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STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis

Nature volume 440, pages 935–939 (2006)Cite this article

Abstract

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter1. Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (∼40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED)2 to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.

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Figure 1: STED microscopy resolves synaptic vesicles in individual boutons of primary cultured hippocampal neurons.
Figure 2: Comparison between surface-exposed and internalized pools of synaptotagmin shows that the protein remains clustered in the presynaptic plasma membrane.
Figure 3: Dot sizes of surface-exposed and internalized synaptotagmin pools do not show major differences, and neither dot brightness nor dot sizes change upon stimulation of exocytosis.

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Acknowledgements

The authors thank E. Neher for helpful comments. S.O.R. acknowledges fellowships from the European Molecular Biology Organization and from the Human Frontier Science Program. This work was partly supported by a grant from the Leibniz Program of the Deutsche Forschungsgemeinschaft awarded to R.J., a grant from the German Ministry of Research and Education to S.W.H., and by the DFG-Centre for Molecular Physiology of the Brain. We thank I. Herefort, M. Wienisch and J. Klingauf for assistance with cell culturing, and A. Schönle for help with his software ImSpector.

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  1. Katrin I. Willig and Silvio O. Rizzoli: *These authors contributed equally to this work

Authors and Affiliations

  1. Departments of NanoBiophotonics,

    Katrin I. Willig, Volker Westphal & Stefan W. Hell

  2. Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany

    Silvio O. Rizzoli & Reinhard Jahn

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  1. Katrin I. Willig
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Corresponding author

Correspondence to Reinhard Jahn.

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Supplementary information

Supplementary Figure S1

Bright (putatively surface) fluorescent dots persist in permeabilized preparations. (JPG 27 kb)

Supplementary Figure S2

Quantification of dot half-width using different dot-selection approaches. (JPG 47 kb)

Supplementary Figure S3

Alternative models for the recycling of synaptotagmin. (JPG 21 kb)

Supplementary Figure S4

The pool of surface synaptotagmin can undergo vesicle recycling. (JPG 21 kb)

Supplementary Figure Legends

This file contains full legends for Supplementary Figures S1–S4. (DOC 22 kb)

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Willig, K., Rizzoli, S., Westphal, V. et al. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006). https://doi.org/10.1038/nature04592

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