A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila

Active zones (AZs) are presynaptic membrane domains mediating synaptic vesicle fusion opposite postsynaptic densities (PSDs). At the Drosophila neuromuscular junction, the ELKS family member Bruchpilot (BRP) is essential for dense body formation and functional maturation of AZs. Using a proteomics approach, we identified Drosophila Syd-1 (DSyd-1) as a BRP binding partner. In vivo imaging shows that DSyd-1 arrives early at nascent AZs together with DLiprin-α, and both proteins localize to the AZ edge as the AZ matures. Mutants in dsyd-1 form smaller terminals with fewer release sites, and release less neurotransmitter. The remaining AZs are often large and misshapen, and ectopic, electron-dense accumulations of BRP form in boutons and axons. Furthermore, glutamate receptor content at PSDs increases because of excessive DGluRIIA accumulation. The AZ protein DSyd-1 is needed to properly localize DLiprin-α at AZs, and seems to control effective nucleation of newly forming AZs together with DLiprin-α. DSyd-1 also organizes trans-synaptic signaling to control maturation of PSD composition independently of DLiprin-α.     

Active zones and receptor surfaces of freeze-fractured crayfish phasic and tonic motor synapses

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15–40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.     

Molecular organization and plasticity of the cytomatrix at the active zone

Regulated neurotransmitter release from presynaptic boutons is crucial for the functioning of chemical synapses, what in turn governs the functional performance of the nervous system. Release occurs at the active zone (AZ), a specialized region of the presynaptic plasma membrane that is defined by a unique and complex meshwork of proteins--the cytomatrix at the AZ (CAZ). Important functions of CAZ proteins include recruitment, docking and priming of synaptic vesicles as well as appropriate localization of voltage-gated calcium channels near vesicle docking sites. We will discuss recent progress in the understanding of the topological localization and the molecular functions of characteristic CAZ proteins as well as emerging molecular mechanisms underlying presynaptic plasticity that involve significant structural CAZ remodeling.     

Molecular determinants of presynaptic active zones

The presynaptic cytoskeletal matrix (cytomatrix) assembled at active zones has been implicated in defining neurotransmitter release sites. Munc13, Rim, Bassoon and Piccolo/Aczonin are recently identified presynaptic cytomatrix proteins. These multidomain proteins are thought to organize the exocytotic and endocytotic machinery precisely at active zones.     

Localization of smg p25A/rab3A p25, a small GTP-binding protein, at the active zone of the rat neuromuscular junction.

smg p25A is a small G protein which has been suggested to regulate neurotransmitter release from the synapses. We investigated here the ultrastructural localization of this small G protein in the rat neuromuscular junction by an immunoperoxidase method. The results showed that smg p25A was distributed non-uniformly on the presynaptic plasma membrane and among the synaptic vesicles with the focal accumulation on the discrete presynaptic sites which corresponded to the active zones, the regions of the presynaptic plasma membrane specialized for the exocytosis of the synaptic vesicles. This unique distribution of smg p25A suggests that it plays an important role in the attachment and fusion of the synaptic vesicles with the active zones.     

Measuring Ca2+-induced structural changes in lipid monolayers: implications for synaptic vesicle exocytosis

Synaptic vesicles (SVs) are small, membrane-bound organelles that are found in the synaptic terminal of neurons. Although tremendous progress has been made in understanding the protein machinery that drives fusion of SVs with the presynaptic membrane, little progress has been made in understanding changes in the membrane structure that accompany this process. We used lipid monolayers of defined composition to mimic biological membranes, which were probed by x-ray reflectivity and grazing incidence x-ray diffraction. These techniques allowed us to successfully monitor structural changes in the membranes at molecular level, both in response to injection of SVs in the subphase below the monolayer, as well as to physiological cues involved in neurotransmitter release, such as increases in the concentration of the membrane lipid PIP₂, or addition of physiological levels of Ca²⁺. Such structural changes may well modulate vesicle fusion in vivo.     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis

Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II-dependent and mediated through the modulation of Synapsin1a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.     

Neurotransmitter release: the dark side of the vacuolar-H+ATPase

Vacuolar-H⁺ATPase (V-ATPase) is a complex enzyme with numerous subunits organized in two domains. The membrane domain V0 contains a proteolipid hexameric ring that translocates protons when ATP is hydrolysed by the catalytic cytoplasmic sector (V1). In nerve terminals, V-ATPase generates an electrochemical proton gradient that is acid and positive inside synaptic vesicles. It is used by specific neurotransmitter-proton antiporters to accumulate neurotransmitters inside their storage organelles. During synaptic activity, neurotransmitters are released from synaptic vesicles docked at specialized portions of the presynaptic plasma membrane, the active zones. A fusion pore opens that allows the neurotransmitter to be released from the synaptic vesicle lumen into the synaptic cleft. We briefly review experimental data suggesting that the membrane domain of V-ATPase could be such a fusion pore.We also discuss the functional implications for quantal neurotransmitter release of the sequential use of the same V-ATPase membrane domain in two different events, neurotransmitter accumulation in synaptic vesicles first, and then release from these organelles during synaptic activity.     

Unwebbing the presynaptic web

The release of neurotransmitter from nerve terminals occurs at a specialized region of the presynaptic plasma membrane called the active zone. A dense matrix of proteins associated with the active zone, called the presynaptic web, is thought to play a fundamental role in defining these neurotransmitter release sites. In this issue of Neuron, Phillips et al. have identified conditions for the biochemical purification of the presynaptic web and show that the web is comprised of proteins involved in the docking, fusion, and recycling of synaptic vesicles.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles

Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses-the Piccolo-Bassoon transport vesicle (PTV)-carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.     

