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GPR133 (ADGRD1) is an adhesion G-protein-coupled receptor that signals through Gαs/cyclic AMP (cAMP) and is required for the growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca 2+ -dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca 2+ -sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca 2+ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM slows tumor growth, suggesting tumorigenic functions of ESYT1. Our findings demonstrate a mechanism for the modulation of GPR133 signaling by increased cytosolic Ca 2+ , which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels., Competing Interests: Declaration of interests D.G.P. and NYU Grossman School of Medicine own an EU and Hong Kong patent titled “Method for treating high-grade gliomas” on the use of GPR133 as a treatment target in glioma. D.G.P. and collaborators at NYU Grossman School of Medicine have filed a patent application titled “Anti-CD97 antibodies and antibody-drug conjugates”. D.G.P. has received consultant fees from Tocagen, Synaptive Medical, Monteris, Robeaute, Advantis, and Servier Pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca 2+ -triggered exocytosis of synaptic vesicles (SVs). In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P 2 -triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Synaptotagmin (syt) 1, a Ca 2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca 2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca 2+ -sensitivity of syt1. Moreover, Ca 2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca 2+ ] i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca 2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS., (© 2024. The Author(s).)

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca 2+ 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 Ca 2+ 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 Ca 2+ 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., Competing Interests: ZW, GK, MB, SR, KI, AW, EC, SW No competing interests declared, (© 2023, Wu, Kusick et al.)

Insulin secretion depends on the Ca 2+ -regulated fusion of granules with the plasma membrane. A recent model of Ca 2+ -triggered exocytosis in secretory cells proposes that lipids in the plasma membrane couple the calcium sensor Syt1 to the membrane fusion machinery (Kiessling et al. , 2018). Specifically, Ca 2+ -mediated binding of Syt1's C2 domains to the cell membrane shifts the membrane-anchored SNARE syntaxin-1a to a more fusogenic conformation, straightening its juxtamembrane linker. To test this model in live cells and extend it to insulin secretion, we enriched INS1 cells with a panel of lipids with different acyl chain compositions. Fluorescence lifetime measurements demonstrate that cells with more disordered membranes show an increase in fusion efficiency, and vice versa. Experiments with granules purified from INS1 cells and recombinant SNARE proteins reconstituted in supported membranes confirmed that lipid acyl chain composition determines SNARE conformation and that lipid disordering correlates with increased fusion. Addition of Syt1's C2AB domains significantly decreased lipid order in target membranes and increased SNARE-mediated fusion probability. Strikingly, Syt's action on both fusion and lipid order could be partially bypassed by artificially increasing unsaturated phosphatidylserines in the target membrane. Thus, plasma membrane lipids actively participate in coupling Ca 2+ /synaptotagmin-sensing to the SNARE fusion machinery in cells.

Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been proposed to act as a 'pre-assembly' mechanism for endocytosis that ensures high-fidelity retrieval of SV cargo. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1 K326A/K328A ) and SV2A knockdown. SV2A co-clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2A T84A ). Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin-1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

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.

