ELKS active zone proteins as multitasking scaffolds for secretion.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

An action potential triggers neurotransmitter release from synaptic vesicles docking to a specialized release site of the presynaptic plasma membrane, the active zone. The active zone is a highly organized structure with proteins that serves as a platform for synaptic vesicle exocytosis, mediated by SNAREs complex and Ca2+ sensor proteins, within a sub-millisecond opening of nearby Ca2+ channels with the membrane depolarization. In response to incoming neuronal signals, each active zone protein plays a role in the release-ready site replenishment with synaptic vesicles for sustainable synaptic transmission. The active zone release apparatus provides a possible link between neuronal activity and plasticity. This review summarizes the mostly physiological role of active zone protein interactions that control synaptic strength, presynaptic short-term plasticity, and homeostatic synaptic plasticity.

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones.SIGNIFICANCE STATEMENT Retinal ribbon synapses are a specialized type of chemical synapse adapted for the regulated fast and tonic release of neurotransmitter. The hallmark of retinal ribbon synapses is the plate-shaped synaptic ribbon, which extends from the release site into the terminals' cytoplasm and tethers hundreds of synaptic vesicles. Here, we show that Piccolino, the synaptic ribbon specific splice variant of Piccolo, interacts with RIBEYE, the main component of synaptic ribbons. This interaction occurs via several PxDLS-like motifs located at the C terminus of Piccolino, which can connect multiple RIBEYE molecules. Loss of Piccolino disrupts the characteristic plate-shaped structure of synaptic ribbons, indicating a role of Piccolino in synaptic ribbon assembly.

Assembling the Presynaptic Active Zone: A Characterization of an Active Zone Precursor Vesicle

During synaptogenesis, macromolecular protein complexes assemble at the pre- and postsynaptic membrane. Extensive literature identifies numerous transmembrane molecules sufficient to induce synapse formation and several intracellular scaffolding molecules responsible for assembling active zones and recruiting synaptic vesicles. However, little is known about the molecular mechanisms coupling membrane receptors to active zone molecules during development. Using C.elegans, we identify an F-actin network present at nascent presynaptic terminals required for presynaptic assembly. We unravel a sequence of events where specificity-determining adhesion molecules define the location of developing synapses and locally assemble F-actin. Next, an adaptor protein NAB-1/Neurabin binds to F-actin and recruits active zone proteins, SYD-1 and SYD-2/Liprin-α by forming a tripartite complex. NAB-1 localizes transiently to synapses during development and is required for presynaptic assembly. Together, we identify a role for the actin cytoskeleton during presynaptic development and characterize a molecular pathway where NAB-1 links synaptic partner recognition to active zone assembly.

Presynaptic Biogenesis Requires Axonal Transport of Lysosome-Related Vesicles

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of β subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.

Synaptic vesicles (SVs) transmit signals by releasing neurotransmitters from specialized synaptic regions of neurons. In the synaptic region, SVs are tightly clustered around small structures called active zones. The motor KIF1A transports SVs outward through axons until they are captured in the synaptic region. This transport must be guided in the forward direction because it is opposed by the dynein motor, which causes SVs to reverse direction multiple times en route. The core synapse stability (CSS) system contributes to both guided transport and capture of SVs. We identified Sentryn as a CSS protein that contributes to the synaptic localization of SVs in Caenorhabditis elegans. Like the CSS proteins SAD Kinase and SYD-2 (Liprin-α), Sentryn also prevents dynein-dependent accumulation of lysosomes in dendrites in strains lacking JIP3. Genetic analysis showed that Sentryn and SAD Kinase each have at least one nonoverlapping function for the stable accumulation of SVs at synapses that, when combined with their shared functions, enables most of the functions of SYD-2 (Liprin-α) for capturing SVs. Also like other CSS proteins, Sentryn appears enriched at active zones and contributes to active zone structure, suggesting that it is a novel, conserved active zone protein. Sentryn is recruited to active zones by a process dependent on the active zone-enriched CSS protein SYD-2 (Liprin-α). Our results define a specialized group of active zone enriched proteins that can affect motorized transport throughout the neuron and that have roles in both guided transport and capture of SVs.

The end of bulb of Held in the rostral ventral cochlear nucleus of the chinchilla and guinea pig was studied with the freeze-fracture technique. The end bulb has multiple, small active zones which are uniformly distributed within the calyceal portion of this terminal. Single or small groups of active zones are surrounded by enlarged channels of extracellular space often containing processes of astrocytes. Small plasmalemmal deformations occur at these active zones. The number of these deformations is thought to be indicative of exocytotic transmitter release because they are more frequent in animals fixed in a noisy environment compared to animals fixed in a quiet environment. Thus, our study provides a basis for the quantitative study of changes in transmitter secretion at a central nervous system synapse driven by a controllable natural stimulus. The postsynaptic active zone at end bulbs resembles other excitatory synapses in the central nervous system in having an aggregate of large particles on the external membrane leaflet. This junctional aggregate of particles is coextensive with the presynaptic active zone and with the postsynaptic density seen in thin sections. Several perisynaptic aggregates of particles are deployed around each active zone on the external membrane leaflet. These irregularly-shaped aggregates occur preferentially opposite the channels of enlarged extracellular space and along the edge of the end bulb and are not components of intercellular junctions or plasmalemmal contacts with cytoplasmic organelles. Although the function of the different particle aggregates on the postsynaptic membrane is not clear, our findings provide a basis for studying the factors controlling and maintaining their structure as well as more evidence that a consistent relationship exists between types of synaptic action and structure of the postsynaptic membrane.