Molecular anatomy and physiology of exocytosis in sensory hair cells

Hair cells mediate our senses of hearing and balance by synaptic release of glutamate from somatic active zones (AZs). They share conserved mechanisms of exocytosis with neurons and other secretory cells of diverse form and function. Concurrently, AZs of these neuro-epithelial hair cells employ several processes that differ remarkably from those of neuronal synaptic terminals of the brain. Their unique molecular anatomy enables them to better respond to small, graded changes in membrane potential and to produce unsurpassed rates of exocytosis. Here, we focus on the AZs of cochlear inner hair cells (IHCs). As in other hair cells, these AZs are occupied by a cytoplasmic extension of the presynaptic density, called the synaptic ribbon: a specialized protein complex required for normal physiological function. Some proteins found at IHC synapses are uniquely expressed or enriched there, where their disruption can beget deafness in humans and in animal models. Other proteins, essential for regulation of conventional neuronal Ca(2+)-triggered fusion, are apparently absent from IHCs. Certain common synaptic proteins appear to have extra significance at ribbon-type AZs because of their interactions with unique molecules, their unusual concentrations, or their atypical localization and regulation. We summarize the molecular-anatomical specializations that underlie the unique synaptic physiology of hair cells.     

Liprin-α/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans

Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for our understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, we show that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. We also demonstrate that DP size is determined by the activity of the AZ protein SYD-2/Liprin-α. Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, our data suggest that a main role of SYD-2/Liprin-α in synaptogenesis is to regulate the polymerization of DPs.     

Molecular organization of the presynaptic active zone

The exocytosis of neurotransmitter-filled synaptic vesicles is under tight temporal and spatial control in presynaptic nerve terminals. The fusion of synaptic vesicles is restricted to a specialized area of the presynaptic plasma membrane: the active zone. The protein network that constitutes the cytomatrix at the active zone (CAZ) is involved in the organization of docking and priming of synaptic vesicles and in mediating use-dependent changes in release during short-term and long-term synaptic plasticity. To date, five protein families whose members are highly enriched at active zones (Munc13s, RIMs, ELKS proteins, Piccolo and Bassoon, and the liprins-α), have been characterized. These multidomain proteins are instrumental for the diverse functions performed by the presynaptic active zone.In our laboratories, work on the molecular organization of the active zone is supported by the Deutsche Forschungsgemeinschaft (Emmy Noether Fellowship, SFB645/A4 to S.S., SFB426/A1 to E.D.G.), the European Commission (SynScaff Consortium), the Land Sachsen-Anhalt (LSA-N2), the Fonds der Chemischen Industrie, and a Max Planck Research Award by the Max Planck Society, the Alexander von Humboldt Society, and local funding (BONFOR to S.S.).     

RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction

At a synapse, fast synchronous neurotransmitter release requires localization of Ca(2+) channels to presynaptic active zones. How Ca(2+) channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ domain of the active-zone protein RIM with the C termini of presynaptic N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all multidomain RIM isoforms. Deletion of RIM proteins ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca(2+) channels. Strikingly, rescue of the decreased Ca(2+)-channel localization required the RIM PDZ domain, whereas rescue of vesicle priming required the RIM N terminus. We propose that RIMs tether N- and P/Q-type Ca(2+) channels to presynaptic active zones via a direct PDZ-domain-mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.     

Endocytic proteins: An expanding repertoire of presynaptic functions

From a presynaptic perspective, neuronal communication mainly relies on two interdependent events: The fast Ca²⁺-triggered fusion of neurotransmitter-containing synaptic vesicles (SVs) and their subsequent high-fidelity reformation. To allow rapid neurotransmission, SVs have evolved into fascinating molecular nanomachines equipped with a well-defined set of proteins. However, upon exocytosis, SVs fully collapse into the presynaptic plasma membrane leading to the dispersal of their molecular components. While the canonical function of endocytic proteins at the presynapse was believed to be the retrieval of SV proteins via clathrin-mediated endocytosis, it is now evident that clathrin-independent endocytic mechanisms predominate. We will highlight in how far these mechanisms still rely on the classical endocytic machinery and discuss the emerging functions of endocytic proteins in release site clearance and SV reformation from endosomal-like vacuoles.     

The GIT family of proteins forms multimers and associates with the presynaptic cytomatrix protein Piccolo

The cytoskeletal matrix assembled at active zones (CAZ) is implicated in defining neurotransmitter release sites. However, little is known about the molecular mechanisms by which the CAZ is organized. Here we report a novel interaction between Piccolo, a core component of the CAZ, and GIT proteins, multidomain signaling integrators with GTPase-activating protein activity for ADP-ribosylation factor small GTPases. A small region (approximately 150 amino acid residues) in Piccolo, which is not conserved in the closely related CAZ protein Bassoon, mediates a direct interaction with the Spa2 homology domain (SHD) domain of GIT1. Piccolo and GIT1 colocalize at synaptic sites in cultured neurons. In brain, Piccolo forms a complex with GIT1 and various GIT-associated proteins, including betaPIX, focal adhesion kinase, liprin-alpha, and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, betaPIX, and focal adhesion kinase, suggesting that these proteins bind to the SHD by different mechanisms. Intriguingly, GIT proteins form homo- and heteromultimers through their C-terminal G-protein-coupled receptor kinase-binding domain in a tail-to-tail fashion. This multimerization enables GIT1 to simultaneously interact with multiple SHD-binding proteins including Piccolo and betaPIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ.     

Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.