Objective: Perisynaptic astrocytic processes have been suggested as sites for the regulated release of neuroactive substances. However, very little is known about the molecular properties of regulated exocytosis in these processes. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins mediate synaptic vesicle exocytosis from neuronal cells and might be candidates for regulated exocytosis also from astrocytic processes. The expression of SNARE proteins in astrocytes, however, is not clarified. Thus, we aimed to investigate the localization and relative concentrations of neuronal SNARE proteins syntaxin-1, synaptosomal nerve-associated protein 25 (SNAP-25), vesicle-associated membrane protein 2 (VAMP-2) (synaptobrevin-2) and calcium sensor synaptotagmin 1 in perisynaptic astrocytic processes compared to nerve terminals and dendrites., Methods: We used quantitative immunogold electron microscopy of the rat hippocampus to investigate the localization and concentration of neuronal SNARE proteins., Results: As expected, analysis of the immunogold data revealed a lower labeling density of SNARE proteins in the perisynaptic astrocytic processes than in presynaptic terminals. The same was also true when compared to dendrites. Contrary to VAMP-2, labeling intensities for syntaxin-1, SNAP-25 and synaptotagmin 1 were not distinguishable from background labeling in the processes. The relative concentration of VAMP-2 stands out, as the mean perisynaptic astrocytic process concentration of the protein was only 68 % lower than in presynaptic terminals and still 32 % higher than in dendrites. VAMP-2 was associated with small vesicles in the processes. Some gold particles were located over the astrocytic plasma membrane., Conclusion: VAMP-2 is expressed in perisynaptic astrocytic processes, with a concentration higher than in the dendrites. Our results are compatible with the role of VAMP-2 in exocytosis from perisynaptic astrocytic processes., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract Reactive oxygen species (ROS)‐induced oxidative DNA damages have been considered the main cause of mutations in genes, which are highly related to carcinogenesis and tumour progression. Extracellular vesicles play an important role in cancer metastasis. However, the precise role of DNA oxidative damage in extracellular vesicles (EVs)‐mediated cancer cell migration and invasion remains unclear. Here, we reveal that ROS‐mediated DNA oxidative damage signalling promotes tumour metastasis through increasing EVs release. Mechanistically, 8‐oxoguanine DNA glycosylase (OGG1) recognises and binds to its substrate 8‐oxo‐7,8‐dihydroguanine (8‐oxoG), recruiting NF‐κB to the synaptotagmin 7 (SYT7) promoter and thereby triggering SYT7 transcription. The upregulation of SYT7 expression leads to increased release of E‐cadherin‐loaded EVs, which depletes intracellular E‐cadherin, thereby inducing epithelial‐mesenchymal transition (EMT). Notably, Th5487, the inhibitor of DNA binding activity of OGG1, blocks the recognition and transmission of oxidative signals, alleviates SYT7 expression and suppresses EVs release, thereby preventing tumour progression in vitro and in vivo. Collectively, our study illuminates the significance of 8‐oxoG/OGG1/SYT7 axis‐driven EVs release in oxidative stress‐induced tumour metastasis. These findings provide a deeper understanding of the molecular basis of cancer progression and offer potential avenues for therapeutic intervention.

The Synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain is found in a group of ER-resident lipid transfer proteins that are recruited to membrane contact sites (MCSs) by adaptors. Deciphering the molecular basis underlying the recruitment of SMP proteins to specific MCS sheds light not only on their cellular localization but also on their biological functions at these sites. Here we summarize recent advances in SMP domain-containing lipid transfer proteins, focusing on a recent study showing the localization, regulation and cellular function of a specific SMP protein named testis expressed protein 2 (Tex2). TMEM55, a potential PIP phosphatase on late endosome/lysosomal (LE/lys) membranes, was identified as an adaptor that enables the recruitment of Tex2 to ER- LE/lys MCS. In addition, we have summarized several important questions about the regulation and physiological functions of Tex2 that remained unanswered.

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among the vertebrate synaptotagmin-like protein (Slp) family. Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function., (© 2023 The Protein Society.)

The Ca 2+ sensor synaptotagmin-1 (Syt1) triggers neurotransmitter release together with the neuronal sensitive factor attachment protein receptor (SNARE) complex formed by syntaxin-1, SNAP25, and synaptobrevin. Moreover, Syt1 increases synaptic vesicle (SV) priming and impairs spontaneous vesicle release. The Syt1 C 2 B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of Syt1 and the mechanism underlying Ca 2+ triggering of release are unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: Binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca 2+ -evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of Syt1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of Syt1 in vesicle priming and clamping of spontaneous release and, importantly, show that Ca 2+ triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with biophysical studies presented in [K. Jaczynska et al. , bioRxiv [Preprint] (2024). https://doi.org/10.1101/2024.06.17.599417 (Accessed 18 June 2024)], our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast SV fusion., Competing Interests: Competing interests statement:The authors declare no competing interest.

Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin mined from the egg transcriptome of spider L. tredecimguttatus, was previously found to promote the release of dopamine from PC12 cells. However, the relevant molecular mechanism has not been fully clear. Here LETX-VI was demonstrated to rapidly penetrate the plasma membrane of PC12 cells via the vesicle exocytosis/endocytosis cycle, during which vesicular transmembrane protein synaptotagmin 1 (Syt1) functions as a receptor, with its vesicle luminal domain interacting with the C-terminal region of LETX-VI. The C-terminal sequence of LETX-VI is the functional region for both entering cells and promoting dopamine release. After gaining entry into the PC12 cells, LETX-VI down-regulated the phosphorylation levels of Syt1 at T201 and T195, thereby facilitating vesicle fusion with plasma membrane and thus promoting dopamine release. The relevant mechanism analysis indicated that LETX-VI has a protein phosphatase 2A (PP2A) activator activity. The present work has not only probed into the Syt1-mediated action mechanism of LETX-VI, but also revealed the structure-function relationship of the toxin, thus suggesting its potential applications in the drug transmembrane delivery and treatment of the diseases related to dopamine release and PP2A activity deficiency., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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-N 59 Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle and plasma membranes to release neurotransmitters. Previous studies suggested the calcium sensor synaptotagmin (Syt) is a facilitator of vesicle docking and both a facilitator and inhibitor of fusion. On phospholipid monolayers, the Syt C2AB domain spontaneously oligomerized into rings that are disassembled by Ca 2+ , suggesting Syt rings may clamp fusion as membrane-separating "washers" until Ca 2+ -mediated disassembly triggers fusion and release [J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 111, 13966-13971 (2014)].). Here, we combined mathematical modeling with experiment to measure the mechanical properties of Syt rings and to test this mechanism. Consistent with experimental results, the model quantitatively recapitulates observed Syt ring-induced dome and volcano shapes on phospholipid monolayers and predicts rings are stabilized by anionic phospholipid bilayers or bulk solution with ATP. The selected ring conformation is highly sensitive to membrane composition and bulk ATP levels, a property that may regulate vesicle docking and fusion in ATP-rich synaptic terminals. We find the Syt molecules hosted by a synaptic vesicle oligomerize into a halo, unbound from the vesicle, but in proximity to sufficiently phosphatidylinositol 4,5-bisphosphate (PIP2)-rich plasma membrane (PM) domains, the PM-bound trans Syt ring conformation is preferred. Thus, the Syt halo serves as landing gear for spatially directed docking at PIP2-rich sites that define the active zones of exocytotic release, positioning the Syt ring to clamp fusion and await calcium. Our results suggest the Syt ring is both a Ca 2+ -sensitive fusion clamp and a high-fidelity sensor for directed docking.

Sociability stands as a crucial factor in the evolutionary success of all mammalian species. Notably, enriched environment (EE) housing has been shown to enhance sociability in mice. However, the precise underlying molecular mechanism remains elusive. In this study, we established an EE paradigm, housing mice for a 14-day period. Both enhanced sociability and an increased spine density in the medial prefrontal cortex (mPFC) of mice subjected to EE were detected. To elucidate the potential molecular pathway, we conducted high-performance liquid chromatography tandem mass spectrometry (HPLC-MS) analysis of the entire mPFC from both EE and home-caged (HC) housed mice. Our analysis identified 16 upregulated and 20 downregulated proteins in the EE group. Among them, Extended Synaptotagmin 1 (ESyt1), an activity-dependent endoplasmic reticulum (ER)-plasma membrane (PM) tethering protein associated with synaptic function and growth, emerged as a potentially key player in the increased synapse formation and enhanced sociability observed in EE-housed mice. Further investigation, involving the knockdown of ESyt1 expression via sh ESyt1 lentivirus in the mPFC, revealed that ESyt1 is crucial for increased spine density of mPFC and enhanced sociability of mice in an enriched environment but not in normal condition. Overall, our findings uncover a novel mechanistic insight into the positive influence of environmental enrichment on social behavior via ESyt1-mediated pathways., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Two recent papers by Mehta et al. and Zhu et al. in this issue (https://doi.org/10.1083/jcb.202311191) discover that synaptotagmin-1, the primary calcium sensor at the synapse, forms biomolecular condensates, identifying a new layer of regulation in calcium-triggered synaptic vesicle exocytosis., (© 2024 Tromm and Milovanovic.)

Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion., (© 2024 Zhu et al.)

Signal transmission between neurons requires exocytosis of neurotransmitters from the lumen of synaptic vesicles into the synaptic cleft. Following an influx of Ca 2+ , this process is facilitated by the Ca 2+ sensor synaptotagmin-1. The underlying mechanisms involve electrostatic and hydrophobic interactions tuning the lipid preferences of the two C2 domains of synaptotagmin-1; however, the details are still controversially discussed. We, therefore, follow a multidisciplinary approach and characterize lipid and membrane binding of the isolated C2A and C2B domains. We first target interactions with individual lipid species, and then study interactions with model membranes of liposomes. Finally, we perform molecular dynamics simulations to unravel differences in membrane binding. We found that both C2 domains, as a response to Ca 2+ , insert into the lipid membrane; however, C2A adopts a more perpendicular orientation while C2B remains parallel. These findings allow us to propose a mechanism for synaptotagmin-1 during membrane fusion., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fusion pore opening is a transient intermediate state of synaptic vesicle exocytosis, which is highly dynamic and precisely regulated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and synaptotagmin-1 (Syt1). Yet, the regulatory mechanism is not fully understood. In this work, using single-channel membrane fusion electrophysiology, we determined that SNAREpins are important for driving fusion pore opening and dilation but incapable of regulating the dynamics. When Syt1 was added, the closing frequency of fusion pores significantly increased, while the radius of fusion pores mildly decreased. In response to Ca2+, SNARE/Syt1 greatly increased the radius of fusion pores and reduced their closing frequency. Moreover, the residue F349 in the C2B domain of Syt1, which mediates Syt1 oligomerization, was required for clamping fusion pore opening in the absence of Ca2+, probably by extending the distance between the two membranes. Finally, in Ca2+-triggered fusion, the primary interface between SNARE and Syt1 plays a critical role in stabilizing and dilating the fusion pore, while the polybasic region of Syt1 C2B domain has a mild effect on increasing the radius of the fusion pore. In summary, our results suggest that Syt1, SNARE, and the anionic membrane synergically orchestrate the dynamics of fusion pore opening in synaptic vesicle exocytosis., (© The Author(s) (2024). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Acute kidney injury (AKI) is common and an independent risk factor for mortality in patients with paraquat (PQ) poisoning. Currently, no specific antidote is available. Synaptotagmin-1 (SYT1) has been identified as a key protein that facilitates PQ efflux in PQ-resistant A549 cells, thereby preventing PQ-induced lung injury. However, the protective effect of STY1 on PQ-induced AKI remains to be elucidated. This study exposed human kidney 2 (HK-2) cells overexpressing SYT1 to PQ. These cells exhibited significantly lower levels of growth inhibition, reactive oxygen species production, early apoptosis, and PQ accumulation compared to the parent HK-2 cells. Transcriptomic screening and Western blot analysis revealed that SYT1 overexpression significantly promoted the expression of glucose transporter 2 (GLUT2). Inhibition of GLUT2 completely abolished the protective effects of SYT1 overexpression in HK-2 cells and restored intracellular PQ concentrations. Further immunoprecipitation-shotgun and RNA interference experiments revealed that SYT1 binds to and stabilizes the protein SERPINE1 mRNA-binding protein 1 (SERBP1), enhancing the stability of GLUT2 mRNA and its protein levels. In summary, SYT1 antagonizes PQ intracellular accumulation and prevents nephrocyte toxicity by up-regulating SERBP1/GLUT2 expression. This study identifies a potential target for the treatment of PQ-induced AKI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Migrasomes, organelles crucial for cell communication, undergo distinct stages of nucleation, maturation, and expansion. The regulatory mechanisms of migrasome formation, particularly through biological cues, remain largely unexplored. This study reveals that calcium is essential for migrasome formation. Furthermore, we identify that Synaptotagmin-1 (Syt1), a well-known calcium sensor, is not only enriched in migrasomes but also indispensable for their formation. The calcium-binding ability of Syt1 is key to initiating migrasome formation. The recruitment of Syt1 to migrasome formation sites (MFS) triggers the swelling of MFS into unstable precursors, which are subsequently stabilized through the sequential recruitment of tetraspanins. Our findings reveal how calcium regulates migrasome formation and propose a sequential interaction model involving Syt1 and Tetraspanins in the formation and stabilization of migrasomes., (© 2024 Han and Yu.)