The active zone defines the area of the presynaptic plasma membrane where synaptic vesicles (SV) dock, mature, and fuse with the cell membrane in a regulated manner. The active zone is defined by an electron-dense cytomatrix of specialized proteins, the cytomatrix assembled at the active zone (CAZ). Some of these proteins, e.g., RIMs, Munc13s, ERC/CASTs as well as Bassoon and Piccolo are exclusive components of the CAZ, and are thought to mediate specific functions, including the localization and regulation of the SV cycle. Recently, major progress has been achieved in studying the assembly of the active zone and the underlying cytoskeletal matrix, suggesting it is not assembled ‘brick-by-brick’ as originally assumed. Rather, mounting evidence suggests that active zone components are preassembled within the neuronal soma, presumably at a trans-Golgi compartment, and then transported on distinct precursor vesicles to primordial presynaptic sites where they fuse with the cell membrane. As Piccolo and Bassoon are unique marker proteins of these vesicles they are called Piccolo-Bassoon transport vesicles (PTVs). The present review summarizes evidence for this active zone precursor vesicle hypothesis, which predicts that presynaptic active zones are assembled in a more or less quantal manner.

Functional synaptic networks are compromised in many neurodevelopmental and neurodegenerative diseases. While the mechanisms of axonal transport and localization of synaptic vesicles and mitochondria are relatively well studied, little is known about the mechanisms that regulate the localization of proteins that localize to active zones. Recent finding suggests that mechanisms involved in transporting proteins destined to active zones are distinct from those that transport synaptic vesicles or mitochondria. Here we report that localization of BRP-an essential active zone scaffolding protein in Drosophila, depends on the precise balance of neuronal Par-1 kinase. Disruption of Par-1 levels leads to excess accumulation of BRP in axons at the expense of BRP at active zones. Temporal analyses demonstrate that accumulation of BRP within axons precedes the loss of synaptic function and its depletion from the active zones. Mechanistically, we find that Par-1 co-localizes with BRP and is present in the same molecular complex, raising the possibility of a novel mechanism for selective localization of BRP-like active zone scaffolding proteins. Taken together, these data suggest an intriguing possibility that mislocalization of active zone proteins like BRP might be one of the earliest signs of synapse perturbation and perhaps, synaptic networks that precede many neurological disorders.

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.

Determining the localization of proteins within a cell and their possible interactions is highly relevant to understand their functionality. Nevertheless, subcellular structures of interest in neurobiology, most importantly synapses with their pre- and postsynaptic compartments, are usually smaller than the resolution limit of conventional light microscopy. Indeed, diffraction of light limits to roughly half of the wavelength of light the resolution of a conventional light microscope. In this regard, super-resolution light microscopy (SRLM) techniques have emerged, achieving even more than ten times the resolution of conventional light microscopy, thus allowing to resolve subsynaptic structures also in situ. Importantly, stimulated emission depletion (STED) microscopy has been extensively used to image in situ the nanoscale organization of presynaptic compartments, such as the area of the presynaptic plasma membrane where synaptic vesicles fuse to release neurotransmitters, the so-called active zone. In this article, we outline a method to determine the localization of active zone scaffolding key players relative to voltage-gated calcium channels within the presynaptic active zone by time-gated STED (gSTED) microscopy in situ.

Liprin-alpha/SYD-2 is a family of multidomain proteins with four known isoforms. One of the reported functions of liprin-alpha is to regulate the development of presynaptic active zones, but the underlying mechanism is poorly understood. Here we report that liprin-alpha directly interacts with the ERC (ELKS-Rab6-interacting protein-CAST) family of proteins, members of which are known to bind RIMs, the active zone proteins that regulate neurotransmitter release. In vitro results indicate that ERC2/CAST, an active zone-specific isoform, interacts with all of the known isoforms of liprin-alpha and that liprin-alpha1 associates with both ERC2 and ERC1b, a splice variant of ERC1 that distributes to both cytosolic and active zone regions. ERC2 colocalizes with liprin-alpha1 in cultured neurons and forms a complex with liprin-alpha1 in brain. Liprin-alpha1, when expressed alone in cultured neurons, shows a partial synaptic localization. When coexpressed with ERC2, however, liprin-alpha1 is redistributed to synaptic sites. Moreover, roughly the first half of ERC2, which contains the liprin-alpha-binding region, is sufficient for the synaptic localization of liprin-alpha1 while the second half is not. These results suggest that the interaction between ERC2 and liprin-alpha may be involved in the presynaptic localization of liprin-alpha and the molecular organization of presynaptic active zones.

Involvement of ELKS, an active zone protein, in exocytotic release from RBL-2H3 cells