, K.A., Hinderliter, A. & Cafiso, D.S. Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains. Biochemistry 45, 9668–9674 (2006).

    Article  CAS  Google Scholar 

  13. Hui, E., Bai, J. & Chapman, E.R. Ca2+-triggered simultaneous membrane penetration of the tandem C2-domains of synaptotagmin I. Biophys. J. 91, 1767–1777 (2008).

    Article  Google Scholar 

  14. Schiavo, G., Gu, Q.M., Prestwich, G.D., Söllner, T.H. & Rothman, J.E. Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin. Proc. Natl. Acad. Sci. USA 93, 13327–13332 (1996).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. Bai, J., Wang, C.T., Richards, D.A., Jackson, M.B. & Chapman, E.R. Fusion pore dynamics are regulated by synaptotagmint-SNARE interactions. Neuron 41, 929–942 (2004).

    Article  CAS  Google Scholar 

  18. Vrljic, M. et al. Molecular mechanism of the synaptotagmin-SNARE interaction in Ca2+-triggered vesicle fusion. Nat. Struct. Mol. Biol. 17, 325–331 (2010).

    Article  CAS  Google Scholar 

  19. Choi, U.B. et al. Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex. Nat. Struct. Mol. Biol. 17, 318–324 (2010).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. van den Bogaart, G. et al. One SNARE complex is sufficient for membrane fusion. Nat. Struct. Mol. Biol. 17, 358–364 (2010).

    Article  CAS  Google Scholar 

  25. Stein, A., Radhakrishnan, A., Riedel, D., Fasshauer, D. & Jahn, R. Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids. Nat. Struct. Mol. Biol. 14, 904–911 (2007).

    Article  CAS  Google Scholar 

  26. Chicka, M.C., Hui, E., Liu, H. & Chapman, E.R. Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+. Nat. Struct. Mol. Biol. 15, 827–835 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Lynch, K.L. et al. Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis. Mol. Biol. Cell 18, 4957–4968 (2007).

    Article  CAS  Google Scholar 

  29. Tucker, W.C., Weber, T. & Chapman, E.R. Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs. Science 304, 435–438 (2004).

    Article  CAS  Google Scholar 

  30. Rhee, J.S. et al. Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1. Proc. Natl. Acad. Sci. USA 102, 18664–18669 (2005).

    Article  CAS  Google Scholar 

  31. Margittai, M., Fasshauer, D., Pabst, S., Jahn, R. & Langen, R. Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling. J. Biol. Chem. 276, 13169–13177 (2001).

    Article  CAS  Google Scholar 

  32. Fasshauer, D. & Margittai, M. A transient N-terminal interaction of SNAP-25 and syntaxin nucleates SNARE assembly. J. Biol. Chem. 279, 7613–7621 (2004).

    Article  CAS  Google Scholar 

  33. Parlati, F. et al. Rapid and efficient fusion of phospholipid vesicles by the alpha-helical core of a SNARE complex in the absence of an N-terminal regulatory domain. Proc. Natl. Acad. Sci. USA 96, 12565–12570 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Siddiqui, T.J. et al. Determinants of synaptobrevin regulation in membranes. Mol. Biol. Cell 18, 2037–2046 (2007).

    Article  CAS  Google Scholar 

  36. Li, F. et al. Energetics and dynamics of SNAREpin folding across lipid bilayers. Nat. Struct. Mol. Biol. 14, 890–896 (2007).

    Article  CAS  Google Scholar 

  37. Holt, M., Riedel, D., Stein, A., Schuette, C. & Jahn, R. Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+. Curr. Biol. 18, 715–722 (2008).

    Article  CAS  Google Scholar 

  38. James, D.J., Khodthong, C., Kowalchyk, J.A. & Martin, T.F. Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion. J. Cell Biol. 182, 355–366 (2008).

    Article  CAS  Google Scholar 

  39. van den Bogaart, G., Hermans, N., Krasnikov, V., de Vries, A.H. & Poolman, B. On the decrease in lateral mobility of phospholipids by sugars. Biophys. J. 92, 1598–1605 (2007).

    Article  CAS  Google Scholar 

  40. Cerjan, C. & Barnett, R.E. The viscosity dependence of a putative diffusion-limited reaction. J. Phys. Chem. 76, 1192–1195 (1971).

