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 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.

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.

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.

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.

Turbocharging synaptic transmission.

Evidence from biochemistry, genetics, and electron microscopy strongly supports the idea that a ring of Synaptotagmin is central to the clamping and release of synaptic vesicles (SVs) for synchronous neurotransmission. Recent direct measurements in cell-free systems suggest there are 12 SNAREpins in each ready-release vesicle, consisting of six peripheral and six central SNAREpins. The six central SNAREpins are directly bound to the Synaptotagmin ring, are directly released by Ca++ , and they initially open the fusion pore. The six peripheral SNAREpins are indirectly bound to the ring, each linked to a central SNAREpin by a bridging molecule of Complexin. We suggest that the primary role of peripheral SNAREpins is to provide additional force to 'turbocharge' neurotransmitter release, explaining how it can occur much faster than other forms of membrane fusion. The SV protein Synaptophysin forms hexamers that bear two copies of the v-SNARE VAMP at each vertex, one likely assembling into a peripheral SNAREpin and the other into a central SNAREpin.

Synaptotagmin-11 Inhibits Synaptic Vesicle Endocytosis via Endophilin A1.

Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca2+-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca2+-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both in vitro and in vivo Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals.SIGNIFICANCE STATEMENT Endocytosis is a key stage of synaptic vesicle (SV) recycling. SV endocytosis retrieves vesicular membrane and protein components precisely to support sustained neurotransmission. However, the molecular mechanisms underlying the regulation of SV endocytosis remain elusive. Here, we reported that Syt11-KO accelerated SV endocytosis and impaired membrane partition of synaptic proteins. EndoA1 was identified as a main inhibitory target of Syt11 during SV endocytosis. Our study reveals a novel inhibitory mechanism of SV endocytosis in preventing hyperactivation of endocytosis, potentially safeguarding the recycling of synaptic proteins during sustained neurotransmission.

Genetic ablation of synaptotagmin-9 alters tomosyn-1 function to increase insulin secretion from pancreatic ß-cells improving glucose clearance.

Stimulus-coupled insulin secretion from the pancreatic islet ß-cells involves the fusion of insulin granules to the plasma membrane (PM) via SNARE complex formation-a cellular process key for maintaining whole-body glucose homeostasis. Less is known about the role of endogenous inhibitors of SNARE complexes in insulin secretion. We show that an insulin granule protein synaptotagmin-9 (Syt9) deletion in mice increased glucose clearance and plasma insulin levels without affecting insulin action compared to the control mice. Upon glucose stimulation, increased biphasic and static insulin secretion were observed from ex vivo islets due to Syt9 loss. Syt9 colocalizes and binds with tomosyn-1 and the PM syntaxin-1A (Stx1A); Stx1A is required for forming SNARE complexes. Syt9 knockdown reduced tomosyn-1 protein abundance via proteasomal degradation and binding of tomosyn-1 to Stx1A. Furthermore, Stx1A-SNARE complex formation was increased, implicating Syt9-tomosyn-1-Stx1A complex is inhibitory in insulin secretion. Rescuing tomosyn-1 blocked the Syt9-knockdown-mediated increases in insulin secretion. This shows that the inhibitory effects of Syt9 on insulin secretion are mediated by tomosyn-1. We report a molecular mechanism by which ß-cells modulate their secretory capacity rendering insulin granules nonfusogenic by forming the Syt9-tomosyn-1-Stx1A complex. Altogether, Syt9 loss in ß-cells decreases tomosyn-1 protein abundance, increasing the formation of Stx1A-SNARE complexes, insulin secretion, and glucose clearance. These outcomes differ from the previously published work that identified Syt9 has either a positive or no effect of Syt9 on insulin secretion. Future work using ß-cell-specific deletion of Syt9 mice is key for establishing the role of Syt9 in insulin secretion.

