Three-Dimensional Structure of the Complexin/SNARE Complex

During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca2+-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel α-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca2+-evoked neurotransmitter release

Synaptic Vesicles Position Complexin to Block Spontaneous Fusion

SummarySynapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin can both promote and inhibit fusion through interactions between its α-helical domain and the SNARE complex. In addition, complexin’s C-terminal half is required for the inhibition of spontaneous fusion in worm, fly, and mouse, although the molecular mechanism remains unexplained. We show here that complexin’s C-terminal domain binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. We propose that the SV pool serves as a platform to sequester and position complexin where it can intercept the rapidly assembling SNAREs and control the rate of spontaneous fusion

Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001

Morphologies of synaptic protein membrane fusion interfaces

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release

Complexin: Does it deserve its name?

Knockout and other perturbations of complexins have provided important insights and elicited controversies about their role in neurotransmitter release. New work by Yang et al. in this issue of Neuron adds important detail and complexity to existing concepts—particularly on the nature of a Ca2+-dependent complexin-synaptotagmin switch for the triggering of exocytosis. But it also provokes thoughts about alternative interpretations, which might result in a simpler model of complexin function

Synaptotagmin 1 oligomers clamp and regulate different modes of neurotransmitter release

Release of neurotransmitters relies on submillisecond coupling of synaptic vesicle fusion to the triggering signal: AP-evoked presynaptic Ca2+ influx. The key player that controls exocytosis of the synaptic vesicle is the Ca2+ sensor synaptotagmin 1 (Syt1). While the Ca2+ activation of Syt1 has been extensively characterized, how Syt1 reversibly clamps vesicular fusion remains enigmatic. Here, using a targeted mutation combined with fluorescence imaging and electrophysiology, we show that the structural feature of Syt1 to self-oligomerize provides the molecular basis for clamping of spontaneous and asynchronous release but is not required for triggering of synchronous release. Our findings propose a mechanistic model that explains how Syt1 oligomers regulate different modes of transmitter release in neuronal synapses

Investigation of SNARE mediated membrane fusion and its regulation by optimized single molecule method

Neurotransmitter release going through synaptic vesicle cycle is one key step how signal is transported in our brains. The mechanism on molecular level has been under development and debated for decades. Many milestones have been made including, the identification of SNARE as core assembly machinery, the clarification of synaptotagmin as calcium sensor, the recognition of NSF and SNAP as disassembly apparatus, the determination of complexin and SM protein as regulatory protein. However, the sequence of their involvement in synaptic vesicle cycle, the relationship between the structure and psychological function, microscale fusion mechanism and are under further investigation. This puzzle is completing with effort from international groups and our group. Complexin as one regulatory protein, has been found owning both inhibitory and facilitatory function. This dual function adds more complication to identify the role of complexin in membrane fusion. Research groups get either inhibitory or facilitatory function based on the experimental condition, which is contradictory. Also, single molecule FRET mixing assay has been adopted widely as one method to isolate membrane fusion system in vitro to give more detailed information on step-by-step mechanism. One major method in single molecule FRET, content mixing, faces obstacles by slow time scale and low fusion percentage. By looking deeper into complexin function, we optimize content mixing and for the first time we observed complexin showing both inhibitory and facilitatory role in a concentration dependent manner

Subcloning and Expression of Complexin Isoforms Involved in Mast Cell Degranulation

Mast cells play an important role in the immune system by releasing chemicals such as chemokines and cytokines once they are stimulated. These products are released after stimulation by a process called mast cell degranulation. Mast cell degranulation is accomplished when vesicles containing the chemicals inside the mast cell fuse with the mast cell membrane via SNARE-mediated (Soluble NSF Attachment Protein Receptors) membrane fusion. This family of proteins consists of syntaxin, SNAP 25-like protein, and synaptobrevin/VAMP (Vesicle Associated Membrane Protein)(2). Comlexin isoforms (complexin 1,2,3,and 4) have been known to regulate this system in a fashion that is still unclear. In order to study the mechanism in which these complexins regulate SNARE-mediated membrane fusion, each isoform was cloned and ligated to the pTYB12 vector to be expressed in E. coli. An induction process using IPTG was used in order to induce production of each isoform via the T7 promoter. In this experiment, we were able to clone all of the complexin isoforms, but only complexin 1 and 3 were successfully expressed