    Article  Google Scholar 

  41. Knight, J.B., Vishwanath, A., Brody, J.P. & Austin, R.H. Hydrodynamic focusing on a silicon chip: mixing nanoliters in microseconds. Phys. Rev. Lett. 80, 3863–3866 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  43. 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).

    Article  CAS  Google Scholar 

  44. Wienken, C.J., Baaske, P., Rothbauer, U., Braun, D. & Duhr, S. Protein-binding assays in biological liquids using microscale thermophoresis. Nat. Commun. 1, 100 (2010).

    Article  Google Scholar 

  45. Duhr, S. & Braun, D. Why do molecules move along a temperature gradient? Proc. Natl. Acad. Sci. USA 103, 19678–19682 (2006).

    Article  CAS  Google Scholar 

  46. 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  Google Scholar 

  47. Borden, C.R., Stevens, C.F., Sullivan, J.M. & Zhu, Y. Synaptotagmin mutants Y311N and K326/327A alter the calcium dependence of neurotransmission. Mol. Cell. Neurosci. 29, 462–470 (2005).

    Article  CAS  Google Scholar 

  48. Marrink, S.J., Risselada, H.J., Yefimov, S., Tieleman, D.P. & de Vries, A.H. The MARTINI force field: coarse grained model for biomolecular simulations. J. Phys. Chem. B 111, 7812–7824 (2007).

    Article  CAS  Google Scholar 

  49. Monticelli, L. et al. The MARTINI coarse grained forcefield: extension to proteins. J. Chem. Theory Comput. 4, 819–834 (2008).

    Article  CAS  Google Scholar 

  50. Sutton, R.B., Ernst, J.A. & Brunger, A.T. Crystal structure of the cytosolic C2A–C2B domains of synaptotagmin III. Implications for Ca2+-independent snare complex interaction. J. Cell Biol. 147, 589–598 (1999).

    Article  CAS  Google Scholar 

  51. Hu, K. et al. Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion. Nature 415, 646–650 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  53. Walter, A.M., Wiederhold, K., Bruns, D., Fasshauer, D. & Sørensen, J.B. Synaptobrevin N-terminally bound to syntaxin-SNAP-25 defines the primed vesicle state in regulated exocytosis. J. Cell Biol. 3, 401–413 (2010).

    Article  Google Scholar 

  54. Borisovska, M. et al. v-SNAREs control exocytosis of vesicles from priming to fusion. EMBO J. 24, 2114–2126 (2005).

    Article  CAS  Google Scholar 

  55. Gerber, S.H. et al. Conformational switch of syntaxin-1 controls synaptic vesicle fusion. Science 321, 1507–1510 (2008).

    Article  CAS  Google Scholar 

  56. Schiavo, G., Stenbeck, G., Rothman, J.E. & Söllner, T.H. Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses. Proc. Natl. Acad. Sci. USA 3, 997–1001 (1997).

    Article  Google Scholar 

  57. Mohrmann, R., de Wit, H., Verhage, M., Neher, E. & Sørensen, J.B. Fast vesicle fusion in living cells requires at least three SNARE complexes. Science 6003, 502–505 (2010).

    Article  Google Scholar 

  58. Ernst, J.A. & Brunger, A.T. High resolution structure, stability, and synaptotagmin binding of a truncated neuronal SNARE complex. J. Biol. Chem. 278, 8630–8636 (2003).

    Article  CAS  Google Scholar 

  59. Cooper McDonald, J. et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 21, 27–40 (2000).

    Article  Google Scholar 

  60. Doeven, M.K. et al. Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles. Biophys. J. 88, 1134–1142 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Stein and U. Ries for protein purification and comments. G.v.d.B. is financed by the Human Frontier Science Program. This work was supported by the US National Institutes of Health (P01 GM072694 to R.J.) and the Deutsche Forschungsgemeinschaft (SFB755 to S.T. and S.H.; SFB803 to K.M., J.H.R., M.H., U.D., H.G. and R.J.).

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S.T. and S.H. performed the flow cytometry experiments. J.H.R. and H.G. performed the MD simulations. M.H. purified the synaptic vesicles. Thermophoresis data were from K.M. and U.D. D.R. performed the EM. G.v.d.B. performed all other experiments. G.v.d.B. and R.J. designed the study and wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Reinhard Jahn.

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The authors declare no competing financial interests.

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van den Bogaart, G., Thutupalli, S., Risselada, J. et al. Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation. Nat Struct Mol Biol 18, 805–812 (2011). https://doi.org/10.1038/nsmb.2061

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