Perspecve

Syntaxin Clustering and Optogenetic Control for Synaptic Membrane Fusion

Miaoling Li 1,†, Teak-Jung Oh 2,†, Huaxun Fan 2,†, Jiajie Diao 3 and Kai Zhang 2,

1 - Key Laboratory of Medical Electrophysiology of Ministry of Education and Medical Electrophysiological Key Laboratory of Sichuan Province, Institute of Cardiovascular Research, Southwest Medical University, Luzhou, Sichuan 646000, China 2 - Department of Biochemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801, USA 3 - Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA

Correspondence to Jiajie Diao and Kai Zhang:Correspondence to: J. Diao, Vontz Center for Molecular Studies, 3125 Eden Avenue, ML 0521, Cincinnati, OH 45267, USA.Correspondence to: K. Zhang, 600 South Mathews Avenue, 314 B Roger Adams Laboratory, Urbana, IL 61801, USA. [email protected], [email protected] https://doi.org/10.1016/j.jmb.2020.07.005

Abstract

Membrane fusion during synaptic transmission mediates the trafficking of chemical signals and neuronal communication. The fast kinetics of membrane fusion on the order of millisecond is precisely regulated by the assembly of SNAREs and accessory . It is believed that the formation of the SNARE complex is a key step during membrane fusion. Little is known, however, about the molecular machinery that mediates the formation of a large pre-fusion complex, including multiple SNAREs and accessory proteins. , a transmembrane on the plasma membrane, has been observed to undergo oligomerization to form clusters. Whether this clustering plays a critical role in membrane fusion is poorly understood in live cells. Optogenetics is an emerging biotechnology armed with the capacity to precisely modulate protein–protein interaction in time and space. Here, we propose an experimental scheme that combines optogenetics with single-vesicle membrane fusion, aiming to gain a better understanding of the molecular mechanism by which the syntaxin cluster regulates membrane fusion. We envision that newly developed optogenetic tools could facilitate the mechanistic understanding of synaptic transmission in live cells and animals. © 2020 Elsevier Ltd. All rights reserved.

Introduction 2), whereas the t-SNARE proteins include syntaxin-1A and SNAP-25 (synaptosomal- mediates neuronal associated protein, 25 kDa) [6]. communication by regulating synaptic transmission In vivo studies demonstrated that the formation of through membrane fusion, protein trafficking [1], and SNARE complex during fast exocytosis completes release [2]. Defective synaptic within a millisecond [7]. To intuitively understand the vesicle exocytosis could lead to neurodegenerative molecular mechanisms of membrane fusion, diseases [3]. Synaptic vesicle exocytosis is precisely researchers confirmed that SNARE complex and regulated by the coordination of calcium and a additional accessory proteins play a synergistic role group of proteins called SNAREs (soluble N-ethyl- in the regulation of the fusion pathway in vivo [8]. maleimide sensitive factor attachment protein re- Although recently developed single- ceptors) [4,5]. SNARE proteins are the core assays [9–24] provides improved ways to track component of the exocytosis machinery and reside synaptic membrane fusion at the single-vesicle level either in the presynaptic vesicles (v-SNARE) or the [25,26], it remains challenging to probe the exact targeted plasma membrane SNARE proteins (t- molecular mechanism underlying the release of SNARE). The v-SNARE protein consists of VAMP2 neurotransmitters from synaptic vesicles [27]. A (vesicle-associated 2, also called counting list of accessory proteins includes the

