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Single-Molecule Studies of the Neuronal SNARE Fusion Machinery

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Single-Molecule Studies of the Neuronal SNARE Fusion Machinery ANRV378-BI78-31 ARI 5 May 2009 14:58 Single-Molecule Studies of the Neuronal SNARE Fusion Machinery Axel T. Brunger,1 Keith Weninger,2 Mark Bowen,3 and Steven Chu4 1The Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science, Stanford University, California 94305; email: [email protected] 2Department of Physics, North Carolina State University, Raleigh, North Carolina 27695-8202; email: [email protected] 3Department of Physiology and Biophysics, Stony Brook University Medical Center, Stony Brook, New York, 11794-8661; email: [email protected] 4Department of Energy, Washington, District of Columbia 20585; email: [email protected] Annu. Rev. Biochem. 2009. 78:903–28 Key Words The Annual Review of Biochemistry is online at FRET, membrane fusion, neurotransmission, synaptic vesicle biochem.annualreviews.org This article’s doi: Abstract 10.1146/annurev.biochem.77.070306.103621 SNAREs are essential components of the machinery for Ca2+-triggered by 72.255.12.226 on 06/04/09. For personal use only. Copyright c 2009 by Annual Reviews. fusion of synaptic vesicles with the plasma membrane, resulting in neu- All rights reserved rotransmitter release into the synaptic cleft. Although much is known 0066-4154/09/0707-0903$20.00 about their biophysical and structural properties and their interactions with accessory proteins such as the Ca2+ sensor synaptotagmin, their Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org precise role in membrane fusion remains an enigma. Ensemble stud- ies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecu- lar insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applica- tions of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological mem- branes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments. 903 ANRV378-BI78-31 ARI 5 May 2009 14:58 ten vesicles are stably docked at the active zone Contents awaiting an action potential (1–4). Exocytosis is triggered within approximately 0.2 ms of INTRODUCTION .................. 904 the Ca2+ influx that follows arrival of an action SINGLE-MOLECULE potential (5, 6). Although extremely rapid, the APPROACHES TO STUDY neurotransmitter release probability has a sig- MEMBRANE PROTEIN nificant heterogeneity in single synaptic release INTERACTIONS sites in hippocampal neurons (7). At most, one AND FUSION .................... 904 synaptic vesicle per synapse undergoes exocy- SNARES ............................. 907 tosis upon depolarization in the central nervous SINGLE-MOLECULE STUDIES system (8). Thus, regulation of neurotransmit- OF THE SYNTAXIN·SNAP-25 ter release occurs at the level of synaptic vesicle BINARY COMPLEX .............. 908 release probability. There is also a background SINGLE-MOLECULE STUDIES rate of fusion of about one per minute per OF THE SYNTAXIN- synapse in the absence of action potentials. SYNAPTOBREVIN BINARY Synaptic vesicle fusion involves a highly con- COMPLEX ....................... 910 served family of proteins termed SNAREs (sol- SINGLE-MOLECULE STUDIES uble N-ethyl maleimide sensitive factor attach- OF THE TERNARY SNARE ment protein receptors) (9–11). SNAREs are COMPLEX: PARALLEL OR directly linked to Ca2+ triggering of exocyto- ANTIPARALLEL? ................ 911 sis in conjunction with a Ca2+ sensor, such as EVIDENCE FOR A TRANS STATE synaptotagmin (12–14). Genetic rescue experi- OF THE TERNARY SNARE ments with mutants of synaptotagmin have now COMPLEX? ...................... 912 firmly established that synaptotagmin is the MODELS OF MEMBRANE Ca2+ sensor for the synchronous component FUSION .......................... 912 of synaptic exocytosis (15), but the mechanism RECONSTITUTION OF of action of the synaptotagmin·SNARE·mem- SNARE-MEDIATED brane fusion machinery remains a matter of MEMBRANE FUSION ........... 913 intense research (14, 16–23). Numerous other NUMBER OF SNARE COMPLEXES auxiliary proteins have been found to be essen- INVOLVED IN SYNAPTIC tial for Ca2+-dependent neurotransmitter re- by 72.255.12.226 on 06/04/09. For personal use only. VESICLE FUSION ............... 916 lease, such as complexin, Munc18, and Munc13. SYNAPTOTAGMIN ................. 917 Thus, SNAREs form only one part, albeit the COMPLEXIN ....................... 918 central part, of the complex system of synap- MUNC18 ............................ 919 tic neurotransmission. In this review, we fo- MUNC13 ............................ 