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Neurons communicate via release of neurotransmitters at synapses, which requires hundreds of different proteins. Here, autophagy plays a key role in protein maintenance and degradation, with vesicles containing the protein ATG9 being crucial for the formation of autophagosomes. We found that these vesicles, while resembling synaptic vesicles in size and density, represent a separate and heterogeneous vesicle population in the synapse. They serve as a platform for the recruitment and assembly of various effector proteins required for interaction with different intracellular organelles. [ABSTRACT FROM AUTHOR]

In neurons, autophagosome biogenesis occurs mainly in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of membrane lipids which requires small vesicles containing ATG9, a lipid scramblase essential for macroautophagy/autophagy. Here, we show that ATG9-containing vesicles are enriched in synapses and resemble synaptic vesicles in size and density. The proteome of ATG9-containing vesicles immuno-isolated from nerve terminals showed conspicuously low levels of trafficking proteins except of the AP2-complex and some enzymes involved in endosomal phosphatidylinositol metabolism. Super resolution microscopy of nerve terminals and isolated vesicles revealed that ATG9-containing vesicles represent a distinct vesicle population with limited overlap not only with synaptic vesicles but also other membranes of the secretory pathway, uncovering a surprising heterogeneity in their membrane composition. Our results are compatible with the view that ATG9-containing vesicles function as lipid shuttles that scavenge membrane lipids from various intracellular membranes to support autophagosome biogenesis. Abbreviations: AP: adaptor related protein complex: ATG2: autophagy related 2; ATG9: autophagy related 9; DNA PAINT: DNA-based point accumulation for imaging in nanoscale topography; DyMIN STED: dynamic minimum stimulated emission depletion; EL: endosome and lysosome; ER: endoplasmic reticulum; GA: Golgi apparatus; iBAQ: intensity based absolute quantification; LAMP: lysosomal-associated membrane protein; M6PR: mannose-6-phosphate receptor, cation dependent; Minflux: minimal photon fluxes; Mito: mitochondria; MS: mass spectrometry; PAS: phagophore assembly site; PM: plasma membrane; Px: peroxisome; RAB26: RAB26, member RAS oncogene family; RAB3A: RAB3A, member RAS oncogene family; RAB5A: RAB5A, member RAS oncogene family; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment receptor; SVs: synaptic vesicles; SYP: synaptophysin; TGN: trans-Golgi network; TRAPP: transport protein particle; VTI1: vesicle transport through interaction with t-SNAREs. [ABSTRACT FROM AUTHOR]

In the secretory pathway the destination of trafficking vesicles is determined by specific proteins that, with the notable exception of SNAREs, are recruited from soluble pools. Previously we have shown that microinjected proteoliposomes containing early or late endosomal SNAREs, respectively, are targeted to the corresponding endogenous compartments, with targeting specificity being dependent on the recruitment of tethering factors by some of the SNAREs. Here, we show that targeting of SNARE-containing liposomes is refined upon inclusion of polyphosphoinositides and Rab5. Intriguingly, targeting specificity is dependent on the concentration of PtdIns(3)P, and on the recruitment of PtdIns(3)P binding proteins such as rabenosyn-5 and PIKfyve, with conversion of PtdIns(3)P into PtdIns(3,5)P2 re-routing the liposomes towards late endosomes despite the presence of GTP-Rab5 and early endosomal SNAREs. Our data reveal a complex interplay between permissive and inhibitory targeting signals that sharpen a basic targeting and fusion machinery for conveying selectivity in intracellular membrane traffic.Targeting of transport vesicles requires specific proteins and membrane lipids. Here, authors microinjected liposomes with a predetermined composition to show that targeting by SNAREs is refined when polyphosphoinositides and Rab GTPases are included. [ABSTRACT FROM AUTHOR]

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle–plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step. [ABSTRACT FROM AUTHOR]

