title: Dance of the SNAREs: assembly and rearrangements detected with FRET at neuronal synapses.
abstract: Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane on activation by calcium binding to synaptotagmin. In the present study, we used fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy between fluorescently labeled SNARE proteins expressed in cultured rat hippocampal neurons to detect resting SNARE complexes, their conformational rearrangement on exocytosis, their disassembly before endocytosis of vesicular proteins, and SNARE assembly at newly docked vesicles. Assembled SNAREs are not only present in docked vesicles; unexpected residual "orphan SNARE complexes" also reside in para-active zone regions. Real-time changes in FRET between N-terminally labeled SNAP-25 and VAMP reported a reorientation of the SNARE motif upon exocytosis, SNARE disassembly in the active zone periphery, and SNARE reassembly in newly docked vesicles. With VAMP labeled C-terminally, decreased fluorescence in C-terminally labeled syntaxin (extracellular) reported trans-cis-conformational changes in SNAREs on vesicle fusion. After fusion SNAP-25 and syntaxin disperse along with VAMP, as well as the FRET signal itself, indicating diffusion of intact SNAREs after vesicle fusion but before their peripheral disassembly. Our measurements of spatiotemporal dynamics of SNARE conformational changes and movements refine models of SNARE function. Technical advances required to detect tiny changes in fluorescence in small fractions of labeled proteins in presynaptic boutons on a time scale of seconds permit the detection of rapid intermolecular interactions between small proportions of protein partners in cellular subcompartments.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.
authors.forename: Vadim
authors.initials: V
authors.lastname: Degtyar
authors.forename: Ismail M
authors.initials: IM
authors.lastname: Hafez
authors.forename: Christopher
authors.initials: C
authors.lastname: Bray
authors.forename: Robert S
authors.initials: RS
authors.lastname: Zucker
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002569:Cerulenin / Q000378:metabolism; D004558:Electric Stimulation; D004622:Embryo, Mammalian; D005089:Exocytosis / Q000187:drug effects / Q000235:genetics; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006624:Hippocampus / Q000166:cytology*; D007091:Image Processing, Computer-Assisted; D008297:Male; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000166:cytology*; D017711:Nonlinear Dynamics; D018408:Patch-Clamp Techniques; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D013569:Synapses / Q000187:drug effects / Q000502:physiology*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism
chemical_list: C092350:FM 4-64; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; D049452:Green Fluorescent Proteins; D002569:Cerulenin
references.citation: Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13310-5
references.pmid: 8917587
references.citation: Biochemistry. 2004 Jan 20;43(2):526-32
references.pmid: 14717608
references.citation: Biophys J. 2002 Dec;83(6):3652-64
references.pmid: 12496132
references.citation: Neuron. 2006 Jul 20;51(2):179-86
references.pmid: 16846853
references.citation: Nat Cell Biol. 2000 Apr;2(4):197-204
references.pmid: 10783237
references.citation: Trends Cell Biol. 2006 Jul;16(7):339-50
references.pmid: 16698267
references.citation: Gene Ther. 2004 Sep;11(17):1303-11
references.pmid: 15229630
references.citation: J Neurophysiol. 2007 Dec;98(6):3221-9
references.pmid: 17942621
references.citation: Neuron. 1995 May;14(5):983-9
references.pmid: 7748565
references.citation: J Neurocytol. 2000 Jan;29(1):67-77
references.pmid: 11068335
references.citation: Neuron. 1997 Jun;18(6):995-1008
references.pmid: 9208866
references.citation: J Biol Chem. 2001 Jan 19;276(3):1766-71
references.pmid: 11035043
references.citation: J Biomed Opt. 2008 May-Jun;13(3):031203
references.pmid: 18601527
references.citation: PLoS One. 2011 Mar 29;6(3):e17896
references.pmid: 21479270
references.citation: Neuron. 2001 Aug 30;31(4):593-605
references.pmid: 11545718
references.citation: Biophys J. 2003 Jun;84(6):3992-4010
references.pmid: 12770904
references.citation: Nature. 1998 Jul 9;394(6689):192-5
references.pmid: 9671304
references.citation: Neuron. 1997 Nov;19(5):1087-94
references.pmid: 9390521
references.citation: Nature. 2003 Aug 21;424(6951):943-7
references.pmid: 12931189
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):14950-7
references.pmid: 20668236
references.citation: Nat Neurosci. 2011 Jun 12;14(7):833-9
references.pmid: 21666673
references.citation: Science. 2004 Apr 9;304(5668):289-92
references.pmid: 15016962
references.citation: Methods Enzymol. 2000;327:472-500
references.pmid: 11045004
references.citation: Science. 2004 Nov 5;306(5698):1042-6
references.pmid: 15528447
references.citation: Dev Cell. 2003 Mar;4(3):295-305
references.pmid: 12636912
references.citation: Science. 2002 May 3;296(5569):913-6
references.pmid: 11988576
references.citation: J Biol Chem. 2001 Aug 3;276(31):29188-94
references.pmid: 11387331
references.citation: Nat Biotechnol. 2004 Apr;22(4):445-9
references.pmid: 14990965
references.citation: J Neurosci. 1997 Aug 1;17(15):5858-67
references.pmid: 9221783
references.citation: Neuropharmacology. 2003 Nov;45(6):857-62
references.pmid: 14529723
references.citation: Nature. 1998 Sep 24;395(6700):347-53
references.pmid: 9759724
references.citation: Nat Neurosci. 2006 Aug;9(8):1019-27
references.pmid: 16845386
references.citation: Biochemistry. 2007 Sep 18;46(37):10685-93
references.pmid: 17718519
references.citation: J Biol Chem. 2009 Nov 13;284(46):32158-66
references.pmid: 19759010
references.citation: Cell. 2006 Nov 17;127(4):831-46
references.pmid: 17110340
references.citation: Science. 2004 Apr 16;304(5669):435-8
references.pmid: 15044754
references.citation: Microsc Microanal. 2006 Jun;12(3):238-54
references.pmid: 17481360
references.citation: Neuron. 1999 Dec;24(4):1017-28
references.pmid: 10624963
references.citation: Nature. 2012 Oct 11;490(7419):201-7
references.pmid: 23060190
references.citation: Cell. 1998 Mar 20;92(6):759-72
references.pmid: 9529252
references.citation: Methods. 2004 Aug;33(4):287-94
references.pmid: 15183177
references.citation: Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14658-63
references.pmid: 19706552
references.citation: J Biol Chem. 2004 Dec 31;279(53):55924-36
references.pmid: 15489225
references.citation: EMBO J. 1996 Dec 16;15(24):6951-9
references.pmid: 9003771
references.citation: Neuron. 2001 Apr;30(1):161-70
references.pmid: 11343652
references.citation: Q Rev Biophys. 2005 Feb;38(1):1-47
references.pmid: 16336742
references.citation: Science. 1997 Apr 11;276(5310):259-63
references.pmid: 9092476
references.citation: Nat Rev Mol Cell Biol. 2001 Feb;2(2):98-106
references.pmid: 11252968
references.citation: EMBO J. 2006 Mar 8;25(5):955-66
references.pmid: 16498411