Role of synaptotagmin 13 (SYT13) in promoting breast cancer and signaling pathways

Purpose Breast cancer is one of the leading causes of tumor death worldwide in female, and the five-year overall survival of breast cancer patients remains poor. It is an urgent need to seek novel target for its treatment. Synaptotagmin 13 (SYT13) is a synaptic vesicle transporting protein that regulates the malignant phenotypes of various cancers. However, its role in breast cancer is still unclear. The current study aimed to investigate the effects of SYT13 on the progression of breast cancer. Methods Twenty-five pairs of breast cancer tissues and non-tumor tissues were obtained to assess the expression of SYT13. We manually modified the expression of SYT13 in MCF-7 and MDA-MB-231 cells. CCK-8 assay, EdU staining, and cell cycle analysis were carried out to measure the proliferated ability of cells. Annexin V/PI and TUNEL assays were used to detect the apoptotic ability of cells. Wound healing and transwell assays were employed to evaluate the migrated and invasive ability of breast cancer cells. Results The results revealed that the mRNA and protein levels of SYT13 were higher in breast cancer tissues and cell lines. Knockdown of SYT13 inhibited the cell proliferation and induced cell cycle arrest in G1 phase of MCF-7 cells by downregulating cyclin D1 and CDK4, as well as upregulating p21. The migration and invasion of MCF-7 cells were repressed by the loss of SYT13 via the gain of E-cadherin and the loss of vimentin. Overexpression of SYT13 in MDA-MB-231 cells led to the opposite effects. Silencing of SYT13 induced the apoptosis ability of MCF-7 cells by the upregulation of bax and the downregulation of bcl-2. Moreover, we found that SYT13 depletion suppressed the FAK/AKT signaling pathway. PF573228 (a FAK inhibitor) and MK2206 (an AKT inhibitor) reversed the SYT13 overexpression-induced promotion of proliferation, migration, and invasion of MDA-MB-231 cells (AU)

Insights into membrane association of the SMP domain of extended synaptotagmin.

The Synaptotagmin-like Mitochondrial-lipid-binding Protein (SMP) domain is a newly identified lipid transfer module present in proteins that regulate lipid homeostasis at membrane contact sites (MCSs). However, how the SMP domain associates with the membrane to extract and unload lipids is unclear. Here, we performed in vitro DNA brick-assisted lipid transfer assays and in silico molecular dynamics simulations to investigate the molecular basis of the membrane association by the SMP domain of extended synaptotagmin (E-Syt), which tethers the tubular endoplasmic reticulum (ER) to the plasma membrane (PM). We demonstrate that the SMP domain uses its tip region to recognize the extremely curved subdomain of tubular ER and the acidic-lipid-enriched PM for highly efficient lipid transfer. Supporting these findings, disruption of these mechanisms results in a defect in autophagosome biogenesis contributed by E-Syt. Our results suggest a model that provides a coherent picture of the action of the SMP domain at MCSs.

Synaptotagmin-3 interactions with GluA2 mediate brain damage and impair functional recovery in stroke.

Synaptotagmin III (Syt3) is a Ca2+-dependent membrane-traffic protein that is highly concentrated in synaptic plasma membranes and affects synaptic plasticity by regulating post-synaptic receptor endocytosis. Here, we show that Syt3 is upregulated in the penumbra after ischemia/reperfusion (I/R) injury. Knockdown of Syt3 protects against I/R injury, promotes recovery of motor function, and inhibits cognitive decline. Overexpression of Syt3 exerts the opposite effects. Mechanistically, I/R injury augments Syt3-GluA2 interactions, decreases GluA2 surface expression, and promotes the formation of Ca2+-permeable AMPA receptors (CP-AMPARs). Using a CP-AMPAR antagonist or dissociating the Syt3-GluA2 complex via TAT-GluA2-3Y peptide promotes recovery from neurological impairments and improves cognitive function. Furthermore, Syt3 knockout mice are resistant to cerebral ischemia because they show high-level expression of surface GluA2 and low-level expression of CP-AMPARs after I/R. Our results indicate that Syt3-GluA2 interactions, which regulate the formation of CP-AMPARs, may be a therapeutic target for ischemic insults.