We study the formation of vesicle condensates induced by the protein synapsin, as a cell-free model system mimicking vesicle pool formation in the synapse. The system can be considered as an example of liquid–liquid phase separation (LLPS) in biomolecular fluids, where one phase is a complex fluid itself consisting of vesicles and a protein network. We address the pertinent question why the LLPS is self-limiting and stops at a certain size, i.e., why macroscopic phase separation is prevented. Using fluorescence light microscopy, we observe different morphologies of the condensates (aggregates) depending on the protein-to-lipid ratio. Cryogenic electron microscopy then allows us to resolve individual vesicle positions and shapes in a condensate and notably the size and geometry of adhesion zones between vesicles. We hypothesize that the membrane tension induced by already formed adhesion zones then in turn limits the capability of vesicles to bind additional vesicles, resulting in a finite condensate size. In a simple numerical toy model we show that this effect can be accounted for by redistribution of effective binding particles on the vesicle surface, accounting for the synapsin-induced adhesion zone. [ABSTRACT FROM AUTHOR]

Synapse diversity has been described from different perspectives, ranging from the specific neurotransmitters released, to their diverse biophysical properties and proteome profiles. However, synapse diversity at the transcriptional level has not been systematically identified across all synapse populations in the brain. To quantify and identify specific synaptic features of neuronal cell types we combined the SynGO (Synaptic Gene Ontology) database with single-cell RNA sequencing data of the mouse neocortex. We show that cell types can be discriminated by synaptic genes alone with the same power as all genes. The cell type discriminatory power is not equally distributed across synaptic genes as we could identify functional categories and synaptic compartments with greater cell type specific expression. Synaptic genes, and specific SynGO categories, belonged to three different types of gene modules: gradient expression over all cell types, gradient expression in selected cell types and cell class- or type-specific profiles. This data provides a deeper understanding of synapse diversity in the neocortex and identifies potential markers to selectively identify synapses from specific neuronal populations. [ABSTRACT FROM AUTHOR]

Vacuolar-type adenosine triphosphatases (V-ATPases)1–3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyse ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by about one V-ATPase molecule per synaptic vesicle6,7. To shed light on this bona fide single-molecule biological process, we investigated electrogenic proton-pumping by single mammalian-brain V-ATPases in single synaptic vesicles. Here we show that V-ATPases do not pump continuously in time, as suggested by observing the rotation of bacterial homologues8 and assuming strict ATP–proton coupling. Instead, they stochastically switch between three ultralong-lived modes: proton-pumping, inactive and proton-leaky. Notably, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. ATP regulates V-ATPase activity through the switching probability of the proton-pumping mode. By contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. A direct consequence of mode-switching is all-or-none stochastic fluctuations in the electrochemical gradient of synaptic vesicles that would be expected to introduce stochasticity in proton-driven secondary active loading of neurotransmitters and may thus have important implications for neurotransmission. This work reveals and emphasizes the mechanistic and biological importance of ultraslow mode-switching.Single-molecule measurements of synaptic vesicles show that V-ATPases do not pump continuously in time but instead stochastically switch between ultralong-lived proton-pumping, inactive and proton-leaky modes. [ABSTRACT FROM AUTHOR]

The size, polydispersity, and electron density profile of synaptic vesicles (SVs) can be studied by small-angle X-ray scattering (SAXS), i.e. by X-ray diffraction from purified SV suspensions in solution. Here we show that size and shape transformations, as they appear in the functional context of these important synaptic organelles, can also be monitored by SAXS. In particular, we have investigated the active uptake of neurotransmitters, and find a mean vesicle radius increase of about 12% after the uptake of glutamate, which indicates an unusually large extensibility of the vesicle surface, likely to be accompanied by conformational changes of membrane proteins and rearrangements of the bilayer. Changes in the electron density profile (EDP) give first indications for such a rearrangement. Details of the protein structure are screened, however, by SVs polydispersity. To overcome the limitations of large ensemble averages and heterogeneous structures, we therefore propose serial X-ray diffraction by single free electron laser pulses. Using simulated data for realistic parameters, we show that this is in principle feasible, and that even spatial distances between vesicle proteins could be assessed by this approach. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets. Cytotoxic T cells have specialised granules that are important for mediating their killing function. Here the authors characterise two types of cytotoxic granules and indicate different functions and temporal release of mediators at the immunological synapse. [ABSTRACT FROM AUTHOR]

