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Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment

Nature Structural & Molecular Biology volume 20, pages 679–686 (2013)Cite this article

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Abstract

Synaptic-vesicle exocytosis is mediated by the vesicular Ca2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ through PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+ influx bringing the vesicle membrane close enough for membrane fusion.

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Figure 1: Synaptotagmin-1 binds syntaxin-1 clusters in inverted sheets of PC12 cell–derived plasma membranes.
Figure 2: Synaptotagmin-1 binding to syntaxin-1 clusters is mediated by PIP2.
Figure 3: Synaptotagmin-1 binds the syntaxin-1 clusters through the conserved polybasic patch.
Figure 4: Synaptotagmin-1 binding to PIP2–syntaxin-1A clusters in artificial stacked supported membranes generated by spin-coating on glass surfaces.
Figure 5: Synaptotagmin-1 binds PIP2 clusters induced by the polybasic linker region of syntaxin-1 in artificial membranes.
Figure 6: Ca2+-dependent recruitment of liposomes to the plasma membrane by synaptotagmin-1.
Figure 7: Two membrane binding modes of synaptotagmin-1.
Figure 8: Lipid binding modes of the C2B domain.

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Acknowledgements

We thank V. Belov (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) for the KK114-maleimide dye and D. Cafiso (University of Virginia, Charlottesville, West Virginia, USA) for 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 (SFB803). We thank A. Schönle (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) for the software ImSpector. X-ray diffraction data were collected at beamline X10SA at the Swiss Light Source, and we thank the beamline staff for their help during data collection.

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  1. Alf Honigmann and Geert van den Bogaart: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Alf Honigmann, Veronika Mueller, Stefan W Hell & Christian Eggeling

  2. Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Geert van den Bogaart, Dragomir Milovanovic, Karin Kühnel & Reinhard Jahn

  3. Department of Tumor Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

    Geert van den Bogaart

  4. Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland

    Emilio Iraheta & Dirk Fasshauer

  5. Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    H Jelger Risselada & Helmut Grubmüller

  6. Institute for Organic and Biomolecular Chemistry, Georg-August-Universität Göttingen, Göttingen, Germany

    Stefan Müllar & Ulf Diederichsen

  7. Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Christian Eggeling

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Contributions

G.v.d.B., A.H., D.M. and R.J. designed and performed the experiments and wrote the paper. E.I., D.F. and K.K. performed the crystallographic structure determination. H.J.R. and H.G. performed the molecular dynamics simulations. S.M. and U.D. synthesized the peptide. S.W.H., V.M. and C.E. contributed to the microscopy and discussed data. All authors contributed to the manuscript.

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Correspondence to Reinhard Jahn.

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Honigmann, A., van den Bogaart, G., Iraheta, E. et al. Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nat Struct Mol Biol 20, 679–686 (2013). https://doi.org/10.1038/nsmb.2570

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