Short-distance vesicle transport via phase separation.

In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca2+ and via its C2A domain-mediated Ca2+ sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.

Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses.

Neuromodulators bind to pre- and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca2+ levels, and are thought to play important roles in neuropsychiatric and neurodegenerative diseases. Here, we discovered in human neurons an unanticipated presynaptic mechanism that acutely changes synaptic ultrastructure and regulates synaptic communication. Activation of neuromodulator receptors bidirectionally controlled synaptic vesicle numbers within nerve terminals. This control correlated with changes in the levels of cAMP-dependent protein kinase A-mediated phosphorylation of synapsin-1. Using a conditional deletion approach, we reveal that the neuromodulator-induced control of synaptic vesicle numbers was largely dependent on synapsin-1. We propose a mechanism whereby non-phosphorylated synapsin-1 "latches" synaptic vesicles to presynaptic clusters at the active zone. cAMP-dependent phosphorylation of synapsin-1 then removes the vesicles. cAMP-independent dephosphorylation of synapsin-1 in turn recruits vesicles. Synapsin-1 thereby bidirectionally regulates synaptic vesicle numbers and modifies presynaptic neurotransmitter release as an effector of neuromodulator signaling in human neurons.

Saturated Reconstruction of a Volume of Neocortex.

We describe automated technologies to probe the structure of neural tissue at nanometer resolution and use them to generate a saturated reconstruction of a sub-volume of mouse neocortex in which all cellular objects (axons, dendrites, and glia) and many sub-cellular components (synapses, synaptic vesicles, spines, spine apparati, postsynaptic densities, and mitochondria) are rendered and itemized in a database. We explore these data to study physical properties of brain tissue. For example, by tracing the trajectories of all excitatory axons and noting their juxtapositions, both synaptic and non-synaptic, with every dendritic spine we refute the idea that physical proximity is sufficient to predict synaptic connectivity (the so-called Peters' rule). This online minable database provides general access to the intrinsic complexity of the neocortex and enables further data-driven inquiries.

Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogues synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin-knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin-knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

Heterogeneity of glutamatergic synapses: cellular mechanisms and network consequences.

Chemical synapses are commonly known as a structurally and functionally highly diverse class of cell-cell contacts specialized to mediate communication between neurons. They represent the smallest "computational" unit of the brain and are typically divided into excitatory and inhibitory as well as modulatory categories. These categories are subdivided into diverse types, each representing a different structure-function repertoire that in turn are thought to endow neuronal networks with distinct computational properties. The diversity of structure and function found among a given category of synapses is referred to as heterogeneity. The main building blocks for this heterogeneity are synaptic vesicles, the active zone, the synaptic cleft, the postsynaptic density, and glial processes associated with the synapse. Each of these five structural modules entails a distinct repertoire of functions, and their combination specifies the range of functional heterogeneity at mammalian excitatory synapses, which are the focus of this review. We describe synapse heterogeneity that is manifested on different levels of complexity ranging from the cellular morphology of the pre- and postsynaptic cells toward the expression of different protein isoforms at individual release sites. We attempt to define the range of structural building blocks that are used to vary the basic functional repertoire of excitatory synaptic contacts and discuss sources and general mechanisms of synapse heterogeneity. Finally, we explore the possible impact of synapse heterogeneity on neuronal network function.

Activity-driven local ATP synthesis is required for synaptic function.

Cognitive function is tightly related to metabolic state, but the locus of this control is not well understood. Synapses are thought to present large ATP demands; however, it is unclear how fuel availability and electrical activity impact synaptic ATP levels and how ATP availability controls synaptic function. We developed a quantitative genetically encoded optical reporter of presynaptic ATP, Syn-ATP, and find that electrical activity imposes large metabolic demands that are met via activity-driven control of both glycolysis and mitochondrial function. We discovered that the primary source of activity-driven metabolic demand is the synaptic vesicle cycle. In metabolically intact synapses, activity-driven ATP synthesis is well matched to the energetic needs of synaptic function, which, at steady state, results in ∼10(6) free ATPs per nerve terminal. Despite this large reservoir of ATP, we find that several key aspects of presynaptic function are severely impaired following even brief interruptions in activity-stimulated ATP synthesis.

Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates.

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy.

Liquid-liquid phase separation in presynaptic nerve terminals.