Membrane traffic in eukaryotic cells is mediated by transport vesicles that bud from a precursor compartment and are transported to their destination compartment where they dock and fuse. To reach their intracellular destination, transport vesicles contain targeting signals such as Rab GTPases and polyphosphoinositides that are recognized by tethering factors in the cytoplasm and that connect the vesicles with their respective destination compartment. The final step, membrane fusion, is mediated by SNARE proteins. SNAREs are connected to targeting signals and tethering factors by multiple interactions. However, it is still debated whether SNAREs only function downstream of targeting and tethering or whether they also participate in regulating targeting specificity. Here, we review the evidence and discuss recent data supporting a role of SNARE proteins as targeting signals in vesicle traffic. [ABSTRACT FROM AUTHOR]

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells. A variety of artificial cells springs from the functionalization of liposomes with proteins but these models suffer from low durability without repair and replenishment mechanisms. Here, the authors show that synthetic amphiphile membranes undergo SNARE-mediated fusion, and determine bending rigidity and pore edge tension as key parameters for fusion. [ABSTRACT FROM AUTHOR]

Membrane fusion is fundamental to biological processes as diverse as membrane trafficking or viral infection. Proteins catalyzing membrane fusion need to overcome energy barriers to induce intermediate steps in which the integrity of bilayers is lost. Here, we investigate the structural features of tightly docked intermediates preceding hemifusion. Using lipid vesicles in which progression to hemifusion is arrested, we show that the metastable intermediate does not require but is enhanced by divalent cations and is characterized by the absence of proteins and local membrane thickening. Molecular dynamics simulations reveal that thickening is due to profound lipid rearrangements induced by dehydration of the membrane surface. Proteins need to overcome energy barriers to induce intermediate steps in membrane fusion. Using lipid vesicles in which progression to hemifusion is arrested, the authors show that the metastable intermediate is enhanced by divalent cations and is characterized by the absence of proteins and local membrane thickening. Simulations reveal that thickening is induced by dehydration of the membrane surface. [ABSTRACT FROM AUTHOR]

Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly. Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles SNARE complexes in a single unravelling step. Here authors use single-molecule methods to show cooperativity between the NSF and SNARE complex, which prevents ATP consumption without productive disassembly. [ABSTRACT FROM AUTHOR]

In the past decade, we developed various fluorescence-based methods for monitoring membrane fusion, membrane docking, distances between membranes, and membrane curvature. These tools were mainly developed using liposomes as model systems, which allows for the dissection of specific interactions mediated by, for example, fusion proteins. Here, we provide an overview of these methods, including two-photon fluorescence cross-correlation spectroscopy and intramembrane Förster energy transfer, with asymmetric labelling of inner and outer membrane leaflets and the calibrated use of transmembrane energy transfer to determine membrane distances below 10 nm. We discuss their application range and their limitations using examples from our work on protein-mediated vesicle docking and fusion. [ABSTRACT FROM AUTHOR]

Synaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by 'crowding' in the vesicle membrane from stable interaction modules. Synaptic vesicles store neurotransmitters and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. Here authors apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins and describe a protein network of vesicle sub-populations and functional assemblies. [ABSTRACT FROM AUTHOR]

Neuronal circuit assembly requires the fine balance between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM2 and the soluble complement proteins C1q and C3 are recognized as key players, the neuronal molecular components that specify synapses to be eliminated are still undefined. Here, we show that exposed phosphatidylserine (PS) represents a neuronal "eat‐me" signal involved in microglial‐mediated pruning. In hippocampal neuron and microglia co‐cultures, synapse elimination can be partially prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, PS exposure at both hippocampal and retinogeniculate synapses and engulfment of PS‐labeled material by microglia occurs during established developmental periods of microglial‐mediated synapse elimination. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, have elevated presynaptic PS exposure and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial‐mediated synapse pruning and identify a novel role of developmentally regulated neuronal PS exposure that is common among developing brain structures. Synopsis: Microglia help refine developing neural circuits through the elimination of supernumerary synapses. Here we show that exposed phosphatidylserine on pre‐ and postsynaptic membranes functions as an "eat‐me" signal contributing to microglial‐mediated synapse pruning. Phosphatidylserine exposure at both hippocampal and retinogeniculate synapses coincides with the onset of synapse elimination and PS engulfment by microglia. Microglia‐mediated synapse elimination is dependent on TREM2 and exposed phosphatidylserine in vitro.Exposed phosphatidylserine is developmentally regulated across periods of pruning in both hippocampus and visual system.In vivo developmental phosphatidylserine exposure is not caspase 3‐dependent.Loss of C1q leads to elevated phosphatidylserine‐positive presynaptic inputs and reduced microglia engulfment. [ABSTRACT FROM AUTHOR]

