In vitro system capable of differentiating fast Ca ²⁺ -triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release

Abstract

Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca 2þ triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca 2þ injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca 2þ injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca 2þ sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca 2þ triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.fast content mixing | single-vesicle fusion assay | membrane fusion | lipid mixing N euronal communication is made possible by the release of neurotransmitters, which in turn depends on the fusion of neurotransmitter-containing vesicles with the active zone in axonal terminals. Synaptic vesicle fusion is triggered by an influx of Ca 2þ ions into the neuron upon depolarization. Neurotransmitter release is quantized (1); that is, it involves a few to tens of individual synaptic fusion events. The process of individual synaptic vesicle fusion is in turn controlled by a set of relatively few proteins, such as the SNARE proteins (2-5), the Ca 2þ sensor for fast synchronous release synaptotagmin 1 (6-8), and the modulator complexin (9-11). Thus, neurotransmitter release is a macroscopic biological phenomenon that is ultimately controlled by a few individual molecules. The understanding of the underlying molecular mechanisms thus requires methods that are inherently capable of observing single vesicles and single molecules (12,13).Ideally, observations of single vesicles and single molecules would be performed in live neurons. Although progress for such studies has been made (14), they currently only provide limited information because the necessary genetic manipulations or labeling techniques may not provide the spatial and time resolution required for studying the dynamics of neurotransmitt...

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

“…To study the molecular mechanisms of Cpx, several reconstituted systems have been developed (19,(39)(40)(41)(42). As we reported previously, the C-terminal domain is important for suppression of Ca 2+ -independent fusion in a single-vesicle content mixing assay with reconstituted full-length neuronal SNAREs, and synaptotagmin-1 (19,43,44). Our fusion assay (Fig.…”

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking

“…Furthermore, a bulk lipid-mixing assay cannot distinguish between effects caused by differences in docking, hemifusion, or complete fusion. Our single-vesicle alternating-laser excitation (ALEX) lipid-mixing method (discussed below) can distinguish between docking and hemifusion/fusion (38), although it still cannot distinguish between hemifusion and complete fusion (35)(36)(37).…”

“…Using the purified large α-Syn oligomers, we first tested whether large α-Syn oligomers inhibited neuronal SNARE-mediated lipid mixing (32,33) with an in vitro lipid-mixing assay using proteoliposomes (34). Lipid mixing in such a bulk assay may result from hemifusion as well as fusion (35)(36)(37), but in any case requires trans-SNARE complex formation. Furthermore, a bulk lipid-mixing assay cannot distinguish between effects caused by differences in docking, hemifusion, or complete fusion.…”

Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes

“…This form of syntaxin-1a can be assembled either with a soluble form of SNAP-25 in DPC or with a lipidated form of SNAP-25 in lipid bilayers. Although SNAP-25 is multipally palmitoylated in eukaryotic cells (16), this posttranslational modification has previously not usually been included in attempts to reconstitute SNARE-mediated fusion in vitro (12). The exact number of palmitates that are attached to each SNAP-25 may be less than four, and may vary depending on physiological conditions; our procedure quantitatively attaches four dodecyl chains, which, however, are shorter by four carbons than the native 16-carbon palmitates.…”

“…Traditionally, syntaxin-1a is purified in bOG (octyl-b-D-glucoside) (2,(11)(12)(13)(14) or sodium cholate (3,4,9), and allowed to assemble into an acceptor complex with soluble SNAP-25a during coexpression in Escherichia coli or postexpression in cholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or bOG. Depending on input stoichiometries and other conditions, this results, to different degrees, in the formation of a 2:1 syntaxin-1a:SNAP-25a complex that does not readily bind to synaptobrevin-2 (4,5).…”