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B + vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

Plant synaptotagmins structurally resemble animal synaptotagmins and extended-synaptotagmins. Animal synaptotagmins are well-characterized calcium sensors in membrane trafficking, and extended-synaptotagmins mediate lipid transfer at the endoplasmic reticulum—plasma membrane contact sites. Here, we characterize SYNAPTOTAGMIN 4 (SYT4), which belongs to the six-member family in Arabidopsis. Fluorometric GUS assay showed that the SYT4 promoter was strongest in roots and the least active in rosettes and cauline leaves, which was confirmed by qPCR. In seedlings, promoter activity was influenced by several factors, such as plant growth regulators, mannitol, sucrose, polyethylene glycol and cold. GUS histochemistry revealed SYT4 promoter activity in the phloem of all organs and even almost exclusively in sieve element precursors and differentiating sieve elements. Accordingly, the SYT-GFP fusion protein also accumulated in these cells with maximal abundance in sieve element precursors. The protein formed a network in the cytoplasm, but during sieve tube differentiation, it deposited at the cell periphery and disappeared from mature tubes. Using photoconvertible fluorescence technology, we showed that a high abundance of SYT4 protein in meristematic protophloem cells was due to its extensive synthesis. SYT4 protein synthesis was interrupted in differentiating sieve elements, but protein degradation was also reduced. In addition to phloem, the fusion protein was detected in shoot and root stem cell niche as early as the late heart stage of the embryo. We isolated and molecularly and biologically characterized five syt4 T-DNA insertion alleles and subjected them to phenotype analysis. The allele with the C2B domain interrupted by an T-DNA insertion exhibits increased sensitivity to factors such as auxins, osmotics, salicylic acid, sodium chloride, and the absence of sucrose in the root growth test.

Abstract Endometrial cancer (EC) is one of the most common gynecologic malignancies. Identification of potential EC biomarkers is essential to improve the prognosis and development of therapies against EC. Synaptotagmin-like protein 1 (SYTL1), as a small GTPase Rab27 effector, mainly plays a role in vesicle trafficking and cytotoxic granule exocytosis in lymphocytes. However the role of SYTL1 in EC remains uncertain. We performed a comprehensive assessment of the relationship between SYTL1 and patient diagnosis and prognosis by analysis of EC patients’ data from TCGA. We employed the LinkedOmics and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database to analyze the biological function of SYTL1 in EC. In addition, the correlation between SYTL1 expression and its DNA methylation was performed by using cBioportal, UALCAN, TCGA Wanderer and MethSurv databases. We further assessed the link between SYTL1 and tumor-infiltrating immune cells by using gene set variation analysis (GSVA). Results We found that SYTL1 was highly expressed in EC patients and cell lines. And increased expression of SYTL1 was associated with age, clinical stage, histological type, histological grade and good overall survival (OS).SYTL1 DNA methylation is negatively associated with SYTL1 expression and UCEC patients’ OS. SYTL1 expression is closely correlated with immune infiltration. Furthermore, we carried out in vitro experiments to verify the results of bioinformatic analysis. Conclusion Our results demonstrated that the elevation of SYTL1 expression is associated with good OS and SYTL1 might be a potential diagnostic and prognostic marker in EC.