The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2. However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis. [ABSTRACT FROM AUTHOR]

Large dense-core vesicles (LDCVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRNAs (miRNAs). Isolation of LDCVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against RNase A. This protocol can be useful in research on the biological functions of LDCVs. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilichydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion. [ABSTRACT FROM AUTHOR]

Membrane fusion is an essential process in nature and is often accomplished by the specific interaction of SNARE proteins. SNARE model systems, in which SNARE domains are replaced by small artificial units, represent valuable tools to study membrane fusion in vitro. The synthesis and analysis is presented of SNARE model peptides that exhibit a recognition motif composed of two different types of peptide nucleic acid (PNA) sequences. This novel recognition unit is designed to mimic the SNARE zippering mechanism that initiates SNARE‐mediated fusion. It contains N‐(2‐aminoethyl)glycine‐PNA (aeg‐PNA) and alanyl‐PNA, which both recognize the respective complementary strand but differ in duplex topology and duplex formation kinetics. The duplex formation of PNA hybrid oligomers as well as the fusogenicity of the model peptides in lipid‐mixing assays were characterized and the peptides were found to induce liposome fusion. As an unexpected discovery, peptides with a recognition unit containing only five aeg‐PNA nucleo amino acids were sufficient and most efficient to induce liposome fusion. To set a SNARE: SNARE model peptides with novel peptide nucleic acid (PNA) hybrid sequences as recognition units were synthesized and analyzed. They were made from two topologically different PNAs with the aim of mimicking SNARE zippering. Lipid‐mixing assays revealed that peptides with a pentameric N‐(2‐aminoethyl)glycine (aeg)‐PNA sequence are the most efficient in liposome fusion. [ABSTRACT FROM AUTHOR]

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Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (pi). It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔμH+ with inversely directed pi transport coupled to the Na+ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and pi using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔμH+-dependent fashion, with glutamate preferred over pi. When facing the extracellular space, pi is transported in a Na+-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and pi homeostasis within glutamatergic neurons. [ABSTRACT FROM AUTHOR]

There is still a large gap in our understanding between the functional complexity of cells and the reconstruction of partial cellular functions in vitro from purified or engineered parts. Here we have introduced artificial vesicles of defined composition into living cells to probe the capacity of the cellular cytoplasm in dealingwith foreignmaterial and to develop tools for the directed manipulation of cellular functions. Our data showthat protein-free liposomes, after variable delay times, are captured by the Golgi apparatus that is reached either by random diffusion or, in the case of large unilamellar vesicles, by microtubuledependent transport via a dynactin/dynein motor complex. However, insertion of early endosomal SNARE proteins suffices to convert liposomes into trafficking vesicles that dock and fuse with early endosomes, thus overriding the default pathway to the Golgi. Moreover, such liposomes can be directed tomitochondria expressing simple artificial affinity tags, which can also be employed to divert endogenous trafficking vesicles. In addition, fusion or subsequent acidification of liposomes can be monitored by incorporation of appropriate chemical sensors. This approach provides an opportunity for probing and manipulating cellular functions that cannot be addressed by conventional genetic approaches. We conclude that the cellular cytoplasm has a remarkable capacity for self-organization and that introduction of such macromolecular complexes may advance nanoengineering of eukaryotic cells. [ABSTRACT FROM AUTHOR]

Fusion was measured by dequenching of labeled membrane lipids (lipid mixing)18,22,28 (top). Plasma membrane-mimicking liposomes contain phospholipids labelled with NBD (green fluorescence) and rhodamine (red fluorescence). Fluorescence resonance energy transfer (FRET) between the two fluorophore-labeled lipids is reduced after LDCV fusion due to lipid dilution by unlabeled lipids of LDCVs, thus de-quenching the donor fluorescence. [Extracted from the article]

Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures. Plekhg5 gene inactivation in mice results in a late-onset motoneuron disease, characterized by degeneration of axon terminals. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26. These findings define a mechanism for regulating autophagy in neurons that specifically targets synaptic vesicles. Disruption of this mechanism may contribute to the pathophysiology of several forms of motoneuron disease. [ABSTRACT FROM AUTHOR]

