Amlodipine inhibits Synaptotagmin-4's oncogenic activity on gastric cancer proliferation by targeting calcium signaling.

BACKGROUND: Gastric cancer (GC) remains a leading cause of cancer mortality globally. Synaptotagmin-4 (SYT4), a calcium-sensing synaptic vesicle protein, has been implicated in the oncogenesis of diverse malignancies. PURPOSE: This study delineates the role of SYT4 in modulating clinical outcomes and biological behaviors in GC. METHODS: We evaluated SYT4 expression in GC specimens using bioinformatics analyses and immunohistochemistry. Functional assays included CCK8 proliferation tests, apoptosis assays via flow cytometry, confocal calcium imaging, and xenograft models. Western blotting elucidated MAPK pathway involvement. Additionally, we investigated the impact of the calcium channel blocker amlodipine on cellular dynamics and MAPK pathway activity. RESULTS: SYT4 was higher in GC tissues, and the elevated SYT4 was significantly correlated with adverse prognosis. Both univariate and multivariate analyses confirmed SYT4 as an independent prognostic indicator for GC. Functionally, SYT4 promoted tumorigenesis by fostering cellular proliferation, inhibiting apoptosis, and enhancing intracellular Ca2+ influx, predominantly via MAPK pathway activation. Amlodipine pre-treatment attenuated SYT4-driven cell growth and potentiated apoptosis, corroborated by in vivo xenograft assessments. These effects were attributed to MAPK pathway suppression by amlodipine. CONCLUSION: SYT4 emerges as a potential prognostic biomarker and a pro-oncogenic mediator in GC through a Ca2+-dependent MAPK mechanism. Amlodipine demonstrates significant antitumor effects against SYT4-driven GC, positing its therapeutic promise. This study underscores the imperative of targeting calcium signaling in GC treatment strategies.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release.

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

Establishment of transgenic fluorescent mice for labeling synapses and screening synaptogenic adhesion molecules.

Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato (Syt1-tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1-tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1-tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1-tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the Syt1-tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.

Synaptotagmin-7 Counteracts Short-Term Depression during Phasic Dopamine Release.

Dopamine neurons switch from tonic pacemaker activity to high-frequency bursts in response to salient stimuli. These bursts lead to superlinear increases in dopamine release, and the degree of this increase is highly dependent on firing frequency. The superlinearity and frequency dependence of dopamine release implicate short-term plasticity processes. The presynaptic Ca2+-sensor synaptotagmin-7 (SYT7) has suitable properties to mediate such short-term plasticity and has been implicated in regulating dopamine release from somatodendritic compartments. Here, we use a genetically encoded dopamine sensor and whole-cell electrophysiology in Syt7 KO mice to determine how SYT7 contributes to both axonal and somatodendritic dopamine release. We find that SYT7 mediates a hidden component of facilitation of release from dopamine terminals that can be unmasked by lowering initial release probability or by predepressing synapses with low-frequency stimulation. Depletion of SYT7 increased short-term depression and reduced release during stimulations that mimic in vivo firing. Recordings of D2-mediated inhibitory postsynaptic currents in the substantia nigra pars compacta (SNc) confirmed a similar role for SYT7 in somatodendritic release. Our results indicate that SYT7 drives short-term facilitation of dopamine release, which may explain the frequency dependence of dopamine signaling seen in vivo.

Extended-Synaptotagmin-1 and -2 control T cell signaling and function.

Upon T-cell activation, the levels of the secondary messenger diacylglycerol (DAG) at the plasma membrane need to be controlled to ensure appropriate T-cell receptor signaling and T-cell functions. Extended-Synaptotagmins (E-Syts) are a family of inter-organelle lipid transport proteins that bridge the endoplasmic reticulum and the plasma membrane. In this study, we identify a novel regulatory mechanism of DAG-mediated signaling for T-cell effector functions based on E-Syt proteins. We demonstrate that E-Syts downmodulate T-cell receptor signaling, T-cell-mediated cytotoxicity, degranulation, and cytokine production by reducing plasma membrane levels of DAG. Mechanistically, E-Syt2 predominantly modulates DAG levels at the plasma membrane in resting-state T cells, while E-Syt1 and E-Syt2 negatively control T-cell receptor signaling upon stimulation. These results reveal a previously underappreciated role of E-Syts in regulating DAG dynamics in T-cell signaling.

Synaptotagmin 7 Sculpts Short-Term Plasticity at a High Probability Synapse.