The presynaptic nerve terminal is crucial for transmitting signals to the adjacent cell. To fulfill this role, specific proteins with distinct functions are concentrated in spatially confined areas within the nerve terminals. A recent concept termed liquid-liquid phase separation (LLPS) has provided new insights into how this process may occur. In this review, we aim to summarize the LLPS of proteins in different parts of the presynaptic nerve terminals, including synaptic vesicle (SV) clusters, the active zone (AZ), and the endocytic zone, with an additional focus on neurodegenerative diseases (NDDs), where the functional relevance of these properties is explored. Last, we propose new perspectives and future directions for the role of LLPS in presynaptic nerve terminals.

Histamine synthesis and transport are coupled in axon terminals via a dual quality control system.

Monoamine neurotransmitters generated by de novo synthesis are rapidly transported and stored into synaptic vesicles at axon terminals. This transport is essential both for sustaining synaptic transmission and for limiting the toxic effects of monoamines. Here, synthesis of the monoamine histamine by histidine decarboxylase (HDC) and subsequent loading of histamine into synaptic vesicles are shown to be physically and functionally coupled within Drosophila photoreceptor terminals. This process requires HDC anchoring to synaptic vesicles via interactions with N-ethylmaleimide-sensitive fusion protein 1 (NSF1). Disassociating HDC from synaptic vesicles disrupts visual synaptic transmission and causes somatic accumulation of histamine, which leads to retinal degeneration. We further identified a proteasome degradation system mediated by the E3 ubiquitin ligase, purity of essence (POE), which clears mislocalized HDC from the soma, thus eliminating the cytotoxic effects of histamine. Taken together, our results reveal a dual mechanism for translocation and degradation of HDC that ensures restriction of histamine synthesis to axonal terminals and at the same time rapid loading into synaptic vesicles. This is crucial for sustaining neurotransmission and protecting against cytotoxic monoamines.

Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching.

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.

Fast resupply of synaptic vesicles requires synaptotagmin-3.

Sustained neuronal activity demands a rapid resupply of synaptic vesicles to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by submicromolar presynaptic Ca2+ signals by an as-yet unidentified high-affinity Ca2+ sensor1,2. Here we identify synaptotagmin-3 (SYT3)3,4 as that presynaptic high-affinity Ca2+ sensor, which drives vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibited enhanced short-term depression, and recovery from depression was slower and insensitive to presynaptic residual Ca2+. During sustained neuronal firing, SYT3 accelerated vesicle replenishment and increased the size of the readily releasable pool. SYT3 also mediated short-term facilitation under conditions of low release probability and promoted synaptic enhancement together with another high-affinity synaptotagmin, SYT7 (ref. 5). Biophysical modelling predicted that SYT3 mediates both replenishment and facilitation by promoting the transition of loosely docked vesicles to tightly docked, primed states. Our results reveal a crucial role for presynaptic SYT3 in the maintenance of reliable high-frequency synaptic transmission. Moreover, multiple forms of short-term plasticity may converge on a mechanism of reversible, Ca2+-dependent vesicle docking.

Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG-Syt1-SV2 plasma membrane nanocluster for synaptic vesicle entry.

The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.

Energy matters: presynaptic metabolism and the maintenance of synaptic transmission.

Synaptic activity imposes large energy demands that are met by local adenosine triphosphate (ATP) synthesis through glycolysis and mitochondrial oxidative phosphorylation. ATP drives action potentials, supports synapse assembly and remodelling, and fuels synaptic vesicle filling and recycling, thus sustaining synaptic transmission. Given their polarized morphological features - including long axons and extensive branching in their terminal regions - neurons face exceptional challenges in maintaining presynaptic energy homeostasis, particularly during intensive synaptic activity. Recent studies have started to uncover the mechanisms and signalling pathways involved in activity-dependent and energy-sensitive regulation of presynaptic energetics, or 'synaptoenergetics'. These conceptual advances have established the energetic regulation of synaptic efficacy and plasticity as an exciting research field that is relevant to a range of neurological disorders associated with bioenergetic failure and synaptic dysfunction.

An open-access volume electron microscopy atlas of whole cells and tissues.

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.

Nanoscale architecture of synaptic vesicles and scaffolding complexes revealed by cryo-electron tomography.

The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.

A plant flavonol and genetic suppressors rescue a pathogenic mutation associated with kinesin in neurons.

