Intersectin 1: a versatile actor in the synaptic vesicle cycle

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.

Differences in neuromuscular transmission and neuromuscular junction morphology exist across muscle fiber types. We hypothesized that these fiber-type differences are reflected in the size of the cycling synaptic vesicle pool. Synaptic vesicle cycling at type-identified rat diaphragm neuromuscular junctions was examined by fluorescently labeling presynaptic vesicles with FM4-64. We found that FM4-64 fluorescence uptake was higher at presynaptic terminals of type I/IIa fibers than type IIx/IIb fibers. However, no fiber-type differences in the rate of FM4-64 destaining were found with repetitive nerve stimulation. Synaptic vesicle density at active zones was examined by transmission electron microscopy. In accordance with FM4-64 uptake, synaptic vesicle density was greater at type I/IIa than IIx/IIb fibers. These results demonstrate differences in synaptic vesicle cycling across diaphragm muscle fiber types, which may underlie previously observed differences in neuromuscular transmission across diaphragm muscle fiber types. In the diaphragm, motor units comprising type I and type IIa fibers are most frequently recruited with a duty cycle of approximately 40%. Motor units comprising IIx/IIb fibers are infrequently recruited and only for short durations. The capacity for synaptic vesicle release and cycling at different muscle fiber types matches the functional requirements of these motor units. If the demand for recruitment of motor units comprising IIx/IIb fibers increases, for example, with mechanical loading, there is an increased risk for neuromuscular transmission failure that my relate to the capacity for synaptic vesicle release and cycling. Muscle fiber type-specific adaptations should be considered when examining neuromuscular disorders.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I gamma-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P(2) metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI gamma-p90 associates with both the mu and beta2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI gamma-p90 tail binds to a cognate recognition site on the sandwich subdomain of the beta2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2mu, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI gamma-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI gamma tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2 beta and AP-2mu. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI gamma-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI gamma-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P(2) synthesis during clathrin-mediated SV endocytosis.

Understanding the functional roles of cells in neuronal circuits and behavior requires the ability to control neuronal activity in an acute and precise way. The field of optogenetics offers a variety of molecular tools to excite or inhibit neurons with light. In the last decade, several strategies have been proposed for reversible silencing of neurotransmission. These tools vary widely in their mechanisms: ranging from opsin-based light-driven ion pumps or anion channels, which are known to hyperpolarize the cell, to alter ionic gradients or cellular biochemistry; over metabotropic receptors, to tools damaging the neurotransmitter release machinery, that allow only long-term silencing as their recovery requires de novo synthesis of targeted proteins. Therefore, the optogenetic toolbox still lacks tools that combine fast activation and fast reversibility with the ability of long-term silencing. In this study, the optogenetic tool optoSynC (optogenetic synaptic vesicle clustering) was characterized in depth. optoSynC utilizes the light-induced homo-oligomerization of Arabidopsis thaliana cryptochrome 2 (CRY2) for silencing synaptic transmission via clustering of synaptic vesicles (SVs). CRY2 was targeted to the SV membrane by fusion to the SV transmembrane protein synaptogyrin-1 (SNG-1). The silencing kinetics of optoSynC were determined with analyses of swimming and crawling behavior in Caenorhabditis elegans (C. elegans). Pan-neuronally expressed optoSynC reduced swimming locomotion by 80% within 30 s following photo-stimulation (τon ~7.2 s). Locomotion recovered within 15 min in darkness (τoff ~6.5 min). Analysis of crawling behavior indicated even faster activation within 2 s and an almost complete inhibition of the LITE-1-mediated escape response. This escape response occurs at high blue light intensities and results in an increased velocity, which was not detected after optoSynC activation. However, optoSynC can exert its full effect at significantly lower intensities (25 µW/mm²) and is so light-sensitive that it can even be activated at 1.4 µW/mm². optoSynC could be fully recovered and reactivated at least twice without decreased efficiency. With a combination of pharmacological analysis and optogenetics, it has been demonstrated that optoSynC can inhibit neurotransmitter release for several hours. To realize its full potential, optoSynC should be expressed in an sng-1 mutant background. Expression along with endogenous SNG-1 decreases the effectiveness of optoSynC by 50%. Specific expression of optoSynC in cholinergic or GABAergic neurons could robustly inhibit swimming behavior by 55% and 30%, respectively. Moreover, optoSynC can selectively inhibit individual neurons like the nociceptive neuron pair PVD. When PVD is photoactivated by Chrimson, a red-light activatable channelrhodopsin, forward locomotion increases. Activation of optoSynC reduced this behavior by 50%. Besides my experiments in C. elegans, my cooperation partners Dr. Holger Dill and Yilmaz Arda Ateş demonstrated silencing of neurotransmission with optoSynC in zebrafish, and Dr. Shigeki Watanabe and Brady D. Goulden in murine hippocampal neurons. The clustering of SVs as the mode of action of optoSynC could be confirmed using transmission electron microscopy. Analysis of micrographs of cholinergic synapses stimulated with and without light revealed that distances of neighboring synaptic vesicles in the cytosol were reduced by around 14% after photoactivation. Distances of docked SVs at the plasma membrane remained unaffected. However, near the dense projection, docked SVs accumulated, while other docked SVs were depleted after optoSynC activation. Activation of optoSynC increased the appearances of SVs and dense core vesicles (DCVs) in micrographs. It is unclear whether sizes became physically larger due to lateral pressure by CRY2 aggregates in the SV membrane or if oligomer formation only altered their appearance in micrographs. optoSynC did not accumulate in the plasma membrane to the extent that abnormal structures at the plasma membrane or dense projection were observed. Recycling of SVs remained unaffected by optoSynC as no unusual number of large vesicles was determined. Therefore, optoSynC inhibits neuronal activity mainly by clustering of cytosolic SVs. To study the precise sequence and timing of events of vesicle mobilization, it is necessary to introduce known stops in the synaptic vesicle cycle which can be achieved by optoSynC. The C. elegans mutation dyn-1(ts-) is a temperature-sensitive dynamin mutation that blocks the recycling of SVs from the plasma membrane and early endosomes at temperatures exceeding 25 °C. By expressing optoSynC in dyn-1(ts-) animals, a novel assay was established, enabling the transfer of SVs between different stages in the SV cycle. Cluster formation of reserve pool SVs blocks the SV cycle before the process of docking and priming begins, while the SV cycle is blocked after the fusion of SVs at high temperatures. It could be demonstrated that behavior returned 15 min after optoSynC activation while animals without optoSynC remained immobile. Unfortunately, the time scale of the recovery from inhibition by optoSynC is too long to effectively study vesicle mobilization.