Abstract Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

Milena Barbosa Porto,1 Geovanna da Mata e Castro,1 Samara Socorro Silva Pereira,2 Elza Maria Gonçalves Santos Uchoa,2 Raffael Zatarin,3 Lysa Bernardes Minasi,1 Aparecido D da Cruz1– 3 1Graduate Program in Genetics, School of Medical and Life Sciences, Pontifical Catholic University of Goiás, Goiânia, GO, Brazil; 2Federal University of Goiás, Graduate Program in Genetics and Molecular Biology, Goiânia, GO, Brazil; 3Clinical Genetics Service, Center for Rehabilitation and Readaptation Dr. Henrique Santillo, State Health Secretary of Goiás, Goiânia, GO, BrazilCorrespondence: Aparecido D da Cruz, Email acruz@pucgoias.edu.brAbstract: Baker-Gordon Syndrome (BAGOS) is a genetically determined 4 (NDD), represented by a phenotypic spectrum of moderate to severe intellectual disability, resulting from mutations in the synaptotagmin 1 (SYT1) gene. Its prevalence is estimated at 1:1,000,000 and the known gene variants have indicated complete penetrance with variable expressivity. SYT1 is a membrane trafficking protein in presynaptic vesicles, which exerts a complex influence on synaptic transmission, with fundamental roles in the release of neurotransmitters and facilitators of endocytosis, impacting both neurotransmission and neuron plasticity. The current case report describes the first Brazilian male patient diagnosed at 17-year-old, and the 39th reported case globally using whole-exome sequencing. A de novo heterozygous missense mutation at chr12q:79448958 (NM_005639.2; c.1103T>C; p.Ile368Thr) in the SYT1 was found and classified as a pathogenic variant. The proband’s clinical phenotype was compatible with BAGOS, involving behavioral changes such as irritability and severe intellectual disability. Knowledge about the mechanism of action and the extent of the genotypic and phenotypic presentations of the mutations in the SYT1 is still unfolding. Thus, we aimed to describe additional genotype–phenotype correlation for BAGOS, contributing to the expansion of the existing knowledge of such a heterogeneous ultra-rare syndrome, and, therefore, improve its diagnostic yield, case management, and therapeutic journey for future patients. Keywords: SYT1, hypotonia, congenital malformations, NDD, WES

Summary: GPR133 (ADGRD1) is an adhesion G-protein-coupled receptor that signals through Gαs/cyclic AMP (cAMP) and is required for the growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca2+-dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca2+-sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca2+ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM slows tumor growth, suggesting tumorigenic functions of ESYT1. Our findings demonstrate a mechanism for the modulation of GPR133 signaling by increased cytosolic Ca2+, which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels.

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b-synaptotagmin complex constitutes the high-affinity BoNT/B receptor., Competing Interests: The authors declare no conflict of interest.

Injuries to the central nervous system (CNS) can cause severe neurological deficits. Axonal regrowth is a fundamental process for the reconstruction of compensatory neuronal networks after injury; however, it is extremely limited in the adult mammalian CNS. In this study, we conducted a loss-of-function genetic screen in cortical neurons, combined with a web-resource-based phenotypic screen, and identified synaptotagmin 4 (Syt4) as a novel regulator of axon elongation. Silencing Syt4 in primary cultured cortical neurons inhibits neurite elongation, with changes in gene expression involved in signaling pathways related to neuronal development. In a spinal cord injury model, inhibition of Syt4 expression in cortical neurons prevented axonal sprouting of the corticospinal tract, as well as neurological recovery after injury. These results provide a novel therapeutic approach to CNS injury by modulating Syt4 function. Significance Statement Promoting axonal regrowth has been considered a promising therapeutic target for functional recovery after central nervous system injury. In the present study, we used a web-resource-based phenotypic screening followed by loss-of-functional screening for neurite elongation. We identified a novel role of synaptotagmin 4 (Syt4), a well-characterized regulator of synaptic function, in neurite elongation. In addition, Syt4 knockdown in corticospinal tract inhibited axonal regrowth and functional recovery after spinal cord injury. In this way, we provide a new screening method, which allows us to efficiently find new targets regulating CNS therapeutics., (Copyright © 2024 Higuchi et al.)