0022-2836/© 2020 Elsevier Ltd. All rights reserved. Journal of Molecular Biology (2020) 432, 4773-4782 4774 Optical control of syntaxin for membrane fusion assembly proteins SM (Sec1/Munc18-like proteins), the disassembly factors NSF (N-ethylmaleimide sensitive factor), (a clamp activator), (a Ca2+ sensor) [4,28]. Together with SNARE proteins, these accessory proteins regulate calcium-triggered fast membrane fusion and exocytosis. What remains unclear is the molecular machinery to initiate the assembly of the large pre-fusion protein complex (Figure 1). The emerging idea is that the configuration and distribution of syntaxin, one of the t-SNARE proteins that localize on the target membrane, could mediate the assembly of a pre-fusion complex [29]. As discussed in detail below, syntaxin could oligomerize and form Figure 1. Schematic of protein cluster-induced forma- nanodomains in the plasma membrane [30], although tion of the pre-fusion superstructure for effective fusion. direct evidence has yet been available to prove that SNAREs are classified according to their compartment syntaxin clustering initiates calcium-triggered, fast distribution as v-SNAREs (in transport vesicles), including membrane fusion either in vitro or in vivo.Inthis synaptobrevin 2 (also called vesicle-associated mem- perspective, we briefly review the formation, dynamics, brane protein 2, VAMP2), and t-SNAREs (in the target function and influencing factors of syntaxin cluster membranes) that composed of SNAP-25 and syntaxin-1A. These three SNARE components can form a ternary during vesicle fusion, and proposed to use photoacti- “ vatable protein-based optogenetics to kinetically complex in a 1:1:1 ratio via the association of their SNARE motifs,” which assemble into a four-helix bundle. The manipulate clustering of syntaxin during vesicle fusion. accessory proteins, including complexin (a clamp activa- It is our expectation that optogenetics could provide tor), synaptotagmin (a Ca2+ sensor), Munc18, and constructive insights into the molecular mechanism Munc13, form a basic interaction module with individual underlying membrane and protein trafficking during SNARE complexes. synaptic transmission.

