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  Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS.

Cypionka, A., Stein, A., Hernandez, M., Hippchen, H., Jahn, R., & Walla, P. J. (2009). Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS. Proceedings of the National Academy of Sciences of the United States of America, 106(44), 18575-18580. doi:10.1073/pnas.0906677106.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-D703-A Version Permalink: http://hdl.handle.net/21.11116/0000-0000-3381-4
Genre: Journal Article

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 Creators:
Cypionka, A.1, Author              
Stein, A.2, Author              
Hernandez, M.3, Author              
Hippchen, H.1, Author              
Jahn, R.3, Author              
Walla, P. J.1, Author              
Affiliations:
1Research Group of Biomolecular Spectroscopy and Single-Molecule Detection, MPI for biophysical chemistry, Max Planck Society, ou_578565              
2Research Group of Membrane Protein Biochemistry, MPI for Biophysical Chemistry, Max Planck Society, ou_2149675              
3Department of Neurobiology, MPI for biophysical chemistry, Max Planck Society, ou_578595              

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Free keywords: fluorescence cross-correlation spectroscopy; fluorescence lifetime analysis; fusion intermediate; single-particle detection; SNAREs
 Abstract: Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is well-established that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.

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Language(s): eng - English
 Dates: 2009-11-03
 Publication Status: Published in print
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: eDoc: 439672
DOI: 10.1073/pnas.0906677106
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Title: Proceedings of the National Academy of Sciences of the United States of America
Source Genre: Journal
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Pages: - Volume / Issue: 106 (44) Sequence Number: - Start / End Page: 18575 - 18580 Identifier: -