Synapses with high release probability (Pr ) tend to exhibit short-term synaptic depression. According to the prevailing model, this reflects the temporary depletion of release-ready vesicles after an initial action potential (AP). At the high-Pr layer 4 to layer 2/3 (L4-L2/3) synapse in rodent somatosensory cortex, short-term plasticity appears to contradict the depletion model: depression is absent at interstimulus intervals (ISIs) <50 ms and develops to a maximum at ∼200 ms. To understand the mechanism(s) underlying the biphasic time course of short-term plasticity at this synapse, we used whole-cell electrophysiology and two-photon calcium imaging in acute slices from male and female juvenile mice. We tested several candidate mechanisms including neuromodulation, postsynaptic receptor desensitization, and use-dependent changes in presynaptic AP-evoked calcium. We found that, at single L4-L2/3 synapses, Pr varies as a function of ISI, giving rise to the distinctive short-term plasticity time course. Furthermore, the higher-than-expected Pr at short ISIs depends on expression of synaptotagmin 7 (Syt7). Our results show that two distinct vesicle release processes summate to give rise to short-term plasticity at this synapse: (1) a basal, high-Pr release mechanism that undergoes rapid depression and recovers slowly (τ = ∼3 s) and (2) a Syt7-dependent mechanism that leads to a transient increase in Pr (τ = ∼100 ms) after the initial AP. We thus reveal how these synapses can maintain a very high probability of neurotransmission for multiple APs within a short time frame. Key words : depression; facilitation; short-term plasticity; synaptotagmin 7.

miR-93 and synaptotagmin-7: two novel players in the regulation of autophagy during cardiac hypertrophy.

The molecular mechanisms involved in the transition of cardiac hypertrophy to heart failure (HF) are not fully characterized. Autophagy is a catabolic, self-renewal intracellular mechanism, which protects the heart during HF. In the heart of a mouse model of angiotensin-II-induced hypertrophy, Sun and colleagues demonstrated that reduced levels of miR-93 lead to synaptotagmin-7 (Syt-7) upregulation and consequent inhibition of autophagy. miR-93 overexpression or syt-7 inhibition rescues autophagy and maladaptive hypertrophy. This research identifies new players in the pathophysiology of cardiac hypertrophy, opening innovative therapeutic perspectives. miR-93 may also be considered in the future as a novel circulating biomarker for patients at high risk to develop HF.

The Zika virus infection remodels the expression of the synaptotagmin-9 secretory protein.

The exact mechanisms involved in flaviviruses virions' release and the specific secretion of viral proteins, such as the Non Structural protein-1 (NS1), are still unclear. While these processes might involve vesicular transport to the cell membrane, NS1 from some flaviviruses was shown to participate in viral assembly and release. Here, we assessed the effect of the Zika virus (ZIKV) NS1 expression on the cellular proteome to identify trafficking-related targets that may be altered in the presence of the viral protein. We detected an increase in the synaptotagmin-9 (SYT9) secretory protein, which participates in the intracellular transport of protein-laden vesicles. We confirmed the effect of NS1 on SYT9 levels by transfection models while also detecting a significant subcellular redistribution of SYT9. We found that ZIKV prM-Env proteins, required for the viral particle release, also increased SYT9 levels and changed its localization. Finally, we demonstrated that ZIKV cellular infection raises SYT9 levels and promotes changes in its subcellular localization, together with a co-distribution with both Env and NS1. Altogether, the data suggest SYT9's implication in the vesicular transport of viral proteins or virions during ZIKV infection, showing for the first time the association of synaptotagmins with the flavivirus' life cycle.

Synaptotagmin-7 mediates cardiac hypertrophy by targeting autophagy.

Sustained cardiac hypertrophy damages the heart and weakens cardiac function, often leading to heart failure and even death. Pathological cardiac hypertrophy has become a central therapeutic target for many heart diseases including heart failure. However, the underlying mechanisms of cardiac hypertrophy, especially the involvement of autophagy program, are still ill-understood. Synaptotagmin-7 (Syt7), a multifunctional and high-affinity calcium sensor, plays a pivotal role in asynchronous neurotransmitter release, synaptic facilitation, and vesicle pool regulation during synaptic transmission. However, little is known about whether Syt7 is expressed in the myocardium and involved in the pathogenesis of heart diseases. Here we showed that Syt7 was significantly upregulated in Ang II-treated hearts and cardiomyocytes. Homozygous syt7 knockout (syt7-/-) mice exhibited significantly attenuated cardiac hypertrophy and fibrosis and improved cardiac function. We further found that Syt7 exerted a pro-hypertrophic effect by suppressing the autophagy process. In exploring the upstream mechanisms, microRNA (miR)-93 was identified to participate in the regulation of Syt7 expression. miR-93 protected hearts against Ang II-induced hypertrophy through targeting Syt7-autophagy pathway. In summary, our data reveal a new cardiac hypertrophy regulator and a novel hypertrophy regulating model composed of miR-93, Syt7 and autophagy program. These molecules may serve as potential therapeutic targets in the treatment of cardiac hypertrophy and heart failure.