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18-1 as a possible acceptor complex for the R- SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18-1 remains tethered by the N-terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1-65) was able to bind to the syntaxin1: SNAP25:Munc18-1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R- SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly. [ABSTRACT FROM AUTHOR]

Accumulation of neurotransmitters in the lumen of synaptic vesicles (SVs) relies on the activity of the vacuolar-type H+-ATPase. This pump drives protons into the lumen, generating a proton electrochemical gradient (Δ μH+) across the membrane. Recent work has demonstrated that the balance between the chemical (ΔpH) and electrical (Δ Ψ) components of Δ μH+ is regulated differently by some distinct vesicle types. As different neurotransmitter transporters use ΔpH and Δ Ψ with different relative efficiencies, regulation of this gradient balance has the potential to influence neurotransmitter uptake. Nevertheless, the underlying mechanisms responsible for this regulation remain poorly understood. In this review, we provide an overview of current neurotransmitter uptake models, with a particular emphasis on the distinct roles of the electrical and chemical gradients and current hypotheses for regulatory mechanisms. [ABSTRACT FROM AUTHOR]

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence-based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high-throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high-throughput MraY assays. [ABSTRACT FROM AUTHOR]

Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomicforce microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed. [ABSTRACT FROM AUTHOR]

Legionella pneumophila, the Gram-negative pathogen causing Legionnaires' disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNAREmimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host's disassembly machinery, thus effectively trapping VAMP4 in an inactive state. [ABSTRACT FROM AUTHOR]

ABSTRACT N-ethylmaleimide sensitive factor (NSF) is a key protein of intracellular membrane traffic. NSF is a highly conserved protein belonging to the ATPases associated with other activities (AAA+ proteins). AAA+ share common domains and all transduce ATP hydrolysis into major conformational movements that are used to carry out conformational work on client proteins. Together with its cofactor SNAP, NSF is specialized on disassembling highly stable SNARE complexes that form after each membrane fusion event. Although essential for all eukaryotic cells, however, the details of this reaction have long been enigmatic. Recently, major progress has been made in both elucidating the structure of NSF/SNARE complexes and in understanding the reaction mechanism. Advances in both cryo EM and single molecule measurements suggest that NSF, together with its cofactor SNAP, imposes a tight grip on the SNARE complex. After ATP hydrolysis and phosphate release, it then builds up mechanical tension that is ultimately used to rip apart the SNAREs in a single burst. Because the AAA domains are extremely well-conserved, the molecular mechanism elucidated for NSF is presumably shared by many other AAA+ ATPases. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 518-531, 2016. [ABSTRACT FROM AUTHOR]

Presynaptic neurotransmitter release is dominated by the synaptic vesicle (SV) cycle and entails the biogenesis, fusion, recycling, reformation or turnover of synaptic vesicles--a process involving bulk movement of membrane and proteins. As key mediators of membrane trafficking, small GTPases from the Rab family of proteins play critical roles in this process by acting as molecular switches that dynamically interact with and regulate the functions of different sets of macromolecular complexes involved in each stage of the cycle. Importantly, mutations affecting Rabs, and their regulators or effectors have now been identified that are implicated in severe neurological and neurodevelopmental disorders. Here, we summarize the roles and functions of presynaptic Rabs and discuss their involvement in the regulation of presynaptic function. [ABSTRACT FROM AUTHOR]

The Ca2+ sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin's binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg2+ completely abrogate synaptotagmin-1 binding to SNAREs regardless of the presence of Ca2+. Altogether, our data indicate that at physiological ion concentrations Ca2+-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles. [ABSTRACT FROM AUTHOR]

Our view of the lateral organization of lipids and proteins in the plasma membrane has evolved substantially in the last few decades. It is widely accepted that many, if not all, plasma membrane proteins and lipids are organized in specific domains. These domains vary widely in size, composition, and stability, and they represent platforms governing diverse cell functions. The presynaptic plasma membrane is a well-studied example of a membrane which undergoes rearrangements, especially during exo- and endocytosis. Many proteins and lipids involved in presynaptic function are known, and major efforts have been made to understand their spatial organization and dynamics. Here, we focus on the mechanisms underlying the organization of SNAREs, the key proteins of the fusion machinery, in distinct domains, and we discuss the functional significance of these clusters. [ABSTRACT FROM AUTHOR]