Sustained neuronal communication relies on the coordinated activity of multiple proteins that regulate synaptic vesicle biogenesis and cycling within the presynaptic terminal. Synaptogyrin and synaptophysin are conserved MARVEL domain-containing transmembrane proteins that are among the most abundant synaptic vesicle constituents, although their role in the synaptic vesicle cycle has remained elusive. To further investigate the function of these proteins, we generated and characterized a synaptogyrin (gyr)-null mutant in Drosophila, whose genome encodes a single synaptogyrin isoform and lacks a synaptophysin homolog. We demonstrate that Drosophila synaptogyrin plays a modulatory role in synaptic vesicle biogenesis at larval neuromuscular junctions. Drosophila lacking synaptogyrin are viable and fertile and have no overt deficits in motor function. However, ultrastructural analysis of gyr larvae revealed increased synaptic vesicle diameter and enhanced variability in the size of synaptic vesicles. In addition, the resolution of endocytic cisternae into synaptic vesicles in response to strong stimulation is defective in gyr mutants. Electrophysiological analysis demonstrated an increase in quantal size and a concomitant decrease in quantal content, suggesting functional consequences for transmission caused by the loss of synaptogyrin. Furthermore, high-frequency stimulation resulted in increased facilitation and a delay in recovery from synaptic depression, indicating that synaptic vesicle exo-endocytosis is abnormally regulated during intense stimulation conditions. These results suggest that synaptogyrin modulates the synaptic vesicle exo-endocytic cycle and is required for the proper biogenesis of synaptic vesicles at nerve terminals.