ESYT1 tethers the ER to mitochondria and is required for mitochondrial lipid and calcium homeostasis.

Mitochondria interact with the ER at structurally and functionally specialized membrane contact sites known as mitochondria-ER contact sites (MERCs). Combining proximity labelling (BioID), co-immunoprecipitation, confocal microscopy and subcellular fractionation, we found that the ER resident SMP-domain protein ESYT1 was enriched at MERCs, where it forms a complex with the outer mitochondrial membrane protein SYNJ2BP. BioID analyses using ER-targeted, outer mitochondrial membrane-targeted, and MERC-targeted baits, confirmed the presence of this complex at MERCs and the specificity of the interaction. Deletion of ESYT1 or SYNJ2BP reduced the number and length of MERCs. Loss of the ESYT1-SYNJ2BP complex impaired ER to mitochondria calcium flux and provoked a significant alteration of the mitochondrial lipidome, most prominently a reduction of cardiolipins and phosphatidylethanolamines. Both phenotypes were rescued by reexpression of WT ESYT1 and an artificial mitochondria-ER tether. Together, these results reveal a novel function for ESYT1 in mitochondrial and cellular homeostasis through its role in the regulation of MERCs.

Risk factors analysis and survival prediction model establishment of patients with lung adenocarcinoma based on different pyroptosis-related gene subtypes.

BACKGROUND: Lung adenocarcinoma (LUAD) is a common cancer with a poor prognosis. Pyroptosis is an important process in the development and progression of LUAD. We analyzed the risk factors affecting the prognosis of patients and constructed a nomogram to predict the overall survival of patients based on different pyroptosis-related genes (PRGs) subtypes. METHODS: The genomic data of LUAD were downloaded from the TCGA and GEO databases, and all data were filtered and divided into TCGA and GEO cohorts. The process of data analysis and visualization was performed via R software. The data were classified based on different PRGs subtypes using the K-means clustering method. Then, the differentially expressed genes were identified between two different subtypes, and risk factors analysis, survival analysis, functional enrichment analysis, and immune cells infiltration landscape analysis were conducted. The COX regression analysis was used to construct the prediction model. RESULTS: Based on the PRGs of LUAD, the patients were divided into two subtypes. We found the survival probability of patients in subtype 1 is higher than that in subtype 2. The results of the logistics analysis showed that gene risk score was closely associated with the prognosis of LUAD patients. The results of GO analysis and KEGG analysis revealed important biological processes and signaling pathways involved in the differentially expressed proteins between the two subtypes. Then we constructed a prediction model of patients' prognosis based on 13 genes, including IL-1A, P2RX1, GSTM2, ESYT3, ZNF682, KCNF1, STK32A, HHIPL2, GDF10, NDC80, GSTA1, BCL2L10, and CCR2. This model was strongly related to the overall survival (OS) and also reflects the immune status in patients with LUAD. CONCLUSION: In our study, we examined LUAD heterogeneity with reference to pyroptosis and found different prognoses between the two subtypes. And a novel prediction model was constructed to predict the OS of LUAD patients based on different PRGs signatures. The model has shown excellent predictive efficiency through validation.

Identification and verification of atrial fibrillation hub genes caused by primary mitral regurgitation.

In the clinic, atrial fibrillation (AF) is a common arrhythmia. Despite constant innovation in treatments for AF, they remain limited by a lack of knowledge of the underlying mechanism responsible for AF. In this study, we examined the molecular mechanisms associated with primary mitral regurgitation (MR) in AF using several bioinformatics techniques. Limma was used to identify differentially expressed genes (DEGs) associated with AF using microarray data from the GSE115574 dataset. WGCNA was used to identify significant module genes. A functional enrichment analysis for overlapping genes between the DEGs and module genes was done and several AF hub genes were identified from a protein-protein interaction (PPI) network. Receiver operating characteristic (ROC) curves were generated to evaluate the validity of the hub genes. We examined 306 DEGs and 147 were upregulated and 159 were downregulated. WGCNA analysis revealed black and ivory modules that contained genes associated with AF. Functional enrichment analysis revealed various biological process terms related to AF. The AUCs for the 8 hub genes screened by the PPI network analysis were > 0.7, indicating satisfactory diagnostic accuracy. The 8 AF-related hub genes included SYT13, VSNL1, GNAO1, RGS4, RALYL, CPLX1, CHGB, and CPLX3. Our findings provide novel insight into the molecular mechanisms of AF and may lead to the development of new treatments.

Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration.

Synaptotagmin-1 and synaptotagmin-7 are two prominent calcium sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding calcium, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examine the biophysical properties of these two synaptotagmin isoforms and compare their interactions with phospholipid membranes. We discover that synaptotagmin-1-membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a super-penetrator. We exploit these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to calcium.

Synaptotagmin-11 regulates immune functions of microglia in vivo.

Membrane trafficking pathways mediate key microglial activities such as cell migration, cytokine secretion, and phagocytosis. However, the underlying molecular mechanism remains poorly understood. Previously, we found that synaptotagmin-11 (Syt11), a non-Ca2+ -binding Syt associated with Parkinson's disease (PD) and schizophrenia, inhibits cytokine release and phagocytosis in primary microglia. Here we reported the in vivo function of Syt11 in microglial immune responses using an inducible microglia-specific Syt11-conditional-knockout (cKO) mouse strain. Syt11-cKO resulted in activation of microglia and elevated mRNA levels of IL-6, TNF-α, IL-1ß, and iNOS in various brain regions under both resting state and LPS-induced acute inflammation state in adult mice. In a PD mouse model generated by microinjection of preformed α-synuclein fibrils into the striatum, a reduced number of microglia migrated toward the injection sites and an enhanced phagocytosis of α-synuclein fibrils by microglia were found in Syt11-cKO mice. To understand the molecular mechanism of Syt11 function, we identified its direct binding proteins vps10p-tail-interactor-1a (vti1a) and vti1b. The linker domain of Syt11 interacted with both proteins and a peptide derived from it competitively inhibited the interaction of Syt11 with vti1a/vti1b in vitro and in cells. Importantly, application of this peptide induced more cytokine secretion in wild-type microglia upon LPS treatment, phenocopying defects in Syt11 knockdown cells. Altogether, we propose that Syt11 inhibits microglial activation in vivo and regulates cytokine secretion through interactions with vti1a and vti1b.

Synaptophysin chaperones the assembly of 12 SNAREpins under each ready-release vesicle.

The synaptic vesicle protein Synaptophysin (Syp) has long been known to form a complex with the Vesicle associated soluble N-ethylmaleimide sensitive fusion protein attachment receptor (v-SNARE) Vesicle associated membrane protein (VAMP), but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully defined reconstitution and single-molecule measurements, we now report that Syp functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Syp directs the assembly of 12 ± 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins. The SNAREpins assemble in successive waves of 6 ± 1 and 5 ± 2 SNAREpins, respectively, tightly linked to oligomerization of and binding to the vesicle Ca++ sensor Synaptotagmin. Templating of 12 SNAREpins by Syp is likely the direct result of its hexamer structure and its binding of VAMP2 dimers, both of which we demonstrate in detergent extracts and lipid bilayers.

Comprehensive analysis of the prognostic values and immune implication of ESYT3 in lung adenocarcinoma.

Few studies have reported the association between ESYT3 and tumors. The purpose of this study was to investigate the molecular features and potential roles of ESYT3 in lung adenocarcinoma (LUAD). In the present study, GEPIA, UALCAN, TCGA databases, and KM Plotter were primarily used to study ESYT3 mRNA expression profiles and prognostic values in patients with LUAD. Then we evaluated co-expressed genes of ESYT3 by cBioPortal online tools and performed enrichment analysis using Metascape. Moreover, the relationship between ESYT3 and immune infiltrating cells was explored via TIMER2, and MethSurv database was used to conduct methylation analysis. We found ESYT3 was downregulated in LUAD tissues based on TCGA and GEPIA databases. Low expression of ESYT3 mRNA was observed to be significantly correlated with N classification and stage classification. GEPIA2, UALCAN databases and KM Plotter showed that low expression levels of ESYT3 was associated with poor survival in LUAD patients. The enrichment analysis indicated that co-expressed genes of ESYT3 were highly enriched in cell division. Then, our study showed ESYT3 was correlated with immune infiltration and immune checkpoints. Additionally, hypomethylation was associated with low ESYT3 expression and poor prognosis in LUAD. In conclusion, this study suggested ESYT3 could be a potential prognostic marker and a promising therapeutic target in LUAD.

