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Neurotransmitters are released from synaptic vesicles, the membrane of which fuses with the plasma membrane upon calcium influx. This membrane fusion reaction is driven by the formation of a tight complex comprising the plasma membrane N‐ethylmaleimide‐sensitive factor attachment receptor (SNARE) proteins syntaxin‐1a and SNAP‐25 with the vesicle SNARE protein synaptobrevin. The neuronal protein Munc18‐1 forms a stable complex with syntaxin‐1a. Biochemically, syntaxin‐1a cannot escape the tight grip of Munc18‐1, so formation of the SNARE complex is inhibited. However, Munc18‐1 is essential for the release of neurotransmitters in vivo. It has therefore been assumed that Munc18‐1 makes the bound syntaxin‐1a available for SNARE complex formation. Exactly how this occurs is still unclear, but it is assumed that structural rearrangements occur. Here, we used a series of mutations to specifically weaken the complex at different positions in order to induce these rearrangements biochemically. Our approach was guided through sequence and structural analysis and supported by molecular dynamics simulations. Subsequently, we created a homology model showing the complex in an altered conformation. This conformation presumably represents a more open arrangement of syntaxin‐1a that permits the formation of a SNARE complex to be initiated while still bound to Munc18‐1. In the future, research should investigate how this central reaction for neuronal communication is controlled by other proteins. [ABSTRACT FROM AUTHOR]

Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines. [ABSTRACT FROM AUTHOR]

Synaptic neurotransmitter release is an evolutionarily conserved process that mediates rapid information transfer between neurons as well as several peripheral tissues. Release of neurotransmitters are ensured by successive events such as synaptic vesicle docking and priming that prepare synaptic vesicles for rapid fusion. These events are orchestrated by interaction of different presynaptic proteins and are regulated by presynaptic calcium. Recent studies have identified various mutations in different components of neurotransmitter release machinery resulting in aberrant neurotransmitter release, which underlie a wide spectrum of psychiatric and neurological symptoms. Here, we review how these genetic alterations in different components of the core neurotransmitter release machinery affect the information transfer between neurons and how aberrant synaptic release affects nervous system function.

Neurotransmitter-filled synaptic vesicles (SVs) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading model posits that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH (retention using selective hooks) system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct transmembrane SV proteins, synaptotagmin 1 and synaptobrevin 2, from the soma of mature cultured rat and mouse neurons. For these studies, the SV reporter constructs were expressed at carefully controlled, very low levels. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons with minimal entry into dendrites. However, even moderate overexpression resulted in the spillover of SV proteins into dendrites, potentially explaining the origin of previous non-polarized transport models, revealing the limited, saturable nature of the direct axonal trafficking pathway. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway, for SV proteins, in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.

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., (© 2024 The Author(s).)

Neurotransmitters are released from synaptic vesicles, the membrane of which fuses with the plasma membrane upon calcium influx. This membrane fusion reaction is driven by the formation of a tight complex comprising the plasma membrane N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins syntaxin-1a and SNAP-25 with the vesicle SNARE protein synaptobrevin. The neuronal protein Munc18-1 forms a stable complex with syntaxin-1a. Biochemically, syntaxin-1a cannot escape the tight grip of Munc18-1, so formation of the SNARE complex is inhibited. However, Munc18-1 is essential for the release of neurotransmitters in vivo. It has therefore been assumed that Munc18-1 makes the bound syntaxin-1a available for SNARE complex formation. Exactly how this occurs is still unclear, but it is assumed that structural rearrangements occur. Here, we used a series of mutations to specifically weaken the complex at different positions in order to induce these rearrangements biochemically. Our approach was guided through sequence and structural analysis and supported by molecular dynamics simulations. Subsequently, we created a homology model showing the complex in an altered conformation. This conformation presumably represents a more open arrangement of syntaxin-1a that permits the formation of a SNARE complex to be initiated while still bound to Munc18-1. In the future, research should investigate how this central reaction for neuronal communication is controlled by other proteins., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Synaptic neurotransmitter release is an evolutionarily conserved process that mediates rapid information transfer between neurons as well as several peripheral tissues. Release of neurotransmitters are ensured by successive events such as synaptic vesicle docking and priming that prepare synaptic vesicles for rapid fusion. These events are orchestrated by interaction of different presynaptic proteins and are regulated by presynaptic calcium. Recent studies have identified various mutations in different components of neurotransmitter release machinery resulting in aberrant neurotransmitter release, which underlie a wide spectrum of psychiatric and neurological symptoms. Here, we review how these genetic alterations in different components of the core neurotransmitter release machinery affect the information transfer between neurons and how aberrant synaptic release affects nervous system function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Uzay and Kavalali.)

Neurotransmitter-filled synaptic vesicles (SVs) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading model posits that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH (retention using selective hooks) system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct transmembrane SV proteins, synaptotagmin 1 and synaptobrevin 2, from the soma of mature cultured rat and mouse neurons. For these studies, the SV reporter constructs were expressed at carefully controlled, very low levels. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons with minimal entry into dendrites. However, even moderate overexpression resulted in the spillover of SV proteins into dendrites, potentially explaining the origin of previous non-polarized transport models, revealing the limited, saturable nature of the direct axonal trafficking pathway. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway, for SV proteins, in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis., Competing Interests: EW, MP, MS, JV, EC No competing interests declared, (© 2023, Watson et al.)

Polarized transport is essential for constructing multiple plasma membrane domains in the cell. Drosophila photoreceptors are an excellent model system to study the mechanisms of polarized transport. Rab11 is the key factor regulating the post-Golgi transport of rhodopsin 1 (Rh1; also known as NinaE), a photoreceptive protein, to the rhabdomere, a photoreceptive plasma membrane. Here, we found that neuronal Synaptobrevin (nSyb) colocalizes with Rab11 on the trans-side of Golgi stacks and post-Golgi vesicles at the rhabdomere base, and nSyb deficiency impairs rhabdomeric transport and induces accumulation of Rh1 and vesicles in the cytoplasm; this is similar to the effects of Rab11 loss. These results indicate that nSyb acts as a post-Golgi SNARE toward rhabdomeres. Surprisingly, in Rab11-, Rip11- and nSyb-deficient photoreceptors, illumination enhances cytoplasmic accumulation of Rh1, which colocalizes with Rab11, Rabenosyn5, nSyb and Arrestin 1 (Arr1). Arr1 loss, but not Rab5 dominant negative (Rab5DN) protein expression, inhibits the light-enhanced cytoplasmic Rh1 accumulation. Rab5DN inhibits the generation of Rh1-containing multivesicular bodies rather than Rh1 internalization. Overall, these results indicate that exocytic Rh1 mingles with endocytosed Rh1 and is then transported together to rhabdomeres., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)