Neurotransmitter release is triggered by a calcium-induced rearrangement in the Synaptotagmin-1/SNARE complex primary interface.

The Ca2+ 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 C2B 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 Ca2+ 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 Ca2+-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 Ca2+ 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.

Tissue-specific knockout in the Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles.

Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity.

Syntaxin 3B: A SNARE Protein Required for Vision.

Syntaxin 3 is a member of a large protein family of syntaxin proteins that mediate fusion between vesicles and their target membranes. Mutations in the ubiquitously expressed syntaxin 3A splice form give rise to a serious gastrointestinal disorder in humans called microvillus inclusion disorder, while mutations that additionally involve syntaxin 3B, a splice form that is expressed primarily in retinal photoreceptors and bipolar cells, additionally give rise to an early onset severe retinal dystrophy. In this review, we discuss recent studies elucidating the roles of syntaxin 3B and the regulation of syntaxin 3B functionality in membrane fusion and neurotransmitter release in the vertebrate retina.

Migfilin promotes autophagic flux through direct interaction with SNAP29 and Vamp8.

Autophagy plays a crucial role in cancer cell survival by facilitating the elimination of detrimental cellular components and the recycling of nutrients. Understanding the molecular regulation of autophagy is critical for developing interventional approaches for cancer therapy. In this study, we report that migfilin, a focal adhesion protein, plays a novel role in promoting autophagy by increasing autophagosome-lysosome fusion. We found that migfilin is associated with SNAP29 and Vamp8, thereby facilitating Stx17-SNAP29-Vamp8 SNARE complex assembly. Depletion of migfilin disrupted the formation of the SNAP29-mediated SNARE complex, which consequently blocked the autophagosome-lysosome fusion, ultimately suppressing cancer cell growth. Restoration of the SNARE complex formation rescued migfilin-deficiency-induced autophagic flux defects. Finally, we found depletion of migfilin inhibited cancer cell proliferation. SNARE complex reassembly successfully reversed migfilin-deficiency-induced inhibition of cancer cell growth. Taken together, our study uncovers a new function of migfilin as an autophagy-regulatory protein and suggests that targeting the migfilin-SNARE assembly could provide a promising therapeutic approach to alleviate cancer progression.

Migratory autolysosome disposal mitigates lysosome damage.

Lysosomes, essential for intracellular degradation and recycling, employ damage-control strategies such as lysophagy and membrane repair mechanisms to maintain functionality and cellular homeostasis. Our study unveils migratory autolysosome disposal (MAD), a response to lysosomal damage where cells expel LAMP1-LC3 positive structures via autolysosome exocytosis, requiring autophagy machinery, SNARE proteins, and cell migration. This mechanism, crucial for mitigating lysosomal damage, underscores the role of cell migration in lysosome damage control and facilitates the release of small extracellular vesicles, highlighting the intricate relationship between cell migration, organelle quality control, and extracellular vesicle release.

Dynamic formation of the protein-lipid prefusion complex.

Synaptic vesicles (SVs) fuse with the presynaptic membrane (PM) to release neuronal transmitters. The SV protein synaptotagmin 1 (Syt1) serves as a Ca2+ sensor for evoked fusion. Syt1 is thought to trigger fusion by penetrating the PM upon Ca2+ binding; however, the mechanistic detail of this process is still debated. Syt1 interacts with the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex, a coiled-coil four-helical bundle that enables the SV-PM attachment. The SNARE-associated protein complexin (Cpx) promotes Ca2+-dependent fusion, possibly interacting with Syt1. We employed all-atom molecular dynamics to investigate the formation of the Syt1-SNARE-Cpx complex interacting with the lipid bilayers of the PM and SVs. Our simulations demonstrated that the PM-Syt1-SNARE-Cpx complex can transition to a "dead-end" state, wherein Syt1 attaches tightly to the PM but does not immerse into it, as opposed to a prefusion state, which has the tips of the Ca2+-bound C2 domains of Syt1 inserted into the PM. Our simulations unraveled the sequence of Syt1 conformational transitions, including the simultaneous docking of Syt1 to the SNARE-Cpx bundle and the PM, followed by Ca2+ chelation and the penetration of the tips of Syt1 domains into the PM, leading to the prefusion state of the protein-lipid complex. Importantly, we found that direct Syt1-Cpx interactions are required to promote these transitions. Thus, we developed the all-atom dynamic model of the conformational transitions that lead to the formation of the prefusion PM-Syt1-SNARE-Cpx complex. Our simulations also revealed an alternative dead-end state of the protein-lipid complex that can be formed if this pathway is disrupted.

A condensates-to-VPS41-associated phagic vacuoles conversion pathway controls autophagy degradation in plants.

Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants.

Vacuolar recruitment of retromer by a SNARE complex enables infection-related trafficking in rice blast.

