The solute carrier SLC17A9 was previously identified as a vesicular nucleotide transporter that is primarily responsible for transporting ATP and related nucleotides into intracellular vesicles, and its deletion in mice resulted in a loss of ATP from intracellular storage organelles. Using recombinant SLC17A9 and functional reconstitution in liposomes we have now attempted to characterize the molecular features of its transport activity. While the purified protein includes the entire open reading frame, forms dimers, and is apparently properly folded and correctly oriented in the liposomes, we were unable to measure any nucleotide transport activity although the electrochemical potential was intact, the liposomes were not leaky, and the related vesicular glutamate transporter (SLC17A57) reconstituted in parallel under the same conditions was active. We also tested whether SLC17A9, similar to related transporters in the SLC17 family, functions as a Na+-dependent phosphate transporter. However, no such activity was detectable, and expression of the transporter in HeLa cells did not result in consistent increases of phosphate uptake. We conclude that the role of SLC17A9 in vesicular nucleotide transport needs further corroboration, possibly requiring additional, hitherto unknown factors.

Loading of synaptic vesicles with neurotransmitters, an essential step in synaptic transmission, is mediated by vesicular neurotransmitter transporters that are fuelled by an electrochemical proton gradient across the vesicle membrane. While advances have recently been made in the structural and mechanistic understanding of these transporters, it is difficult to study transport kinetics and associated ion fluxes, largely due to technical constraints in accessing intracellular transporters. Here we describe a microfluidics platform that enables rapid solution changes, thus resolving solute fluxes across the vesicle membrane. Antibodies directed against general or subclass-specific vesicle proteins are immobilized on functionalized glass slides, followed by immunocapture of synaptic vesicles yielding a vesicle monolayer virtually free of contamination. After mounting in a specially designed microfluidics setup, acidification of vesicles, as measured by fluorescent reporters, can be repetitively switched on and off with a time resolution of seconds, demonstrating the versatility of the system.

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes affected in neural diseases. Monitoring this process, particularly synaptic vesicle recycling, in living cells has been instrumental in revealing the mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from limitations, such as large probe sizes or limited compatibility for human neurons, hampering the quantitative analysis of synaptic pathophysiology. Here, we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes that target the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, noninvasive imaging and functional interrogation of Syt1 exo-endocytosis and trafficking in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed- and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for studying synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes to be affected in neural diseases. Monitoring this process, and in particular synaptic vesicle recycling, in living cells has been instrumental in unraveling mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from major limitations such large probe size or lack of suitability for human neurons, hampering the understanding of human synaptic pathophysiology. Here we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes targeting the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, non-invasive imaging and functional interrogation of synaptic transmission in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed-and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for human synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders. ### Competing Interest Statement Silvio Rizzoli and Felipe Opazo are shareholders of NanoTag Biotechnology GmbH.

Synaptic transmission is mediated by the exocytotic release of neurotransmitters stored in synaptic vesicles (SVs). SVs filled with neurotransmitters preferentially undergo exocytosis, but it is unclear how this is achieved. Here, we show that during transmitter loading, SVs substantially increase in size, which is reversible and requires synaptophysin, an abundant membrane protein with an unclear function. SVs are larger when synaptophysin is knocked out, and conversely, liposomes are smaller when reconstituted with synaptophysin. Moreover, transmitter loading of SVs accelerates fusion in vitro, which is abolished when synaptophysin is lacking despite near normal transmitter uptake. We conclude that synaptophysin functions as a curvature-promoting entity in the SV membrane, allowing for major lateral expansion of the SV membrane during neurotransmitter filling, thus increasing their propensity for exocytosis.

This chapter presents an optimized method for isolating synaptic vesicles (SVs) from neurospheres derived from human induced pluripotent stem cells (hiPSCs). The protocol begins with neurosphere cultivation to achieve mature neurons, which is essential for the functional studies of neuronal activity. Following this, neurosphere-derived synaptosomes are isolated, and SVs are enriched through differential centrifugation. The method culminates in the proteomic analysis of SVs using nano-liquid chromatography coupled with high-resolution tandem mass spectrometry (nanoLC-MS/MS), providing a detailed proteome profile of the isolated vesicles. This protocol can contribute to the understanding of SV molecular heterogeneity and the mechanisms of neurotransmitter uptake and release and be applied to the field of research in neurological and neuropsychiatric disorders.

Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that establish proton gradients across cellular membranes[1][1],[2][2]. Their pharmacological inhibition is currently under active investigation as a therapeutic strategy for cancer, infectious diseases, and autophagy-related disorders[3][3],[4][4]. However, the molecular mechanism underlying V-ATPase inhibition remains poorly understood. Based on ensemble average measurements, it is widely assumed that inhibitors suppress activity by slowing the catalytic transport cycle and reducing proton transport rates[5][5]–[7][6]. Here, we tested this popular notion by directly measuring single-molecule proton pumping in the presence of three potent V-ATPase inhibitors: bafilomycin A1, concanamycin A, and diphyllin. Although all compounds abolish proton gradients in a canonical concentration-dependent manner (IC50 of 0.2 nM, 0.6 nM, and 41 nM, respectively), they leave the proton transport rate of active V-ATPases essentially unchanged. Instead, inhibitors modulate the reversible switching kinetics between ultralong-lived active (pumping) and inactive modes. Distinct inhibitors modulate mode lifetimes in a mode-specific and differentially efficient manner, altering the probability of the pump being in the active mode. Given that mode-switching has been documented across diverse primary[8][7],[9][8] and secondary[10][9]–[13][10] active transporters, our results suggest a novel strategy for therapeutic intervention that targets mode occupancy rather than the canonical transport cycle. ### Competing Interest Statement Dimitrios Stamou is the founder of Atomos Biotech. The other authors declare no competing interests. Novo Nordisk Foundation, NNF17OC0028176 Lundbeck Foundation, R441-2023-360 European Research Council, https://ror.org/0472cxd90 [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-7 [7]: #ref-8 [8]: #ref-9 [9]: #ref-10 [10]: #ref-13

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 indi-vidual 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.