SYT7 is a key player in increasing exosome secretion and promoting angiogenesis in non-small-cell lung cancer.

Lung cancer is the leading cause of cancer-related mortality, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. Our previous study confirmed that synaptotagmin 7 (SYT7) promoted NSCLC metastasis in vivo and in vitro. Studies have shown that SYT7 is an important regulatory molecule of exocytosis in various cells. However, the characteristics of SYT7 across cancers and the function of SYT7 in tumor exosome secretion remain unclear. In this study, we conducted systematic pancancer analyses of SYT7, namely, analyses of expression patterns, diagnostic and prognostic values, genetic alterations, methylation, immune infiltration, and potential biological pathways. Furthermore, we demonstrated that SYT7 increased the secretion of exosomes from A549 and H1299 cells, promoting the migration, proliferation, and tube formation of human umbilical vein endothelial cells (HUVECs). Notably, SYT7 promoted angiogenesis by transferring exosomes containing the molecule centrosomal protein of 55 kDa (CEP55) protein to HUVECs. The CEP55 protein levels was downregulated in STAT1 inhibitor-treating SYT7-overexpresion NSCLC cells. We further found that SYT7 activated the mTOR signaling pathway through the downstream molecule CEP55, thereby promoting the invasion and metastasis of NSCLC cells. SYT7 promoted exosome secretion by NSCLC cells through upregulating syntaxin-1a and syntaxin-3. In vivo, SYT7 promoted the tumorigenesis, angiogenesis and metastasis of A549 cells through the exosome pathway. Our study is of great importance for understanding the mechanism of tumor exosome secretion and the role of exosomes in tumor progression.

Calcium Sensors of Neurotransmitter Release.

Calcium (Ca2+) plays a critical role in triggering all three primary modes of neurotransmitter release (synchronous, asynchronous, and spontaneous). Synaptotagmin1, a protein with two C2 domains, is the first isoform of the synaptotagmin family that was identified and demonstrated as the primary Ca2+ sensor for synchronous neurotransmitter release. Other isoforms of the synaptotagmin family as well as other C2 proteins such as the double C2 domain protein family were found to act as Ca2+ sensors for different modes of neurotransmitter release. Major recent advances and previous data suggest a new model, release-of-inhibition, for the initiation of Ca2+-triggered synchronous neurotransmitter release. Synaptotagmin1 binds Ca2+ via its two C2 domains and relieves a primed pre-fusion machinery. Before Ca2+ triggering, synaptotagmin1 interacts Ca2+ independently with partially zippered SNARE complexes, the plasma membrane, phospholipids, and other components to form a primed pre-fusion state that is ready for fast release. However, membrane fusion is inhibited until the arrival of Ca2+ reorients the Ca2+-binding loops of the C2 domain to perturb the lipid bilayers, help bridge the membranes, and/or induce membrane curvatures, which serves as a power stroke to activate fusion. This chapter reviews the evidence supporting these models and discusses the molecular interactions that may underlie these abilities.

SNARE Proteins in Synaptic Vesicle Fusion.

Neurotransmitters are stored in small membrane-bound vesicles at synapses; a subset of synaptic vesicles is docked at release sites. Fusion of docked vesicles with the plasma membrane releases neurotransmitters. Membrane fusion at synapses, as well as all trafficking steps of the secretory pathway, is mediated by SNARE proteins. The SNAREs are the minimal fusion machinery. They zipper from N-termini to membrane-anchored C-termini to form a 4-helix bundle that forces the apposed membranes to fuse. At synapses, the SNAREs comprise a single helix from syntaxin and synaptobrevin; SNAP-25 contributes the other two helices to complete the bundle. Unc13 mediates synaptic vesicle docking and converts syntaxin into the permissive "open" configuration. The SM protein, Unc18, is required to initiate and proofread SNARE assembly. The SNAREs are then held in a half-zippered state by synaptotagmin and complexin. Calcium removes the synaptotagmin and complexin block, and the SNAREs drive vesicle fusion. After fusion, NSF and alpha-SNAP unwind the SNAREs and thereby recharge the system for further rounds of fusion. In this chapter, we will describe the discovery of the SNAREs, their relevant structural features, models for their function, and the central role of Unc18. In addition, we will touch upon the regulation of SNARE complex formation by Unc13, complexin, and synaptotagmin.

Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.