Role of calcium-sensor proteins in cell membrane repair.

Cell membrane repair is a critical process used to maintain cell integrity and survival from potentially lethal chemical, and mechanical membrane injury. Rapid increases in local calcium levels due to a membrane rupture have been widely accepted as a trigger for multiple membrane-resealing models that utilize exocytosis, endocytosis, patching, and shedding mechanisms. Calcium-sensor proteins, such as synaptotagmins (Syt), dysferlin, S100 proteins, and annexins, have all been identified to regulate, or participate in, multiple modes of membrane repair. Dysfunction of membrane repair from inefficiencies or genetic alterations in these proteins contributes to diseases such as muscular dystrophy (MD) and heart disease. The present review covers the role of some of the key calcium-sensor proteins and their involvement in membrane repair.

Palmitoylation of the Parkinson's disease-associated protein synaptotagmin-11 links its turnover to α-synuclein homeostasis.

Synaptotagmin-11 (Syt11) is a vesicle-trafficking protein that is linked genetically to Parkinson's disease (PD). Likewise, the protein α-synuclein regulates vesicle trafficking, and its abnormal aggregation in neurons is the defining cytopathology of PD. Because of their functional similarities in the same disease context, we investigated whether the two proteins were connected. We found that Syt11 was palmitoylated in mouse and human brain tissue and in cultured cortical neurons and that this modification to Syt11 disrupted α-synuclein homeostasis in neurons. Palmitoylation of two cysteines adjacent to the transmembrane domain, Cys39 and Cys40, localized Syt11 to digitonin-insoluble portions of intracellular membranes and protected it from degradation by the endolysosomal system. In neurons, palmitoylation of Syt11 increased its abundance and enhanced the binding of α-synuclein to intracellular membranes. As a result, the abundance of the physiologic tetrameric form of α-synuclein was decreased, and that of its aggregation-prone monomeric form was increased. These effects were replicated by overexpression of wild-type Syt11 but not a palmitoylation-deficient mutant. These findings suggest that palmitoylation-mediated increases in Syt11 amounts may promote pathological α-synuclein aggregation in PD.

Role of synaptotagmin 13 (SYT13) in promoting breast cancer and signaling pathways.

PURPOSE: Breast cancer is one of the leading causes of tumor death worldwide in female, and the five-year overall survival of breast cancer patients remains poor. It is an urgent need to seek novel target for its treatment. Synaptotagmin 13 (SYT13) is a synaptic vesicle transporting protein that regulates the malignant phenotypes of various cancers. However, its role in breast cancer is still unclear. The current study aimed to investigate the effects of SYT13 on the progression of breast cancer. METHODS: Twenty-five pairs of breast cancer tissues and non-tumor tissues were obtained to assess the expression of SYT13. We manually modified the expression of SYT13 in MCF-7 and MDA-MB-231 cells. CCK-8 assay, EdU staining, and cell cycle analysis were carried out to measure the proliferated ability of cells. Annexin V/PI and TUNEL assays were used to detect the apoptotic ability of cells. Wound healing and transwell assays were employed to evaluate the migrated and invasive ability of breast cancer cells. RESULTS: The results revealed that the mRNA and protein levels of SYT13 were higher in breast cancer tissues and cell lines. Knockdown of SYT13 inhibited the cell proliferation and induced cell cycle arrest in G1 phase of MCF-7 cells by downregulating cyclin D1 and CDK4, as well as upregulating p21. The migration and invasion of MCF-7 cells were repressed by the loss of SYT13 via the gain of E-cadherin and the loss of vimentin. Overexpression of SYT13 in MDA-MB-231 cells led to the opposite effects. Silencing of SYT13 induced the apoptosis ability of MCF-7 cells by the upregulation of bax and the downregulation of bcl-2. Moreover, we found that SYT13 depletion suppressed the FAK/AKT signaling pathway. PF573228 (a FAK inhibitor) and MK2206 (an AKT inhibitor) reversed the SYT13 overexpression-induced promotion of proliferation, migration, and invasion of MDA-MB-231 cells. CONCLUSION: The results indicated that SYT13 promoted the malignant phenotypes of breast cancer cells by the activation of FAK/AKT signaling pathway.

