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A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis

Nature Structural & Molecular Biology volume 25, pages 911–917 (2018)Cite this article

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Abstract

The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.

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Fig. 1: Correlating conformational changes in SNARE proteins with fusion efficiency during Ca2+-triggered vesicle fusion.
Fig. 2: C2AB induces trans–cis conformational transition of neuronal SNARE complex.
Fig. 3: SNARE complex conformation and DCV fusion depend on the ratio of saturated and unsaturated fatty acids in the target membrane.
Fig. 4: Mutations in C2AB and SNAREs impair trans–cis conformational transition of the SNARE complex and DCV fusion.
Fig. 5: Model for Ca2+-triggered fusion mechanism.

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Data availability

Source data for Figs. 2, 3a,b,d,e, and 4 are available with the paper online. The custom code used to acquire and analyze the data, as well other data in this study, is available from the corresponding author upon reasonable request.

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Acknowledgements

We thank A. Lambacher and P. Fromherz for providing FLIC analysis software and for their help with the production of FLIC substrates. This work was supported by US NIH grant P01 GM72694 to L.K.T., D.S.C., B.L., and V.K.

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Authors and Affiliations

  1. Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA

    Volker Kiessling, Alex J. B. Kreutzberger, Binyong Liang, Patrick Seelheim & Lukas K. Tamm

  2. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA

    Volker Kiessling, Alex J. B. Kreutzberger, Binyong Liang, Patrick Seelheim & Lukas K. Tamm

  3. Department of Chemistry, University of Virginia, Charlottesville, VA, USA

    Sarah B. Nyenhuis & David S. Cafiso

  4. Department of Cell Biology, University of Virginia, Charlottesville, VA, USA

    J. David Castle

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Contributions

V.K. performed and analyzed all of the sdFLIC experiments. A.J.B.K. performed and analyzed all of the DCV fusion experiments. B.L. and S.B.N. prepared protein samples. P.S. prepared shRNA for SytKD DCVs. V.K. designed the work. V.K., D.S.C., J.D.C., and L.K.T. wrote the paper.

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Correspondence to Volker Kiessling.

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Integrated supplementary information

Supplementary Figure 1 SdFLIC example images and evaluation of different SNARE complexes in bPC/bPE/bPS/bPIP2/cholesterol.

a–c, Example sdFLIC images (second column) with a yellow grid of 12 × 12 oxides that were evaluated, extracted intensity data from one set of 16 oxides and FLIC fit curve (third column), and example histogram of fit results (fourth column) obtained from one sample under one condition for Syx*192/SNAP-25 (a), Syx*192/SNAP-25/Syb(1-96) (b), and Syx*192(183–288)/SNAP-25/Syb (c). Images were rotated and scaled for printing and show an area of 201 × 201 μm2.

Supplementary Figure 2 SdFLIC example images and evaluation for Syx*192/SNAP-25/Syb(1-96) in different lipid environments.

a–l, Example sdFLIC images with a yellow grid of 12 × 12 oxides that were evaluated, extracted intensity data from one set of 16 oxides and FLIC fit curve, and example histogram of fit results obtained from one sample under one condition for Syx*192/SNAP-25/Syb(1-96). Images in a–f were taken before and after the addition of Ca2+/C2AB in different lipid headgroup environments, as described in Fig. 1d and indicated in each panel. Images in g–l were taken in different lipid environments, as described in Fig. 2a and indicated in each panel. Images were rotated and scaled for printing and show an area of 201 × 201 μm2.

Supplementary Figure 3 SdFLIC example images and evaluation for Syx*192/SNAP-25/Syb(1-96) in different lipid environments (continued).

a–g, Example sdFLIC images with a yellow grid of 12 × 12 oxides that were evaluated, extracted intensity data from one set of 16 oxides and FLIC fit curve, and example histogram of fit results obtained from one sample under one condition for Syx*192/SNAP-25/Syb(1-96). Images were taken in different lipid environments, as described in Fig. 2a (a–d) and Fig. 2c (e–g), and as indicated in each panel. Images were rotated and scaled for printing and show an area of 201 × 201 μm2.