Most interneuronal communication occurs with high spatiotemporal precision at structurally and molecularly specialized synaptic sites, where neurotransmitter-filled synaptic vesicles (SVs) fuse at dedicated presynaptic active zones (AZs) in response to an action potential (AP). The strength of synaptic communication is dynamically and purposefully regulated in an activity-dependent manner, known as synaptic plasticity, which manifests itself over a wide range of timescales. I investigated the mechanism underlying a form of synaptic plasticity that operates on a millisecond timescale, called short-term facilitation (STF), in which synaptic strength increases with repeated stimulation. In the mammalian brain, hippocampal mossy fiber bouton (hMFB) to CA3 pyramidal cell synapses exhibit a strong presynaptic STF. STF is crucial for the function of the hippocampal network by allowing hMFB-CA3 synapses to operate as conditional detonators (Henze et al., 2002b). Deciphering how STF works is key to understanding information processing in neuronal circuits associated with learning and memory processes. In recent years, considerable progress has been made with respect to the functional characterization of STF at hMFB-CA3 synapses, and several mechanisms have been proposed to contribute to STF, one of them involving the Ca2+ sensor synaptotagmin-7 (Syt-7) (Jackman et al., 2016). However, an ultrastructural characterization assessing the contribution of STF and Syt-7 to SV pool organization at individual hMFB AZs is still missing. This information is critical since SV organization is of fundamental relevance to the functional state of synapses, given that morphologically docked SVs serve as a proxy for molecularly primed SVs (Imig et al., 2014), and that synapse-specific levels of SV organization co-determine certain functional properties of neurotransmitter release at hippocampal synapses (Maus et al., 2020). Conventional electron microscopy (EM) techniques lack the temporal resolution and level of ultrastructural preservation required to monitor ultrastructural changes associated with STF. The development of light stimulation-coupled high-pressure freezing (HPF), also known as flash-and-freeze EM (Watanabe et al., 2013b) is a powerful method for investigating ultrastructure-function relationships associated with short-lived functional states. The goal of my PhD project was to employ optogenetics and flash-and-freeze EM to analyse the dynamic organization and activity-dependent remodelling of AZ-proximal SV pools during STF at hMFB-CA3 synapses, and to dissect the underlying molecular mechanisms. In order to do this, I used optogenetics as a tool to study synaptic structure in the context of different functional states of hMFB-CA3 synapses. To this end, I exploited mouse genetics to (i) selectively express channelrhodopsin 2 (ChR2) in granule cells (GCs) of the hippocampal dentate gyrus, and to (ii) delete Syt-7 protein expression to genetically isolate aspects of activity-dependent SV pool remodelling most relevant to the STF functional state. My data demonstrated that genetic deletion of Syt-7 at hMFB-CA3 synapses leads to an increased size of light stimulation-evoked EPSCs, and to a pronounced reduction in STF compared to Syt-7 WT synapses. Ultrastructural analysis of resting synapses revealed that Syt-7 KO AZs exhibited a SV docking deficit compared to Syt-7 WT AZs from hMFB-CA3 synapses, and that this SV docking deficit was exaggerated following stimulation, indicating a role for Syt-7 in regulating activity-dependent SV docking. This study advances our understanding of Syt-7-mediated STF, which is essential for information processing in hippocampal circuits, and lays the groundwork for a deeper understanding of mediated functions such as working memory and learning.

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.

A critical aim in neuroscience is to obtain a comprehensive view of how regulated neurotransmission is achieved. Our current understanding of synapses relies mainly on data from electrophysiological recordings, imaging, and molecular biology. Based on these methodologies, proteins involved in a synaptic vesicle (SV) formation, mobility, and fusion at the active zone (AZ) membrane have been identified. In the last decade, electron tomography (ET) combined with a rapid freezing immobilization of neuronal samples opened a window for understanding the structural machinery with the highest spatial resolution in situ. ET provides significant insights into the molecular architecture of the AZ and the organelles within the presynaptic nerve terminal. The specialized sensory ribbon synapses exhibit a distinct architecture from neuronal synapses due to the presence of the electron-dense synaptic ribbon. However, both synapse types share the filamentous structures, also commonly termed as tethers that are proposed to contribute to different steps of SV recruitment and exocytosis. In this review, we discuss the emerging views on the role of filamentous structures in SV exocytosis gained from ultrastructural studies of excitatory, mainly central neuronal compared to ribbon-type synapses with a focus on inner hair cell (IHC) ribbon synapses. Moreover, we will speculate on the molecular entities that may be involved in filament formation and hence play a crucial role in the SV cycle.

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a >40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

The likelihood with which an action potential elicits neurotransmitter release, the release probability (p(r)), is an important component of synaptic strength. Regulatory mechanisms controlling several steps of synaptic vesicle (SV) exocytosis may affect p(r), yet their relative importance in determining p(r) and eliciting temporal changes in neurotransmitter release at individual synapses is largely unknown. We have investigated whether the size of the active zone cytomatrix is a major determinant of p(r) and whether changes in its size lead to corresponding alterations in neurotransmitter release. We have used a fluorescent sensor of SV exocytosis, synaptophysin-pHluorin, to measure p(r) at individual synapses with high accuracy and employed a fluorescently labeled cytomatrix protein, Bassoon, to quantify the amount of active zone cytomatrix present at these synapses. We find that, for synapses made by a visually identified presynaptic neuron, p(r) is indeed strongly correlated with the amount of active zone cytomatrix present at the presynaptic specialization. Intriguingly, active zone cytomatrices are frequently subject to synapse-specific changes in size on a time scale of minutes. These spontaneous alterations in active zone size are associated with corresponding changes in neurotransmitter release. Our results suggest that the size of the active zone cytomatrix has a large influence on the reliability of synaptic transmission. Furthermore, they implicate mechanisms leading to rapid structural alterations at active zones in synapse-specific forms of plasticity.