clusters may have distinct functions depending on Dynamic organization of syntaxin on the the size and spatial distribution. For example, STED plasma membrane super-resolution imaging on Drosophila neuro- muscular junctions showed that larger syntaxin Syntaxin is a membrane-anchored protein that plays clusters are more abundant and stable at active a central role in membrane fusion. It contains a zones. Larger clusters could possess a higher cytosolic domain and a type II single-transmembrane capability for vesicle docking, whereas the smaller C-terminal domain. The N-terminal Habc domain of clusters may serve as a reservoir to maintain the syntaxin is believed to interact with Munc18, while the balance of syntaxin distribution between large and SNARE domain can form a four-helix-bundle with small clusters [39]. These results suggest that the SNAP-25 and VAMP2 for membrane fusion (Figure 1). homologous oligomerization of syntaxin proteins Results from super-resolution imaging indicate that plays an essential role in vesicle fusion [40]. syntaxin could undergo a dynamic equilibrium between Super-resolution imaging further unveiled the size clustering and freely diffusive states in the plasma and density of the syntaxin cluster in mammalian membrane [30]. Enhanced clustering causes reduced cells. For example, STED fluorescence microscopy mobility of syntaxin [31,32]. Overexpression of syntaxin resolved that the density of endogenous syntaxin increases the number rather than the size of nanoclus- clusters in PC12 cells is 19.6 clusters/μm2 [30], or ters [33]. Syntaxin could oligomerize via the transmem- 9000 syntaxin clusters per cell. Quantitative immu- brane domain [34] or the cytoplasmic domain [35]. The nostaining estimated the total number of syntaxin per balance between the assembly and disassembly of cell is 830,000, indicating that each cluster contains syntaxin clusters could play a crucial role during about 90 syntaxin molecules. Considering the synaptic transmission [36]. average diameter of the clusters is about 50– Syntaxin clustering could help vesicle tethering 60 nm (resolved by STED), it is expected that and docking prior to membrane fusion. Indeed, it has are densely packed in the cluster. This been shown that syntaxin, together with the acces- estimate remains consistent with another study, sory protein Munc18, recruits secretory vesicles to which demonstrated that the density of syntaxin the docking site on the plasma membrane [37]. It is ranged from 540 molecules/μm2 [121]to2000 likely that the Habc-domain of syntaxin mediates molecules/μm2 in PC12 cells [41]. both cluster formation and vesicle docking because Syntaxin clusters coordinate with secretory vesi- both processes slowed down upon overexpression cles by presenting the docking sites for secretory of the Habc domain [38]. In addition, syntaxin granules in the plasma membrane. Moreover, Optical control of syntaxin for membrane fusion 4775 syntaxin clusters are able to assemble reversibly at additional opening is induced upon Ca2+ entry. It the sites of the granule. However, docking of means that one of the transitions is directly induced by granules on syntaxin could be intermittent because depolarization, and an additional transition is involved syntaxin clusters were also found at sites where in the juxtamembrane region of syntaxin [50]. there are no granules [37]. Furthermore, syntaxin clustering at granules requires Munc18-syntaxin The SNARE motif is essential for the formation binding [42]. Coordination between granule and of syntaxin clusters in the plasma membrane syntaxin clusters could facilitate exocytosis. Besides lipid-syntaxin interaction, inter-syntaxin pro- Lipid factors mediate syntaxin clustering on the tein interactions also contribute to syntaxin cluster plasma membrane formation. Interaction between the SNARE motifs could facilitate this process. The SNARE motif, a homologous Membrane–protein interactions have been shown sequence of 60–70 amino acids where the SNARE to be involved in syntaxin clustering, such as complex is formed, anchors to the plasma membrane cholesterol and phosphoinositides [43]. Cholesterol by a C-terminal transmembrane region (TMR). The could enhance cluster stability on the plasma SNARE domain is next to a large N terminus containing membrane via modulating the hydrophobic mem- the Habc domain via a linker region (Figure 2). The brane thickness in a dose-dependent manner. SNARE motifs of syntaxin induce self-oligomerization Indeed, cholesterol depletion leads to the dispersion at concentrations above 2 μM. Furthermore, the of syntaxin domains and the reduction of syntaxin deletion of the SNARE motif abolished syntaxin clusters in PC12 and pancreatic β cells [44]. It is also homo-oligomerization [35]. Another crucial factor for suggested that cholesterol could contribute to syntaxin clustering is the forces acting on the TMR. SNARE nanodomain formation [45]. Although the TMR of syntaxin could form the clusters In addition to cholesterol, phosphatidylinositol phos- by self-organization [35], cytoplasmic interactions phates (PIPs) are also involved in syntaxin clustering. between syntaxins could recruit free syntaxin into a Phosphatidylinositol-4,5-bisphosphate (PIP2), which cluster and increase the dwelling time of syntaxin in a commonly colocalizes with vesicle docking sites in cluster. Recent work suggested that the domains of the plasma membrane, could form nanodomains by cytoplasmic protein interactions mediate a tightly themselves with about 73 nm in diameter [46]. STED packed state, and the deletions of the cytoplasmic fluorescence microscopy revealed that syntaxin clus- region produced a state of looser packing [51]. tering requires a high concentration of PIP2, and