Fast resupply of synaptic vesicles requires synaptotagmin-3.

Sustained neuronal activity demands a rapid resupply of synaptic vesicles to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by submicromolar presynaptic Ca2+ signals by an as-yet unidentified high-affinity Ca2+ sensor1,2. Here we identify synaptotagmin-3 (SYT3)3,4 as that presynaptic high-affinity Ca2+ sensor, which drives vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibited enhanced short-term depression, and recovery from depression was slower and insensitive to presynaptic residual Ca2+. During sustained neuronal firing, SYT3 accelerated vesicle replenishment and increased the size of the readily releasable pool. SYT3 also mediated short-term facilitation under conditions of low release probability and promoted synaptic enhancement together with another high-affinity synaptotagmin, SYT7 (ref. 5). Biophysical modelling predicted that SYT3 mediates both replenishment and facilitation by promoting the transition of loosely docked vesicles to tightly docked, primed states. Our results reveal a crucial role for presynaptic SYT3 in the maintenance of reliable high-frequency synaptic transmission. Moreover, multiple forms of short-term plasticity may converge on a mechanism of reversible, Ca2+-dependent vesicle docking.

ELK1 suppresses SYTL1 expression by recruiting HDAC2 in bladder cancer progression.

ETS transcription factor (ELK1) stimulates the expression of genes at the onset of the cell cycle and participates in early developmental programming. Here, we investigated whether alterations of ELK1 lead to progression of bladder cancer (BCa), a main neoplasm of urinary tract, and clarified the function of ELK1 in BCa. Using the GEO database, we identified ELK1 as the most significantly overexpressed gene in BCa, which was substantiated in the acquired clinical samples and cells. Silencing of ELK1 inhibited the malignant phenotype of BCa cells. Further analysis revealed that ELK1 synergized with histone deacetylase 2 (HDAC2) to specifically bind to the synaptotagmin like 1 (SYTL1) promoter, thereby repressing SYTL1 transcription and protein expression. Depletion of SYTL1 reversed the repressive effects of ELK1 depletion on the malignant phenotype of BCa cells. Our in vitro findings were reproduced in vivo on a nude mouse tumorigenic model. Together, our results reveal that ELK1, through suppression of SYTL1 via HDAC2, supports the malignant phenotype of BCa cells.

Allosteric stabilization of calcium and phosphoinositide dual binding engages several synaptotagmins in fast exocytosis.

Synaptic communication relies on the fusion of synaptic vesicles with the plasma membrane, which leads to neurotransmitter release. This exocytosis is triggered by brief and local elevations of intracellular Ca2+ with remarkably high sensitivity. How this is molecularly achieved is unknown. While synaptotagmins confer the Ca2+ sensitivity of neurotransmitter exocytosis, biochemical measurements reported Ca2+ affinities too low to account for synaptic function. However, synaptotagmin's Ca2+ affinity increases upon binding the plasma membrane phospholipid PI(4,5)P2 and, vice versa, Ca2+ binding increases synaptotagmin's PI(4,5)P2 affinity, indicating a stabilization of the Ca2+/PI(4,5)P2 dual-bound state. Here, we devise a molecular exocytosis model based on this positive allosteric stabilization and the assumptions that (1.) synaptotagmin Ca2+/PI(4,5)P2 dual binding lowers the energy barrier for vesicle fusion and that (2.) the effect of multiple synaptotagmins on the energy barrier is additive. The model, which relies on biochemically measured Ca2+/PI(4,5)P2 affinities and protein copy numbers, reproduced the steep Ca2+ dependency of neurotransmitter release. Our results indicate that each synaptotagmin engaging in Ca2+/PI(4,5)P2 dual-binding lowers the energy barrier for vesicle fusion by ~5 kBT and that allosteric stabilization of this state enables the synchronized engagement of several (typically three) synaptotagmins for fast exocytosis. Furthermore, we show that mutations altering synaptotagmin's allosteric properties may show dominant-negative effects, even though synaptotagmins act independently on the energy barrier, and that dynamic changes of local PI(4,5)P2 (e.g. upon vesicle movement) dramatically impact synaptic responses. We conclude that allosterically stabilized Ca2+/PI(4,5)P2 dual binding enables synaptotagmins to exert their coordinated function in neurotransmission.