Reactive oxygen species (ROS)-induced oxidative DNA damages have been considered the main cause of mutations in genes, which are highly related to carcinogenesis and tumour progression. Extracellular vesicles play an important role in cancer metastasis. However, the precise role of DNA oxidative damage in extracellular vesicles (EVs)-mediated cancer cell migration and invasion remains unclear. Here, we reveal that ROS-mediated DNA oxidative damage signalling promotes tumour metastasis through increasing EVs release. Mechanistically, 8-oxoguanine DNA glycosylase (OGG1) recognises and binds to its substrate 8-oxo-7,8-dihydroguanine (8-oxoG), recruiting NF-κB to the synaptotagmin 7 (SYT7) promoter and thereby triggering SYT7 transcription. The upregulation of SYT7 expression leads to increased release of E-cadherin-loaded EVs, which depletes intracellular E-cadherin, thereby inducing epithelial-mesenchymal transition (EMT). Notably, Th5487, the inhibitor of DNA binding activity of OGG1, blocks the recognition and transmission of oxidative signals, alleviates SYT7 expression and suppresses EVs release, thereby preventing tumour progression in vitro and in vivo. Collectively, our study illuminates the significance of 8-oxoG/OGG1/SYT7 axis-driven EVs release in oxidative stress-induced tumour metastasis. These findings provide a deeper understanding of the molecular basis of cancer progression and offer potential avenues for therapeutic intervention., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

SYT13 is one of the atypical members of the synaptotagmin (SYT) family whose function has attracted considerable attention in recent years. Although SYT13 has been studied in several types of human cancers, such as lung cancer, its role in esophageal squamous cell carcinoma (ESCC) is still unclear. It was demonstrated that SYT13 is significantly upregulated in ESCC tissues compared with normal ones and correlated with higher degree of malignancy. Knockdown of SYT13 could inhibit ESCC cell proliferation and migration, while promoting cell apoptosis. Meanwhile, ESCC cells with relatively lower SYT13 expression grew slower in vivo and finally formed smaller xenografts. Furthermore, acrosomal vesicular protein 1 was identified as a potential downstream target of SYT13, which regulates cell phenotypes of ESCC cells in cooperation with SYT13. All the in vitro and in vivo results in this study identified that SYT13 silencing could be an effective strategy to inhibit the development of ESCC, which could be considered as a promising therapeutic target in the treatment of ESCC.

The Synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain is found in a group of ER-resident lipid transfer proteins that are recruited to membrane contact sites (MCSs) by adaptors. Deciphering the molecular basis underlying the recruitment of SMP proteins to specific MCS sheds light not only on their cellular localization but also on their biological functions at these sites. Here we summarize recent advances in SMP domain-containing lipid transfer proteins, focusing on a recent study showing the localization, regulation and cellular function of a specific SMP protein named testis expressed protein 2 (Tex2). TMEM55, a potential PIP phosphatase on late endosome/lysosomal (LE/lys) membranes, was identified as an adaptor that enables the recruitment of Tex2 to ER- LE/lys MCS. In addition, we have summarized several important questions about the regulation and physiological functions of Tex2 that remained unanswered., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Cervical squamous cell carcinoma (CESC) ranks among the primary contributors to global cancer-associated mortality. However, the role mediated by synaptotagmin 7 (SYT7) in CESC remains unclear. Our study employed immunohistochemistry to assess the level of SYT7 expression in the tissue microarray. Furthermore, lentiviral shRNA transduction was utilized to establish SYT7 knockdown cell line models based on HeLa and SiHa cell lines. The functional impacts of silencing SYT7 expression in vitro were evaluated. A subcutaneous xenograft model was employed to examine the tumorigenic potential of cells with or without SYT7. The content of SYT7 in CESC tissues was significantly elevated compared to adjacent normal tissues. Functionally, silencing SYT7 in HeLa and SiHa cells suppressed cell proliferation, colony formation ability, and apoptosis enhancement. Additionally, cells with suppressed SYT7 also exhibited inhibited cell migration and invasion. In vivo experiments demonstrated the loss of tumorigenic ability in SYT7 knockdown cells and suppressed tumor growth. Quantitative PCR PrimeView PathArray and apoptosis antibody array analyses revealed that upon elimination of SYT7, there was a significant upregulation observed in Caspase 8, TNF-R1 (TNF receptor superfamily member 1A), and HSPA5 (heat shock protein family A [Hsp70] member 5), while TGFBI (transforming growth factor beta-induced), RPL31 (ribosomal protein L31), LUM (lumican), HSDL2 (hydroxysteroid dehydrogenase-like 2), ITGB5 (integrin subunit beta 5), and Smad2 (SMAD family member2) were downregulated. Overall, we have demonstrated the tumor-promoting functions of SYT7 in CESC.

Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABCD antibodies- ABclonal- Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific., (Copyright: © 2024 Biddle MS et al.)

Synaptotagmin-1 (Syt1) is a presynaptic calcium sensor with two calcium binding domains, C2A and C2B, that triggers action potential-induced synchronous neurotransmitter release, while suppressing asynchronous and spontaneous release. We identified a de novo missense mutation (P401L) in the C2B domain in a patient with developmental delay and autistic symptoms. Expressing the orthologous mouse mutant (P400L) in cultured Syt1 null mutant neurons revealed a reduction in dendrite outgrowth with a proportional reduction in synapses. This was not observed in single Syt1 PL -rescued neurons that received normal synaptic input when cultured in a control network. Patch-clamp recordings showed that spontaneous miniature release events per synapse were increased more than 500% in Syt1 PL -rescued neurons, even beyond the increased rates in Syt1 KO neurons. Furthermore, action potential-induced asynchronous release was increased more than 100%, while synchronous release was unaffected. A similar shift to more asynchronous release was observed during train stimulations. These cellular phenotypes were also observed when Syt1 PL was overexpressed in wild type neurons. Our findings show that Syt1 PL desynchronizes neurotransmission by increasing the readily releasable pool for asynchronous release and reducing the suppression of spontaneous and asynchronous release. Neurons respond to this by shortening their dendrites, possibly to counteract the increased synaptic input. Syt1 PL acts in a dominant-negative manner supporting a causative role for the mutation in the heterozygous patient. We propose that the substitution of a rigid proline to a more flexible leucine at the bottom of the C2B domain impairs clamping of release by interfering with Syt1's primary interface with the SNARE complex. This is a novel cellular phenotype, distinct from what was previously found for other SYT1 disease variants, and points to a role for spontaneous and asynchronous release in SYT1-associated neurodevelopmental disorder., (© 2024. The Author(s).)

Synaptic dysfunction is a core component of the pathophysiology of schizophrenia. However, the genetic risk factors and molecular mechanisms related to synaptic dysfunction are still not fully understood. The Stonin 2 (STON2) gene encodes a major adaptor for clathrin-mediated endocytosis (CME) of synaptic vesicles. In this study, we showed that the C-C (307Pro-851Ala) haplotype of STON2 increases the susceptibility to schizophrenia and examined whether STON2 variations cause schizophrenia-like behaviors through the regulation of CME. We found that schizophrenia-related STON2 variations led to protein dephosphorylation, which affected its interaction with synaptotagmin 1 (Syt1), a calcium sensor protein located in the presynaptic membrane that is critical for CME. STON2 307Pro851Ala knockin mice exhibited deficits in synaptic transmission, short-term plasticity, and schizophrenia-like behaviors. Moreover, among seven antipsychotic drugs, patients with the C-C (307Pro-851Ala) haplotype responded better to haloperidol than did the T-A (307Ser-851Ser) carriers. The recovery of deficits in Syt1 sorting and synaptic transmission by acute administration of haloperidol effectively improved schizophrenia-like behaviors in STON2 307Pro851Ala knockin mice. Our findings demonstrated the effect of schizophrenia-related STON2 variations on synaptic dysfunction through the regulation of CME, which might be attractive therapeutic targets for treating schizophrenia-like phenotypes., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)