decrease of PIP2 by the phosphatase synaptojanin-1 induced reduction of syntaxin clustering. It is also found Current progress for the study of that syntaxin enriches in membrane domains of formation and function of syntaxin phosphatidylinositol-3,4,5-bisphosphate (PIP3) [47]. The reduction of PIP3 disperses syntaxin clusters, clusters whereas adding PIP3 to giant unilamellar vesicles How does syntaxin mobility affect SNARE complex containing syntaxin 1A results in profound syntaxin formation? The decrease in syntaxin mobility could clustering in the syntaxin-juxtamembrane domain [48]. promote its recruitment into nonfunctional SNARE However, PIP2 only accelerates granule priming but complexes and therefore limits vesicle fusion [52]. was not required for secretory granule docking or The function of syntaxin depends on their localization, syntaxin clustering at the release site [49]. The lipid distribution, and recruitment to specific sites in the environment modulates the energetic requirements for fusion. Syntaxin clustering is mediated by electrostatic interactions with PIP2. Syntaxin clustering needs weak homophilic interactions in the plasma membrane. Results from STED super-resolution imaging demonstrated that electrostatic protein–lipid interac- tions are involved in the formation of syntaxin sequestering. Thus, electrostatic protein–lipid interac- tions represent a novel mechanism for the formation of protein nanodomains in the membrane [47]. Furthermore, Ca2+ could modulate the size of the syntaxin cluster by connecting it with PIP2 to form larger-scale domains, as revealed by STED imaging, Figure 2. Syntaxin-1A can form clusters with approxi- Förster (fluorescence) resonance energy transfer mately 70 copies of monomers. The cytoplasmic region of (FRET), and atomic force microscopy [34]. FRET syntaxin contains Habc and SNARE domains. PIP2 analysis proved that partially opening of SNARE enriches with syntaxin cluster and can facilitate the complex occurs in the absence of Ca2+ entry, and an membrane fusion. 4776 Optical control of syntaxin for membrane fusion plasma membrane. Super-resolution imaging and the local concentration of syntaxin (if syntaxin cluster computational models demonstrated that free and localizes within the fusion site). clustered syntaxin molecules keep a mobile fraction Meanwhile, the formation mechanism of syntaxin in the plasma membrane, in which precisely shifts clusters is unknown. Syntaxin clustering could be between free and clustered syntaxin molecules con- regulated by its SNARE domain [35], as well as lipid trolled the magnitude of the syntaxin interaction energy molecules such as PIP2 and cholesterol [43,45,47]. of Ea values [39]. Clusters of syntaxin in active-zone are Thus, it is crucial to determine the spatial localization more stable, abundant, and larger than that outside of of syntaxin cluster within the membrane prior to active zones, which provide regions to facilitate docking membrane fusion. To achieve this goal, new tools and fusion, whereas the smaller clusters outside the that allow for active modulation of syntaxin clustering may serve as flexible reservoir or sites of in live cells are on-demand [55]. spontaneous transmitter release. The cluster- ing of syntaxin might also facilitate the efficient recycling of cis-SNARE complexes at the fusion sites. The Optogenetics controls protein activity computational model could predict the dynamics of and with light syntaxin clusters for their dissociation and aggregation rate. The reduction of the dissociation rate causes a In the following sections, we propose a scheme larger cluster size. Interactional energy increasing (a that utilizes the emerging non-neuronal optogenetics fraction of 1 kBT) will lead to stronger clustering. Slight to study syntaxin clustering in live cells. Optoge- decreasing the energy of syntaxin assembly results in netics uses light to control protein activities and losing large clusters [39]. empowers new ways to control protein conformation The evidence mentioned above suggests that in live cells. These advantages initially enabled the syntaxin manifests as either diffusive monomers or interrogation of neuronal firing by light-sensitive organized clusters, which is mediated by lipid-protein synthetic ion channels [56] and channel rhodopsin and protein–protein interactions. The size, composi- [57,58], which led to the coining of the term tion, and dynamics of syntaxin clusters are expected to “optogenetics” [59]. Recently, optogenetics has play crucial roles in fast membrane fusion. Not extended its application to control protein–protein surprisingly, conformation and mobility of syntaxin interactions in live cells [60–66]. To contrast from could be serves as a pharmaceutical target for the optogenetic techniques that control the activation intervention of neurological diseases. For instance, of excitable cells, this extended modality has general anesthetics, such as propofol, could affect often been referred to as non-neuronal optogenetics presynaptic release mechanisms by restricting syn- [67]. Core components of non-neuronal optoge- taxin mobility, as revealed by single-molecule imaging netics are a group of photoactivatable proteins that microscopy [31]. Authors proposed that at a clinically undergo light-mediated conformational change, relevant concentration of propofol and etomidate, which modulates their intra- or inter-molecular syntaxin mobility is reduced. In contrast, non- interaction [58,61,62,64–66,68,69]. Multiple lines anesthetic analogs produce the opposite effect and of research, including ours, have applied non- increase syntaxin mobility [31]. However, a most recent neuronal optogenetics in a variety of biological study showed that general anesthesia