The retromer complex is a conserved sorting machinery that maintains cellular protein homeostasis by transporting vesicles containing cargo proteins to defined destinations. It is known to sort proteins at the vacuole membranes for retrograde trafficking, preventing their degradation in the vacuole. However, the detailed mechanism of retromer recruitment to the vacuole membrane has not yet been elucidated. Here, we show that the vacuolar SNARE complex MoPep12-MoVti1-MoVam7-MoYkt6 regulates retromer-mediated vesicle trafficking by recruiting the retromer to the vacuole membrane, which promotes host invasion in Magnaporthe oryzae. Such recruitment is also essential for the retrieval of the autophagy regulator MoAtg8 and enables appressorium-mediated host penetration. Furthermore, the vacuolar SNARE subunits are involved in suppressing the host defense response by regulating the deployment of retromer-MoSnc1-mediated effector secretion. Altogether, our results provide insights into the mechanism of vacuolar SNAREs-dependent retromer recruitment which is necessary for pathogenicity-related membrane trafficking events in the rice blast fungus.

A look beyond the QR code of SNARE proteins.

Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor (SNARE) proteins catalyze the fusion process of vesicles with target membranes in eukaryotic cells. To do this, they assemble in a zipper-like fashion into stable complexes between the membranes. Structural studies have shown that the complexes consist of four different helices, which we subdivide into Qa-, Qb-, Qc-, and R-helix on the basis of their sequence signatures. Using a combination of biochemistry, modeling and molecular dynamics, we investigated how the four different types are arranged in a complex. We found that there is a matching pattern in the core of the complex that dictates the position of the four fundamental SNARE types in the bundle, resulting in a QabcR complex. In the cell, several different cognate QabcR-SNARE complexes catalyze the different transport steps between the compartments of the endomembrane system. Each of these cognate QabcR complexes is compiled from a repertoire of about 20 SNARE subtypes. Our studies show that exchange within the four types is largely tolerated structurally, although some non-cognate exchanges lead to structural imbalances. This suggests that SNARE complexes have evolved for a catalytic mechanism, a mechanism that leaves little scope for selectivity beyond the QabcR rule.

VAMP2 chaperones α-synuclein in synaptic vesicle co-condensates.

α-Synuclein (α-Syn) aggregation is closely associated with Parkinson's disease neuropathology. Physiologically, α-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of α-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)-a component of the SNARE complex that resides on SVs-directly interacts with the carboxy-terminal region of α-Syn through charged residues to regulate α-Syn's function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, α-Syn and other components. Moreover, VAMP2 binding protects α-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains α-Syn's function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson's disease.

Intermediate steps in the formation of neuronal SNARE complexes.

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature-dependent with a reduced concentration at 37 °C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25 and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.

Synaptotagmin-1 undergoes phase separation to regulate its calcium-sensitive oligomerization.

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.

Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

Exploring the structural dynamics of the vesicle priming machinery.

Various cell types release neurotransmitters, hormones and many other compounds that are stored in secretory vesicles by exocytosis via the formation of a fusion pore traversing the vesicular membrane and the plasma membrane. This process of membrane fusion is mediated by the Soluble N-ethylmaleimide-Sensitive Factor Attachment Proteins REceptor (SNARE) protein complex, which in neurons and neuroendocrine cells is composed of the vesicular SNARE protein Synaptobrevin and the plasma membrane proteins Syntaxin and SNAP25 (Synaptosomal-Associated Protein of 25 kDa). Before a vesicle can undergo fusion and release of its contents, it must dock at the plasma membrane and undergo a process named 'priming', which makes it ready for release. The primed vesicles form the readily releasable pool, from which they can be rapidly released in response to stimulation. The stimulus is an increase in Ca2+ concentration near the fusion site, which is sensed primarily by the vesicular Ca2+ sensor Synaptotagmin. Vesicle priming involves at least the SNARE proteins as well as Synaptotagmin and the accessory proteins Munc18, Munc13, and Complexin but additional proteins may also participate in this process. This review discusses the current views of the interactions and the structural changes that occur among the proteins of the vesicle priming machinery.

A matter of timing.

A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.

LKS4-mediated SYP121 phosphorylation participates in light-induced stomatal opening in Arabidopsis.

