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

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.

[1]  Peter Walla.  Modern Biophysical Chemistry , 2009 .

[2]  J. Malsam,et al.  The carboxy-terminal domain of complexin I stimulates liposome fusion , 2009, Proceedings of the National Academy of Sciences.

[3]  S. Boxer,et al.  Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides , 2009, Proceedings of the National Academy of Sciences.

[4]  J. Rothman,et al.  Alternative Zippering as an On-Off Switch for SNARE-Mediated Fusion , 2009, Science.

[5]  G. Gould,et al.  Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro , 2008, PloS one.

[6]  J. Rizo,et al.  Synaptic vesicle fusion , 2008, Nature Structural &Molecular Biology.

[7]  H. McMahon,et al.  Mechanisms of membrane fusion: disparate players and common principles , 2008, Nature Reviews Molecular Cell Biology.

[8]  T. Ha,et al.  Complexin and Ca2+ stimulate SNARE-mediated membrane fusion , 2008, Nature Structural &Molecular Biology.

[9]  R. Jahn,et al.  Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids , 2007, Nature Structural &Molecular Biology.

[10]  S. Munro,et al.  An elaborate classification of SNARE proteins sheds light on the conservation of the eukaryotic endomembrane system. , 2007, Molecular biology of the cell.

[11]  J. Rothman,et al.  Selective Activation of Cognate SNAREpins by Sec1/Munc18 Proteins , 2007, Cell.

[12]  R. Jahn,et al.  Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies , 2007, The EMBO journal.

[13]  T. Ha,et al.  Multiple intermediates in SNARE-induced membrane fusion , 2006, Proceedings of the National Academy of Sciences.

[14]  Helmut Grubmüller,et al.  Molecular Anatomy of a Trafficking Organelle , 2006, Cell.

[15]  Reinhard Jahn,et al.  SNAREs — engines for membrane fusion , 2006, Nature Reviews Molecular Cell Biology.

[16]  Alexander Stein,et al.  N- to C-Terminal SNARE Complex Assembly Promotes Rapid Membrane Fusion , 2006, Science.

[17]  Y. Shin,et al.  Hemifusion arrest by complexin is relieved by Ca2+–synaptotagmin I , 2006, Nature Structural &Molecular Biology.

[18]  Demet Araç,et al.  Unraveling the mechanisms of synaptotagmin and SNARE function in neurotransmitter release. , 2006, Trends in cell biology.

[19]  J. Rizo,et al.  SNARE-mediated lipid mixing depends on the physical state of the vesicles. , 2006, Biophysical journal.

[20]  H. Hellmuth,et al.  A Two‐Photon Fluorescence‐Correlation Study of Lectins Interacting with Carbohydrated 20 nm Beads , 2006, Chembiochem : a European journal of chemical biology.

[21]  Edwin R Chapman,et al.  SNARE-driven, 25-millisecond vesicle fusion in vitro. , 2005, Biophysical journal.

[22]  A. Brunger,et al.  Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs). , 2004, Biophysical journal.

[23]  J. Rothman,et al.  Imaging single membrane fusion events mediated by SNARE proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  T. Weber,et al.  Reconstitution of Ca2+-Regulated Membrane Fusion by Synaptotagmin and SNAREs , 2004, Science.

[25]  C. Seidel,et al.  Determinants of liposome fusion mediated by synaptic SNARE proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[26]  N. Düzgüneş.  Preparation and quantitation of small unilamellar liposomes and large unilamellar reverse-phase evaporation liposomes. , 2003, Methods in enzymology.

[27]  J. Rigaud,et al.  Reconstitution of membrane proteins into liposomes. , 2003, Methods in enzymology.

[28]  J. Rothman,et al.  Regulation of membrane fusion by the membrane-proximal coil of the t-SNARE during zippering of SNAREpins , 2002, The Journal of cell biology.

[29]  P. Schwille,et al.  Two-photon fluorescence cross-correlation spectroscopy. , 2001, Chemphyschem : a European journal of chemical physics and physical chemistry.

[30]  R. Rigler,et al.  Fluorescence correlation spectroscopy , 2001 .

[31]  J. Rothman,et al.  Close Is Not Enough , 2000, The Journal of cell biology.

[32]  J. Rothman,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. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[33]  W. Antonin,et al.  Mixed and Non-cognate SNARE Complexes , 1999, The Journal of Biological Chemistry.

[34]  A. Brunger,et al.  Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Benedikt Westermann,et al.  SNAREpins: Minimal Machinery for Membrane Fusion , 1998, Cell.

[36]  A T Brünger,et al.  Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation* , 1997, The Journal of Biological Chemistry.

[37]  P. Schwille,et al.  Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. , 1997, Biophysical journal.

[38]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[39]  D. Hoekstra,et al.  Use of resonance energy transfer to monitor membrane fusion. , 1981, Biochemistry.

[40]  S. Aragon,et al.  Fluorescence correlation spectroscopy and Brownian rotational diffusion , 1975 .

[41]  W. Webb,et al.  Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy , 1972 .

[42]  M. Smoluchowski.  Versuch einer mathematischen Theorie der Koagulationskinetik kolloider Lösungen , 1918 .