significantly systems such as yeast [61,69–71], mammalian changed the configuration of lipid rafts on the plasma cells [62–66,68,72–88], primary neurons [89–91], membrane, which mainly influences channels [53]. fruit fly [92,93], zebrafish [94–96], frog [97], and Meanwhile, another study also demonstrated that mouse [89,98–100]. propofol reduces synaptic transmission by inhibiting sodium and calcium channels at nerve terminals [54]. Highlight optogenetic dimerization and However, pharmacological approaches (e.g., using oligomerization propofol) lack the flexibility and spatiotemporal accuracy to study syntaxin clustering. An important group of optogenetic tools involves the inter-molecular dimerization and oligomerization. Cryptochrome 2 (CRY2) was initially shown to Unsolved problems undergo blue light-mediated heterodimerization with cryptochrome-Interacting-Basic-helix–loop–helix Syntaxin clusters modulate the motile and immo- (CIB1) [101]. It was then found that CRY2PHR (the tile form of syntaxin, but the physiological functions PHR domain of CRY2, amino acid 1–498) [64]also of these clusters remain unclear. Indeed, syntaxin homo-oligomerizes upon blue light illumination [77]. clustering could negatively regulate membrane The extent of oligomerization could be enhanced by the fusion by sequestering active monomeric syntaxin protein engineering of CRY2, which led to the (if syntaxin cluster localizes outside the fusion site). development of CRY2olig (CRY2PHR E490G) [78] On the other hand, the syntaxin cluster could and CRY2clust (CRY2 with 9 amino acid substitutions positively regulate membrane fusion by increasing at 499–507) [102]. The Vaucheria frigida aureochrome1 Optical control of syntaxin for membrane fusion 4777 protein with LOV domain (VfAu1-LOV, also called the incorporation of the iLID-micro heterodimeriza- AuLOV) and its unique sequence-specific basic tion system, 5-phosphatase was recruited to PM region/leucine zipper (bZIP) DNA binding domain was upon the pulse of blue light. For vesicle docking discovered about a decade ago [103]. Several studies control, the CRY2–CIBN system with 5-phosphatase characterized the photophysical and photochemical was equipped to selectively convert PIP2 to PIP1 at properties of AuLOV for light-induced dimerization the vesicle docking site in the PM for indirect control mechanism and transcriptional regulation [103–105]. of syntaxin cluster. Long-term PIP2 disruption Amongst those studies, the bZIP domain-intact AuLOV showed perturbation of intracellular calcium concen- variant (113ZL) contributed stabilization of AuLOV tration and signaling without syntaxin clustering protein in the monomeric form at the dark state and inhibition. In contrast, selective and transient PIP2 dimeric form upon blue light irradiation. Surprisingly, the reduction at the vesicle docking sites promoted bZIP truncated LOV domain variant (204LOV) showed significant vesicle undocking without affecting cyto- weaker but similar photophysical properties as 113ZL solic calcium concentration and signaling [113]. protein [105]. AuLOV has been engineered to control A recent development in optogenetics allows for receptor dimerization and has been used in vitro and in precise control of protein level and activity at the vivo [103–108]. Photophysical properties of these light- single-synapse level. As shown in a recent study, the activated oligomerizers and dimerizers are listed in level of AMPA receptors can be precisely modulated Table 1. close to postsynaptic density via the optical dimer- ization pair CRY2 and CIB1. Light-induced AMPA receptor recruitment to PSD increases the synaptic Utilization of optogenetic dimer and strength at the exposed synaptic sites [114]. This oligomers to modulate protein–protein work suggests that non-neuronal optogenetics could interactions access individual synapse and delineate synaptic transmission and plasticity at the subcellular level. The optogenetic tools mentioned above provides Another study demonstrated the power of temporal an opportunity to control membrane dynamics. regulation of optogenetics. Ephrin type-B receptor 2 Membrane tethering and fusion govern vital cellular (EphB2) is indispensable for normal brain develop- processes and communications such as endoplas- ment and function. The outcome of EphB2 activa- mic reticulum (ER)–Golgi protein and tion, including enhanced synaptic transmission and release via its vesicle and plasma gene expression, suggests that it is involved in membrane (PM) fusion. Several studies proved that memory formation and consolidation. To investigate optogenetics unsealed the dynamic membrane the roles of EphB2, Alapin and co-workers devel- fusion control and tethering control within organelle oped an optoEphB2 by fusing its cytoplasmic or between organelles. For instance, the light- domain with Cry2olig [115]. Blue light stimulation inducible tethering (LIT) system granted the control- induces EphB2 oligomerization. Applying this lable tethering between the ER and mitochondria technique in a transgenic mouse model with the (MT). The light-oxygen-voltage 2 (LOV2) domain auditory fear conditioning paradigm reveals that derived blue light activatable heterodimerization photoactivation of EphB2 during the training process protein system, iLID (SsrA–SspB), was used to leads to enhanced long-term memory as well as induce either local or global ER-MT oligomerization activation of cAMP/Ca2+-responsive element bind- in various cell types including primary neuron [112]. ing (CREB) protein. On the contrary, EphB2 activa- Vesicle docking, as we discussed previously, tion after training shows no such influence. requires delicate and exhaustive control. Through Moreover, light-induced activation of EphB2 is able