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.

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.

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.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of small conserved eukaryotic proteins that mediate membrane fusion between organelles and with the plasma membrane. SNAREs are directly or indirectly anchored to membranes. Prior to fusion, complementary SNAREs assemble between membranes with the aid of accessory proteins that provide a scaffold to initiate SNARE zippering, pulling the membranes together and mediating fusion. Recent advances have enabled the construction of detailed models describing bilayer transitions and energy barriers along the fusion pathway and have elucidated the structures of SNAREs complexed in various states with regulatory proteins. In this Review, we discuss how these advances are yielding an increasingly detailed picture of the SNARE-mediated fusion pathway, leading from first contact between the membranes via metastable non-bilayer intermediates towards the opening and expansion of a fusion pore. We describe how SNARE proteins assemble into complexes, how this assembly is regulated by accessory proteins and how SNARE complexes overcome the free energy barriers that prevent spontaneous membrane fusion.

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.

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.

The neuronal Q-SNARE protein SNAP25a (isoform 2) forms part of the SNARE complex eliciting synaptic vesicle fusion during neuronal exocytosis. While the post-fusion cis-SNARE complex has been studied extensively, little is known about the pre-fusion conformation of SNAP25a. Here we analyze SNAP25a in its monomeric pre-fusion conformation by NMR spectroscopy and find it intrinsically disordered and highly dynamic. While from residue L35 onwards, intrinsic disorder dominates (including the second SNARE motif, SN2), region A5 to L35 (comprising the N-terminus of the first SNARE motif, SN1) shows strong α-helical propensity. Our findings suggest that the N-terminus of SN1 may act as a nucleation site for SNARE acceptor complex assembly. As a side note, our result of an intrinsic disorder of SNAP25 contrasts with a recent prediction of AlphaFold2 that predicted SN1 and SN2 to be entirely α-helical with high confidence.

The structure of biological vesicles, in particular synaptic vesicles (SVs), as well as synthetic lipid model systems, in particular small unilamellar lipid vesicles (LVs) has been subject of abiding interest. Given the small size of both LVs and SVs (R≈20 nm), high spatial resolution is required to identify the distribution of lipids and protein constituents. Cryogenic electron microscopy studies of synaptic vesicles have revealed the outer and inner layer of proteins around the lipid bilayer. However, the samples have to be cryogenically fixed for this technique and throughput is limited. Another standard technique for structural characterization is solution small angle x-ray scattering (SAXS), which enables the measurement of vesicles in a quasi-physiological environment combined with a high spatial resolution. However, due to the average over an extremely large ensemble, SAXS yields information only about the average structure (size and electron density profile). The distribution function of structural parameters is not accessible, and many structural details are lost or screened by polydispersity, as well as by powder averaging. To overcome these limitations, we now have performed coherent diffractive imaging experiments on single vesicles using single femtosecond x-ray free electron laser (XFEL) pulses. For these measurements, single vesicles surrounded by a thin buffer layer are delivered into a nano-focused XFEL beam by an aerosol injector. By the ‘diffract-before-destroy’ principle, the individual vesicles can be probed without radiation damage. This approach leads to the measurement of thousands of diffraction patterns that can now be analyzed and reconstructed. The (preliminary) results of this analysis will be presented.

In the ear, inner hair cells (IHCs) employ sophisticated glutamatergic ribbon synapses with afferent neurons to transmit auditory information to the brain. The presynaptic machinery responsible for neurotransmitter release in IHC synapses includes proteins such as the multi-C2-domain protein otoferlin and the vesicular glutamate transporter 3 (VGluT3). Yet, much of this likely unique molecular machinery remains to be deciphered. The scarcity of material has so far hampered biochemical studies which require large amounts of purified samples. We developed a subcellular fractionation workflow combined with immunoisolation of VGluT3-containing membrane vesicles, allowing for the enrichment of glutamatergic organelles that are likely dominated by synaptic vesicles (SVs) of IHCs. We have characterized their protein composition in mice before and after hearing onset using mass spectrometry and confocal imaging and provide a fully annotated proteome with hitherto unidentified proteins. Despite the prevalence of IHC marker proteins across IHC maturation, the profiles of trafficking proteins differed markedly before and after hearing onset. Among the proteins enriched after hearing onset were VAMP-7, syntaxin-7, syntaxin-8, syntaxin-12/13, SCAMP1, V-ATPase, SV2, and PKCα. Our study provides an inventory of the machinery associated with synaptic vesicle-mediated trafficking and presynaptic activity at IHC ribbon synapses and serves as a foundation for future functional studies.

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.