KIF1A, a microtubule-based motor protein responsible for axonal transport, is linked to a group of neurological disorders known as KIF1A-associated neurological disorder (KAND). Current therapeutic options for KAND are limited. Here, we introduced the clinically relevant KIF1A(R11Q) variant into the Caenorhabditis elegans homolog UNC-104, resulting in uncoordinated animal behaviors. Through genetic suppressor screens, we identified intragenic mutations in UNC-104's motor domain that rescued synaptic vesicle localization and coordinated movement. We showed that two suppressor mutations partially recovered motor activity in vitro by counteracting the structural defect caused by R11Q at KIF1A's nucleotide-binding pocket. We found that supplementation with fisetin, a plant flavonol, improved KIF1A(R11Q) worms' movement and morphology. Notably, our biochemical and single-molecule assays revealed that fisetin directly restored the ATPase activity and processive movement of human KIF1A(R11Q) without affecting wild-type KIF1A. These findings suggest fisetin as a potential intervention for enhancing KIF1A(R11Q) activity and alleviating associated defects in KAND.

Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

Different states of synaptic vesicle priming explain target cell type-dependent differences in neurotransmitter release.

Pronounced differences in neurotransmitter release from a given presynaptic neuron, depending on the synaptic target, are among the most intriguing features of cortical networks. Hippocampal pyramidal cells (PCs) release glutamate with low probability to somatostatin expressing oriens-lacunosum-moleculare (O-LM) interneurons (INs), and the postsynaptic responses show robust short-term facilitation, whereas the release from the same presynaptic axons onto fast-spiking INs (FSINs) is ~10-fold higher and the excitatory postsynaptic currents (EPSCs) display depression. The mechanisms underlying these vastly different synaptic behaviors have not been conclusively identified. Here, we applied a combined functional, pharmacological, and modeling approach to address whether the main difference lies in the action potential-evoked fusion or else in upstream priming processes of synaptic vesicles (SVs). A sequential two-step SV priming model was fitted to the peak amplitudes of unitary EPSCs recorded in response to complex trains of presynaptic stimuli in acute hippocampal slices of adult mice. At PC-FSIN connections, the fusion probability (Pfusion) of well-primed SVs is 0.6, and 44% of docked SVs are in a fusion-competent state. At PC-O-LM synapses, Pfusion is only 40% lower (0.36), whereas the fraction of well-primed SVs is 6.5-fold smaller. Pharmacological enhancement of fusion by 4-AP and priming by PDBU was recaptured by the model with a selective increase of Pfusion and the fraction of well-primed SVs, respectively. Our results demonstrate that the low fidelity of transmission at PC-O-LM synapses can be explained by a low occupancy of the release sites by well-primed SVs.

Single-vesicle imaging reveals actin-dependent spatial restriction of vesicles at the active zone, essential for sustained transmission.

Synaptic-vesicle (SV) recruitment is thought to maintain reliable neurotransmitter release during high-frequency signaling. However, the mechanism underlying the SV reloading for sustained neurotransmission at central synapses remains unknown. To elucidate this, we performed direct observations of SV reloading and mobility at a single-vesicle level near the plasma membrane in cerebellar mossy fiber terminals using total internal reflection fluorescence microscopy, together with simultaneous recordings of membrane fusion by capacitance measurements. We found that actin disruption abolished the rapid SV recruitment and reduced sustained release. In contrast, induction of actin polymerization and stabilization did not affect vesicle recruitment and release, suggesting that the presence of actin filaments, rather than actin dynamics, was required for the rapid recruitment. Single-particle tracking experiments of quantum dot-labeled vesicles, which allows nanoscale resolution of vesicle mobility, revealed that actin disruption caused vesicles to diffuse more rapidly. Hidden Markov modeling with Bayesian inference revealed that SVs had two diffusion states under normal conditions: free-diffusing and trapped. After disruption of the actin filament, vesicles tended to have only the free-diffusing state. F-actin staining showed that actin filaments were localized outside the active zones (AZs) and surrounded some SV trajectories. Perturbation of SV mobility, possibly through interference with biomolecular condensates, also suggested that the restricted diffusion state determined the rate of SV recruitment. We propose that actin filaments confined SVs near the AZ to achieve rapid and efficient recruitment followed by priming and sustained synaptic transmission.

Overlapping role of synaptophysin and synaptogyrin family proteins in determining the small size of synaptic vesicles.

Members of the synaptophysin and synaptogyrin family are vesicle proteins with four transmembrane domains. In spite of their abundance in synaptic vesicle (SV) membranes, their role remains elusive and only mild defects at the cellular and organismal level are observed in mice lacking one or more family members. Here, we show that coexpression with synapsin in fibroblasts of each of the four brain-enriched members of this family-synaptophysin, synaptoporin, synaptogyrin 1, and synaptogyrin 3-is sufficient to generate clusters of small vesicles in the same size range of SVs. Moreover, mice lacking all these four proteins have larger SVs. We conclude that synaptophysin and synaptogyrin family proteins play an overlapping function in the biogenesis of SVs and in determining their small size.