Supplementary Figure 4 SdFLIC and DCV fusion results obtained with different SNARE complex constructs and supported membrane control.

a, sdFLIC results from Syx*/SNAP-25/Syb(1-96) in bPC/bPE/bPS/bPIP2/cholesterol, labeled at residues 192 and 249 for comparison in EDTA (solid bars) and after addition of Ca2+/C2AB (open bars). b, sdFLIC results from Syb*28(1-96) after it was added to reconstituted Syx1a/SNAP-25 acceptor complex30 in different lipid environments as indicated in EDTA (solid bars) and after addition of Ca2+/C2AB (open bars). c, sdFLIC results from Syb*28(1-96) after binding to reconstituted Syx1a/SNAP-25 acceptor complex at different bPS concentrations with and without 1 mol% PIP2 as indicated. d, Distance of the lipid-bilayer surface from substrate as determined by FLIC in EDTA and after addition of Ca2+/C2AB. e, sdFLIC results from Syx*192/SNAP-25/Syb(1-96) in bPC/bPE/bPS/bPIP2/cholesterol in EDTA (solid bar) and after addition of C2AB/EDTA, Ca2+ and EDTA in successive order. f, sdFLIC and g, fusion probabilities when complexin, Munc18, or both have been added to form the ‘trigger-ready’ primed prefusion state19. When complexin is added to Syx*/SNAP-25/Syb(1-96), the complex moves slightly further away from the membrane. Adding Ca2+/C2AB straightens the complex in the same way as without complexin. When complexin is added to acceptor complex in the supported membrane, it inhibits Syb binding and DCV docking in the absence of Ca2+ (ref. 22). In the presence of Ca2+/C2AB, Syt-deficient DCVs dock and proceed to fusion with the supported membrane, similar to the WT DCV fusion in the presence of Ca2+ (ref. 19). When Munc18 and soluble Syb2 (Syb1-96) is added to reconstituted Syx*192/SNAP-25 (Fig. 1c), the measured distance of Syx*192 within this complex increases by more than 6 nm. When Ca2+/C2AB is added to this in the membrane assembled complex, its structure becomes more upright although the distance does not increase to the same height as in the isolated preassembled SNARE complex. Fusion of SytKD-DCVs in the absence of Ca2+ is increased by Munc18 and is further stimulated by Ca2+/C2AB. The height of Syx*192 within a complex that has been assembled by adding Munc18, complexin, and Syb1-96 to Syx*192/SNAP-25, is raised to 9.5 nm. When Ca2+/C2AB is added, the distance of Syx*192 from the membrane increases further to 11.6 nm, about the same as that of the cis-SNARE complex. Adding Munc18 and complexin at the same time to Syx1a/SNAP-25 in the supported membrane results in a ‘trigger competent’ prefusion state that allows DCV docking in the absence of Ca2+ without significant fusion19. Adding Ca2+/C2AB to the docked DCVs after a 15-min incubation time increases the fusion probability of the SytKD-DCVs from almost zero to 58%. Note that when multiprotein complexes are assembled on the membrane, the measured distance of Syx*192 most likely represents an average originating from different multiprotein assemblies. All data represent means from at least five independent experiments. Error bars represent s.e.m.

Supplementary Figure 5 SNARE and Ca2+/C2AB-dependent fusion in proteo-liposome/DCV fusion assay and Syx*192-membrane distance changes induced by C2AB-WT or the C2AB-KAKA mutant with and without PIP2.

a–d, Example (NBD) fluorescence dequenching curves after WT-DCVs have been added to Syx/SNAP-25-containing proteoliposomes in EDTA (black curves) or in the presence of Ca2+/C2AB. Syx/SNAP-25 is reconstituted into DP (a), PO (b), DO (c), or brain lipids (d). e, Average fluorescence increase due to NBD dequenching after proteoliposome/DCV fusion in EDTA (solid bars) and in the presence of Ca2+/C2AB. The lipid headgroup composition is PC/PE/PS/PIP2/cholesterol (34/30/15/1/20) in all cases, and the acyl chain composition is as indicated. Averages represent means from three repeats, and error bars represent s.d. f, Syx*192-membrane distance changes in response to C2AB-WT with (red) and without (orange) 1 mol% PIP2 and C2AB-KAKA with (light blue) and without (dark blue) 1 mol% PIP2. Data points represent means from at least five independent experiments. Error bars represent s.e.m.

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Kiessling, V., Kreutzberger, A.J.B., Liang, B. et al. A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis. Nat Struct Mol Biol 25, 911–917 (2018). https://doi.org/10.1038/s41594-018-0130-9

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