For our brains and nervous systems to work properly, the nerve cells within them must be able to 'talk' to each other. They do this by releasing chemical signals called neurotransmitters which other cells can detect and respond to. Neurotransmitters are packaged in tiny membrane-bound spheres called vesicles. When a cell of the nervous system needs to send a signal to its neighbours, the vesicles fuse with the outer membrane of the cell, discharging their chemical contents for other cells to detect. The initial trigger for neurotransmitter release is a short, fast increase in the amount of calcium ions inside the signalling cell. One of the main proteins that helps regulate this process is synaptotagmin which binds to calcium and gives vesicles the signal to start unloading their chemicals. Despite acting as a calcium sensor, synaptotagmin actually has a very low affinity for calcium ions by itself, meaning that it would not be efficient for the protein to respond alone. Synpatotagmin is more likely to bind to calcium if it is attached to a molecule called PIP₂, which is found in the membranes of cells The effect also occurs in reverse, as the binding of calcium to synaptotagmin increases the protein's affinity for PIP₂. However, how these three molecules ­ synaptotagmin, PIP₂, and calcium ­ work together to achieve the physiological release of neurotransmitters is poorly understood. To help answer this question, Kobbersmed, Berns et al. set up a computer simulation of 'virtual vesicles' using available experimental data on synaptotagmin's affinity with calcium and PIP₂. In this simulation, synaptotagmin could only trigger the release of neurotransmitters when bound to both calcium and PIP₂. The model also showed that each 'complex' of synaptotagmin/calcium/PIP₂ made the vesicles more likely to fuse with the outer membrane of the cell ­ to the extent that only a handful of synaptotagmin molecules were needed to start neurotransmitter release from a single vesicle. These results shed new light on a biological process central to the way nerve cells communicate with each other. In the future, Kobbersmed, Berns et al. hope that this insight will help us to understand the cause of diseases where communication in the nervous system is impaired.

Synaptotagmin-13 orchestrates pancreatic endocrine cell egression and islet morphogenesis.

During pancreas development endocrine cells leave the ductal epithelium to form the islets of Langerhans, but the morphogenetic mechanisms are incompletely understood. Here, we identify the Ca2+-independent atypical Synaptotagmin-13 (Syt13) as a key regulator of endocrine cell egression and islet formation. We detect specific upregulation of the Syt13 gene and encoded protein in endocrine precursors and the respective lineage during islet formation. The Syt13 protein is localized to the apical membrane of endocrine precursors and to the front domain of egressing endocrine cells, marking a previously unidentified apical-basal to front-rear repolarization during endocrine precursor cell egression. Knockout of Syt13 impairs endocrine cell egression and skews the α-to-ß-cell ratio. Mechanistically, Syt13 is a vesicle trafficking protein, transported via the microtubule cytoskeleton, and interacts with phosphatidylinositol phospholipids for polarized localization. By internalizing a subset of plasma membrane proteins at the front domain, including α6ß4 integrins, Syt13 modulates cell-matrix adhesion and allows efficient endocrine cell egression. Altogether, these findings uncover an unexpected role for Syt13 as a morphogenetic driver of endocrinogenesis and islet formation.

Molecular landscape of BoNT/B bound to a membrane-inserted synaptotagmin/ganglioside complex.

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.