920 Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org cus on single-molecule studies of the neuronal SUMMARY AND FUTURE SNAREs, i.e., syntaxin, synaptobrevin, SNAP- ISSUES ........................... 920 25, some of their binding partners and com- plexes, and their role in synaptic vesicle docking and fusion. INTRODUCTION Synaptic neurotransmitter release involves the Ca2+-triggered fusion of synaptic vesicles with SINGLE-MOLECULE SNARE: soluble the plasma membrane in the presynaptic ter- APPROACHES TO STUDY N-ethyl maleimide minal, releasing the neurotransmitter into the MEMBRANE PROTEIN sensitive factor synaptic cleft. Synaptic vesicles are recruited to INTERACTIONS AND FUSION attachment protein receptors the active zone in the presynaptic membrane One of the key advantages of single-molecule but do not readily fuse. Instead, an average of experiments is that they allow one to study the 904 Brunger et al. ANRV378-BI78-31 ARI 5 May 2009 14:58 behavior of a single or countable number of Quantitative interpretation of FRET effi- molecules, or molecular complexes, and their ciencies in terms of absolute distances is not action on an individual molecule or particle straightforward. It requires careful measure- smFRET: single- such as a synaptic vesicle. This is especially use- ment of fluorophore dynamics to correct the molecule fluorescence ful for systems that show significant variability anisotropy and quantum yield terms in the resonance energy of individual molecular “trajectories” for bio- Forster¨ radius (29b). Interpreting quantitative transfer logical or chemical processes, such as protein smFRET values in terms of macromolecu- FRET: fluorescence folding, protein synthesis, or protein-assisted lar structure is a developing field (29c–29f). resonance energy membrane fusion. The ability to observe in- An ideal fluorophore attachment site has free, transfer dividual events therefore removes the require- isotropic rotation so there is a high degree of ment of synchronizing events in ensemble ex- uncertainty when correlating fluorophore sep- periments that monitor the average behavior of aration to protein structure. Recently, molec- many individual molecules or particles (often on ular dynamics simulations of protein-attached the order of Avogadro’s number), and it allows dyes show some promise in obtaining a con- one to perform statistical analysis of a popula- version function between FRET efficiency and tion of individual trajectories that would not be absolute FRET distance (29g). possible in bulk owing to ensemble averaging. The sample is illuminated with laser light There are several recent reviews that discuss using total internal reflection in order to re- applications of single-molecule techniques to strict illumination to the region near the surface biological systems (24–28). (the electric field intensity is restricted to a sur- Here, we briefly discuss the principles face layer with decay length ∼100 nm) and thus and focus primarily on single-molecule flu- has reduced background fluorescence. Two or orescence approaches such as the particu- more lasers emitting at different wavelengths lar experiment shown in Figure 1a. Protein- are used to study colocalization and FRET protein interactions are monitored between the between the fluorescent dyes. A similar setup membrane-bound syntaxin·SNAP-25 complex was used to study single-vesicle interactions and synaptobrevin, which is introduced above between protein-containing liposomes and de- the supported bilayer (29). Fluorescent labels posited bilayers while simultaneously monitor- are covalently attached at different positions in ing content mixing (30). the individual proteins. These dyes are planar An alternative to the supported bilayer ge- aromatic ring structures that range in size from ometry is to tether “acceptor” liposomes to an by 72.255.12.226 on 06/04/09. For personal use only. around 0.5 to 1.2 kDa and may be charged as inert surface and then to monitor the inter- well. Because any covalent modification of a action of these liposomes with “donor” lipo- macromolecule with such a dye can affect the somes in solution. This geometry has been used energtic and kinetic properties of the system, for single-vesicle studies (31). Both geometries Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org it is important to study this effect. In our ex- have advantages and disadvantages. The sup- periments, we therefore tested for the potential ported bilayers mimic the geometry of synap- influence of the labels by repeating them with tic vesicles
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