A genetic analysis of complexin function in neurotransmitter release and synaptic plasticity

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references.Information transfer at neuronal synapses requires rapid fusion of docked synaptic vesicles in response to calcium influx during action potentials. The molecular nature of the fusion clamp machinery that prevents exocytosis of synaptic vesicles in the absence of a calcium signal is still unclear. Here we show that complexin, a small alpha-helical protein that binds fully assembled SNARE complexes, functions as the synaptic vesicle fusion clamp in vivo. Drosophila has a single complexin homolog that is abundantly expressed in presynaptic nerve terminals. Animals lacking complexin die throughout development, with adult escapers showing severe locomotion defects and a loss of visual function. Electrophysiological analysis at neuromuscular junctions in complexin null mutants reveals a dramatic increase in spontaneous synaptic vesicle fusion that is independent of nerve stimulation or extracellular calcium. High frequency stimulation at high calcium concentrations shows that the readily releasable pool in complexin mutants is severely depleted. Thus, complexin is required for maintenance of the readily releasable pool of vesicles at the synapse, and without it vesicles exocytose directly after priming. These data indicate that complexin interacts with assembled SNARE complexes to prevent premature vesicle fusion in the absence of calcium entry. In addition, a preliminary analysis of synaptotagmin 1; complexin double mutants reveals that the elevated mini frequency in complexin single mutants is dependent on synaptotagmin 1. This finding suggests that the dominant function of complexin at the synapse is to prevent synaptotagmin 1 from triggering fusion in the absence of calcium. Further analysis of synaptotagmin 1; complexin double mutants may reveal new aspects of the mechanism of the calcium-regulated vesicle fusion reaction. Minis have long been thought to represent background noise at the synapse, but there is now growing evidence that mini frequency is important in synaptic maintenance and plasticity. Complexin mutants display a substantial synaptic overgrowth phenotype. We hypothesized that the enhanced mini frequency in complexin mutants drives synaptic overgrowth and that complexin is phosphorylated by PKA to regulate mini frequency at Drosophila synapses in an activity-dependent retrograde signaling pathway that mediates a large increase in mini frequency and a concomitant induction of synaptic growth. Like complexin mutants, a syntaxin mutant with elevated mini frequency also displays enhanced synaptic growth, providing further evidence that an increase in mini frequency drives synaptic plasticity. S126 in complexin is phosphorylated by PKA in vitro. Future results may reveal that S126 is phosphorylated by PKA in vivo to regulate mini frequency in an activity-dependent manner. These results have the potential to reveal a new role for minis in local synaptic plasticity in response to neuronal activity.by Sarah Huntwork-Rodriguez.Ph.D

A single molecular study of the regulation of SNARE-mediated membrane fusion

The presynaptic membrane fusion is mediated by a protein set called SNARE (Soluble NSF Attachment protein REceptor) proteins. SNARE proteins form a ternary SNARE-complex that comprises minimal machinery for membrane fusion; the complex consists of three SNARE proteins: Syntaxin 1, SNAP-25 and Vamp 2, also called Synaptobrevin 2. The SNARE complex is a four-helix coiled coil with four SNARE motifs; two come from SNARE-25 and one each from Syntaxin 1 and Vamp 2. It is believed that a regulatory protein Complexin binds tightly to the SNARE complex and stabilizes the complex, preventing it from driving toward fusion. However, the detailed mechanism of fusion clamping is still unclear. In our work, we constructed a single-molecule lipid-mixing assay on a supported lipid bilayer to investigate the role of Complexin 1, one of the important regulatory proteins. Moreover, we found that Synaptotagmin 1, a calcium sensor for Ca2+-triggered fusion, plays a role along with Complexin in fusion clamping. Furthermore, the supported lipid bilayer was also incorporated into a photo-bleaching assay to investigate the role of various lipids on Syntaxin 1 clustering