Brain synapses pose special challenges on the quality control of their protein machineries as they are far away from the neuronal soma, display a high potential for plastic adaptation and have a high energy demand to fulfill their physiological tasks. This applies in particular to the presynaptic part where neurotransmitter is released from synaptic vesicles, which in turn have to be recycled and refilled in a complex membrane trafficking cycle. Pathways to remove outdated and damaged proteins include the ubiquitin-proteasome system acting in the cytoplasm as well as membrane-associated endolysosomal and the autophagy systems. Here we focus on the latter systems and review what is known about the spatial organization of autophagy and endolysomal processes within the presynapse. We provide an inventory of which components of these degradative systems were found to be present in presynaptic boutons and where they might be anchored to the presynaptic apparatus. We identify three presynaptic structures reported to interact with known constituents of membrane-based protein-degradation pathways and therefore may serve as docking stations. These are (i) scaffolding proteins of the cytomatrix at the active zone, such as Bassoon or Clarinet, (ii) the endocytic machinery localized mainly at the peri-active zone, and (iii) synaptic vesicles. Finally, we sketch scenarios, how presynaptic autophagic cargos are tagged and recruited and which cellular mechanisms may govern membrane-associated protein turnover in the presynapse.

At conventional synapses of the central nervous system (CNS), fast synaptic transmission is mediated by the release of neurotransmitters (NTs) upon Ca2+-triggered synaptic vesicle (SV) exocytosis. Upon exocytosis SV proteins have to be resorted and retrieved from the surface by compensatory endocytosis in order to replenish the pool of NT-filled SVs. For my thesis I used pH-switchable dyes, both genetically encoded as well as new exogenous ones, for optically analysing SV protein molecules necessary for fusion as well as their retrieval. Exocytosis is mediated by the assembly of low-energy SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes formed by the coil-coiling of three SNARE proteins: Synaptosomal associated protein - 25 (SNAP-25), Syntaxin 1A (Syx1A), and Synaptobrevin 2 (Syb2). However, it is unknown how many SNARE complexes are minimally needed for SV priming and fusion at CNS synapses. To resolve this issue, single vesicle fusion events were optically measured in real time using the genetically encoded probe SynaptopHluorin (SpH), a pH-sensitive green fluorescent protein (GFP), pHluorin (pHl) fused to the luminal domain of the SV SNARE Syb2. Fluorescence responses upon fusion displayed a quantal distribution of SpH molecules into SVs. Quantitative single molecule experiments revealed that the quantal size corresponds to single SpH molecule fluorescence. Surprisingly, when overexpressed on a genetic null background, SpH could fully rescue evoked SV fusion. However, SVs expressing only one copy of SpH were unable to rapidly fuse upon stimulation. Taken together, the first part of the study demonstrates that two copies of SpH and hence two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission.In order to maintain a steady-state rate of synaptic transmission the fused SV constituents are retrieved for further rounds of use by a compensatory process of endocytosis. Although clathrin-mediated endocytosis (CME) is thought to be the predominant mechanism of SV recycling, it seems too slow to account for fast recycling. Therefore, it has been suggested that a pre-sorted and pre-assembled pool of SV proteins on the presynaptic membrane might support the first phase of CME. In the second part of this study the spatial and temporal dynamics of such a readily retrievable pool (RRetP) of SV proteins at the presynaptic membrane of hippocampal neurons was monitored using a novel probe. By applying CypHer 5E, a new cyanine dye-based pH-sensitive exogenous marker, coupled to antibodies against luminal domains of SV proteins, the preferential retrieval of native SV constituents from the RRetP upon exocytosis was demonstrated. The functional size and capacity of this pool was found to closely resemble that of the readily releasable pool (RRP) of docked and primed SVs, suggesting that the RRetP can sustain SV recycling during moderate synaptic activity. Thus, the second part of the thesis demonstrates that small central synapses can avoid SV depletion in response to mild stimulation by having a preassembled pool of ready-to-go SV constituents (RRetP), which efficiently supports compensatory endocytosis to a significant degree without relying on freshly exocytosed SV constituents.

Polyhedral Protein Cages Encase Synaptic Vesicles and Participate in Their Attachment to the Active Zone

The synaptic vesicle (SV) cycle, a trafficking pathway by which SV fuses with the plasma membrane to release neurotransmitters at the neuronal synapse, resides at the heart of neurotransmission. SV fusion consumes vesicle membrane and proteins, whose availability is limited, and these components must be recycled quickly to prevent synaptic fatigue. Biochemical, genetic and physiological approaches over the past five decades have led to a discovery of a large directory of proteins and lipids central to the SV cycle and several models on how these constituents account for the synapse function. The complexity of the SV cycle is starting to be comprehended, which opens new perspectives for our understanding of neuronal physiology and provides mechanistic explanations for several neurological and neurodegenerative diseases. Here, selected classic and recent insights into the mechanisms of two key SV trafficking steps (exocytosis and endocytosis) are reviewed, as well as their links to selected brain pathologies.

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.