The article presents a study revealing that a protein called Rab26 is seen on the surface of the vesicles near synapses and implying that excess vesicles at synapses may be destroyed by autophagy, which begins with the formation of a membrane around the material that needs to be destroyed. Topics discussed include Rab26's properties, its association with pre-autophagosomal compartments, and Atg16L1 being found as an effector of Rab26.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (~40 nm) and large (~100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23-30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions. [ABSTRACT FROM AUTHOR]

Synaptic-vesicle exocytosis is mediated by the vesicular Ca2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ through PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+ influx bringing the vesicle membrane close enough for membrane fusion. [ABSTRACT FROM AUTHOR]

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids L-glutamate and D-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca2+-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both L-glutamate and D-serine. Further-more, they show uptake of L-glutamate and D-serine that is driven by a proton electrochemical gradient. D-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas L-serine is not transported, serine racemase, the synthesizing enzyme for D-serine, is anchored to the membrane of the vesicles, allowing local generation of D-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between D-serine and L-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing D-serine, L-glutamate, and most likely other neuromodulators in an activity-dependent manner. [ABSTRACT FROM AUTHOR]

Formation and normal function of neuronal synapses are intimately dependent on the delivery to and removal of biological materials from synapses by the intracellular transport machinery. Indeed, defects in intracellular transport contribute to the development and aggravation of neurodegenerative disorders. Despite its importance, regulatory mechanisms underlying this machinery remain poorly defined. We recently uncovered a phosphorylation-regulated mechanism that controls FEZ1-mediated Kinesin-1 based delivery of Stx1 into neuronal axons. Using C. elegans as a model organism to investigate transport defects, we show that FEZ1 mutations resulted in abnormal Stx1 aggregation in neuronal cell bodies and axons. This phenomenon closely resembles transport defects observed in neurodegenerative disorders. Importantly, diminished transport due to mutations of FEZ1 and Kinesin-1 were concomitant with increased accumulation of autophagosomes. Here, we discuss the significance of our findings in a broader context in relation to regulation of Kinesin-mediated transport and neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Calcium-dependent exocytosis of synaptic vesicles mediates the release of neurotransmitters. Important proteins in this process have been identified such as the SNAREs, synaptotagmins, complexins, Munc18 and Munc13. Structural and functional studies have yielded a wealth of information about the physiological role of these proteins. However, it has been surprisingly difficult to arrive at a unified picture of the molecular sequence of events from vesicle docking to calcium-triggered membrane fusion. Using mainly a biochemical and biophysical perspective, we briefly survey the molecular mechanisms in an attempt to functionally integrate the key proteins into the emerging picture of the neuronal fusion machine. [ABSTRACT FROM AUTHOR]

Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca2+ sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca2+, but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca2+-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca2+-dependent exocytosis. [ABSTRACT FROM AUTHOR]

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca2+ triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in eis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca2+-dependent and -independent c/s-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca2+-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca2+-depen-dent c/s-binding prevents trans-interactions if the c;'s-membrane contains 12-20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between eis- and trans-binding, which may play an important modulatory role in synaptic transmission. [ABSTRACT FROM AUTHOR]

Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prospective synapse by kinesin-mediated axonal transport. However, precisely how the various cargoes are linked to the motor proteins remains unclear. Here, we report a transport complex linking syntaxin 1a (Stx) and Munc18, two proteins functioning in synaptic vesicle exocytosis at the presynaptic plasma membrane, to the motor protein Kinesin-1 via the kinesin adaptor FEZ1. Mutation of the FEZ1 ortholog UNC-76 in Caenorhabditis elegans causes defects in the axonal transport of Stx. We also show that binding of FEZ1 to Kinesin-1 and Munc18 is regulated by phosphorylation, with a conserved site (serine 58) being essential for binding. When expressed in C. elegans, wild-type but not phosphorylation-deficient FEZ1 (S58A) restored axonal transport of Stx. We conclude that FEZ1 operates as a kinesin adaptor for the transport of Stx, with cargo loading and unloading being regulated by protein kinases. [ABSTRACT FROM AUTHOR]

Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases. [ABSTRACT FROM AUTHOR]