By modulating stomatal opening and closure, plants control gas exchange, water loss, and photosynthesis in response to various environmental signals. During light-induced stomatal opening, the transport of ions and solutes across the plasma membrane (PM) of the surrounding guard cells results in an increase in turgor pressure, leading to cell swelling. Simultaneously, vesicles for exocytosis are delivered via membrane trafficking to compensate for the enlarged cell surface area and maintain an appropriate ion-channel density in the PM. In eukaryotic cells, soluble N-ethylmaleimide-sensitive factor adaptor protein receptors (SNAREs) mediate membrane fusion between vesicles and target compartments by pairing the cognate glutamine (Q)- and arginine (R)-SNAREs to form a core SNARE complex. Syntaxin of plants 121 (SYP121) is a known Q-SNARE involved in stomatal movement, which not only facilitates the recycling of K+ channels to the PM but also binds to the channels to regulate their activity. In this study, we found that the expression of a receptor-like cytoplasmic kinase, low-K+ sensitive 4/schengen 1 (LKS4/SGN1), was induced by light; it directly interacted with SYP121 and phosphorylated T270 within the SNARE motif. Further investigation revealed that LKS4-dependent phosphorylation of SYP121 facilitated the interaction between SYP121 and R-SNARE vesicle-associated membrane protein 722 (VAMP722), promoting the assembly of the SNARE complex. Our findings demonstrate that the phosphorylation of SNARE proteins is an important strategy adopted by plants to regulate the SNARE complex assembly as well as membrane fusion. Additionally, we discovered the function of LKS4/SGN1 in light-induced stomatal opening via the phosphorylation of SYP121.

Localized, highly efficient secretion of signaling proteins by migrasomes.

Migrasomes, enriched with signaling molecules such as chemokines, cytokines and angiogenic factors, play a pivotal role in the spatially defined delivery of these molecules, influencing critical physiological processes including organ morphogenesis and angiogenesis. The mechanism governing the accumulation of signaling molecules in migrasomes has been elusive. In this study, we show that secretory proteins, including signaling proteins, are transported into migrasomes by secretory carriers via both the constitutive and regulated secretion pathways. During cell migration, a substantial portion of these carriers is redirected to the rear of the cell and actively transported into migrasomes, driven by the actin-dependent motor protein Myosin-5a. Once at the migrasomes, these carriers fuse with the migrasome membrane through SNARE-mediated mechanisms. Inhibiting migrasome formation significantly reduces secretion, suggesting migrasomes as a principal secretion route in migrating cells. Our findings reveal a specialized, highly localized secretion paradigm in migrating cells, conceptually paralleling the targeted neurotransmitter release observed in neuronal systems.

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation.

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.

Dual-Ring SNAREpin Machinery Tuning for Fast Synaptic Vesicle Fusion.

During neurotransmission, neurotransmitters are released less than a millisecond after the arrival of the action potential. To achieve this ultra-fast event, the synaptic vesicle must be pre-docked to the plasma membrane. In this primed state, SNAREpins, the protein-coiled coils whose assembly provides the energy to trigger fusion, are partly zippered and clamped like a hairpin and held open and ready to snap close when the clamp is released. Recently, it was suggested that three types of regulatory factors, synaptophysin, synaptotagmins, and complexins act cooperatively to organize two concentric rings, a central and a peripheral ring, containing up to six SNAREpins each. We used a mechanical model of the SNAREpins with two separate states, half-zippered and fully zippered, and determined the energy landscape according to the number of SNAREpins in each ring. We also performed simulations to estimate the fusion time in each case. The presence of the peripheral SNAREpins generally smoothens the energy landscape and accelerates the fusion time. With the predicted physiological numbers of six central and six peripheral SNAREpins, the fusion time is accelerated at least 100 times by the presence of the peripheral SNAREpins, and fusion occurs in less than 10 µs, which is well within the physiological requirements.

Rab8a/SNARE complex activation promotes vesicle anchoring and transport in spinal astrocytes to drive neuropathic pain.

Neuropathic pain (NPP) remains a clinically challenging condition, driven by the activation of spinal astrocytes and the complex release of inflammatory mediators. This study aimed to examine the roles of Rab8a and SNARE complex proteins in activated astrocytes to uncover the underlying mechanisms of NPP. The research was conducted using a rat model with chronic constriction injury (CCI) of the sciatic nerve and primary astrocytes treated with lipopolysaccharide. Enhanced expression of Rab8a was noted specifically in spinal dorsal horn astrocytes through immunofluorescence. Electron microscopy observations showed increased vesicular transport and exocytic activity in activated astrocytes, which was corroborated by elevated levels of inflammatory cytokines such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α detected through quantitative PCR. Western blot analyses confirmed significant upregulation of Rab8a, VAMP2, and Syntaxin16 in these cells. Furthermore, the application of botulinum neurotoxin type A (BONT/A) reduced the levels of vesicle transport-associated proteins, inhibiting vesicular transport in activated astrocytes. These findings suggest that the Rab8a/SNARE pathway in astrocytes enhances vesicle transport and anchoring, increasing the secretion of bioactive molecules that may play a crucial role in the pathophysiology of NPP. Inhibiting this pathway with BONT/A offers a novel therapeutic target for managing NPP, highlighting its potential utility in clinical interventions.