Table 1. Photophysical properties of photoactivatable protein oligomerization and dimerization under blue light stimulation

Optical activation Photoactivatable Proteins Asso. T1/2 (s) Disso. T1/2 (s) Fluo. ratio Kd dark (μM) Kd light (μM) Ref Oligomerization CRY2 – 90–360 0.3 ––[78,102,109] CRY2PHR 145 267 0.01 ––[77,102,110] CRY2olig 15–115 1386 0.4–0.9 ––[78,102,110] CRY2clust 5 230 0.51 ––[102] Homodimerization AuLOV-bZIP –– –131 0.012 [105] AuLOV –– –35 0.05 [105] Heterodimerization CRY2/CIBN b1 300 – 44[64,109] CRY2PHR/CIBN 3.7–186 164–380 –– – [109,111] iLID/nano 2–95 28–64 – 4.7 0.132 [109,111] iLID/micro 1.5–72 23–52 – 47 0.8 [109,111] LOVpep/ePDZb –– –72 12 [109] LOVpep+/ePDZb 25–39 20–54 – 150 18 [109] 4778 Optical control of syntaxin for membrane fusion

Figure 3. A strategy for light-induced oligomerization of SNARE proteins. SNARE proteins can be fused to photoactivatable protein. Light-triggered oligomerization of photoactivatable proteins facilitates the oligomerization of SNARE proteins. When light is shut off, it is expected spontaneous dissociation of the oligomerized photoactivatable proteins could reverse the monomeric state of SNARE proteins. to rescue the impaired long-term memory in a mouse To overcome this challenge, we propose that lacking EphB2 activity [115]. In summary, non- oligomerization-based optogenetic tools could help neuronal optogenetics allows for spatiotemporal determine how syntaxin clustering regulates fuso- control of protein activity in live animals. genic protein assembly and membrane fusion (Figure 3). As discussed before, the current devel- opment of optogenetic toolbox has allowed re- Perspectives searchers to directly control the membrane fusion at the synaptic level. Thus, it is our expectation that Although the conformational change of syntaxin has optogenetic control of syntaxin in live cells could been suggested to regulate membrane fusion, its role in provide insights into the role of syntaxin clusters (i.e., individual fusion steps, i.e., docking, Ca2+-free sponta- “ ” 2+ either as a hot spot to initiate fusion or a reservoir to neous fusion, and Ca -triggered fusion, is unclear. sequester active, monomeric syntaxin) underlying Essentially, it is unknown if membrane fusion depends membrane fusion during synaptic transmission. on the oligomeric or monomeric form of syntaxin. On the one hand, one can propose a working hypothesis that syntaxin clustering serves as a “hot spot” to recruit fusogenic components, including SNAP-25 and Ca2+ channels, to prime membrane fusion [116]. Indeed, the formation of the syntaxin/SNAP-25 dimers (i.e., the Acknowledgments binary SNARE complex), as receptors for VAMP2, is K.Z. would like to thank the support from the the rate-limiting steps for the assemble of ternary School of Molecular and Cellular Biology at UIUC. SNARE complexes [117]. That syntaxin is involved in J.D. would like to thank the support from University membrane fusion is further supported by the observa- of Cincinnati College of Medicine. tion that Ca2+-induced neurotransmitter secretion is inhibited by the neurotoxin BoNT/C, which cleaves syntaxin-1A [6]. Furthermore, the N-terminal peptide Received 15 April 2020; motif of syntaxin is an initiation factor for the assembly Received in revised form 5 July 2020; of the SNARE-Sec1/Munc18 membrane fusion com- Accepted 12 July 2020 plex [118]. Syntaxin exhibits an open conformation in Available online 16 July 2020 the SNARE complex and a closed conformation outside the SNARE complex. The latter controls the Keywords: initiation of the synaptic vesicle fusion reaction [119]. SNAREs; Given that syntaxin presents a nano-cluster to partic- synaptic transmission; ipate in the process of membrane fusion [120], it is cluster; crucial to determine whether and how syntaxin clusters syntaxin; affect membrane fusion and neurotransmitter release. optogenetics Alternatively, if it is the monomeric syntaxin that initiates functional membrane fusion, syntaxin oligomers could †These authors contributed equally to this work. then serve as a reservoir to sequester monomeric syntaxin and reduce membrane fusion capacity, which Abbreviations used: is important in preventing hyper membrane fusion. SNARE, soluble N-ethylmaleimide sensitive factor at- Successful testing of these hypotheses suffers from tachment protein receptor; VAMP2, vesicle-associated challenges arising from a lack of tools to precisely membrane protein 2; NSF, N-ethylmaleimide sensitive control the oligomerization state of syntaxin. factor; SNAP, soluble NSF attachment protein; SM, Sec1/ Optical control of syntaxin for membrane fusion 4779

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