Mutations in the second C2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions.

The synaptic vesicle protein, synaptotagmin, has been hypothesized to mediate several functions in neurotransmitter release including calcium sensing, vesicle recycling, and synaptic vesicle docking. These hypotheses are based on evidence from in vitro binding assays, peptide and antibody injection experiments, and genetic knockout studies. Synaptotagmin contains two domains that are homologous to the calcium ion (Ca(2+))-binding C2 domain of protein kinase C. The two C2 domains of synaptotagmin have broadly differing ligand-binding properties. We have focused on the second C2 domain (C2B) of synaptotagmin I, in particular, on a series of conserved lysine residues on beta-strand 4 of C2B. This polylysine motif binds clathrin-adapter protein AP-2, neuronal calcium channels, and inositol high polyphosphates. It also mediates Ca(2+)-dependent oligomerization. To investigate the importance of these lysine residues in synaptic transmission, we have introduced synaptotagmin I (syt) transgenes harboring specific polylysine motif mutations into flies otherwise lacking the synaptotagmin I protein (syt(null)). Electrophysiological analyses of these mutants revealed that evoked transmitter release is decreased by approximately 36% and that spontaneous release is increased approximately twofold relative to syt(null) flies that express a wild type syt transgene. Synaptotagmin expression in both the mutant and the wild type transgenic lines was equivalent, as measured by semiquantitative Western blot analysis. Thus, the alteration in synaptic transmission was due to the mutation and not to the level of synaptotagmin expression. We conclude that synaptotagmin interactions mediated by the C2 B polylysine motif are required to attain full synaptotagmin function in vivo.     

Drosophila synaptotagmin I null mutants show severe alterations in vesicle populations but calcium-binding motif mutants do not

Synaptotagmin I is a synaptic vesicle protein postulated to mediate vesicle docking, vesicle recycling, and the Ca(2+) sensing required to trigger vesicle fusion. Analysis of synaptotagmin I knockouts (sytI(NULL) mutants) in both Drosophila and mice led to these hypotheses. Although much research on the mechanisms of synaptic transmission in Drosophila is performed at the third instar neuromuscular junction, the ultrastructure of this synapse has never been analyzed in sytI(NULL) mutants. Here we report severe synaptic vesicle depletion, an accumulation of large vesicles, and decreased vesicle docking at sytI(NULL) third instar neuromuscular junctions. Mutations in synaptotagmin I's C(2)B Ca(2+)-binding motif nearly abolish synaptic transmission and decrease the apparent Ca(2+) affinity of neurotransmitter release. Although this result is consistent with disruption of the Ca(2+) sensor, synaptic vesicle depletion and/or redistribution away from the site of Ca(2+) influx could produce a similar phenotype. To address this question, we examined vesicle distributions at neuromuscular junctions from third instar C(2)B Ca(2+)-binding motif mutants and transgenic wild-type controls. The number of docked vesicles and the overall number of synaptic vesicles in the vicinity of active zones was unchanged in the mutants. We conclude that the near elimination of synaptic transmission and the decrease in the Ca(2+) affinity of release observed in C(2)B Ca(2+)-binding motif mutants is not due to altered synaptic vesicle distribution but rather is a direct result of disrupting synaptotagmin I's ability to bind Ca(2+). Thus, Ca(2+) binding by the C(2)B domain mediates a post-docking step in fusion.     

Presynaptic disruption of transmitter release by lead

Low concentrations of inorganic lead ions (Pb2+) disrupt transmitter release by causing aberrant augmentation of spontaneous and suppression of evoked release. These effects result from high affinity interactions of Pb2+ with the voltage-gated calcium channels (VGCC) as well as Ca2+ binding proteins which regulate the synaptic vesicle mobilization, docking, and exocytosis processes. Augmentation of spontaneous release may involve stimulation of vesicle mobilization consequent to Pb2+ activation of CaMKII-dependent phosphorylation of synapsin I and/or stimulation of asynchronous exocytosis via direct Pb2+ activation of the putative exocytotic Ca2+-sensor protein synaptotagmin I. In addition, synergistic stimulation of PLC and DAG/Pb2+-dependent activation of PKC may enhance the secretagogue effects of Pb2+ by increasing metal sensitivity of exocytosis and/or modulating calcium channel activity. In contrast to intracellularly-mediated actions of Pb2+ resulting in augmentation of spontaneous release, the inhibition of evoked transmitter release by Pb2+ is largely attributable to extracellular block of the voltage-gated calcium channels.     

Deconstructing Synaptotagmin-1's Distinct Roles in Synaptic Vesicle Priming and Neurotransmitter Release

Synaptotagmin-1 (SYT1) is a synaptic vesicle resident protein that interacts via its C2 domain with anionic lipids from the plasma membrane in a calcium-dependent manner to efficiently trigger rapid neurotransmitter (NT) release. In addition, SYT1 acts as a negative regulator of spontaneous NT release and regulates synaptic vesicle (SV) priming. How these functions relate to each other mechanistically and what role other synaptotagmin (SYT) isoforms play in supporting and complementing the role of SYT1 is still under intensive investigation. In this work, we analyzed three putative functions of SYT1 in exocytosis by systematically varying its expression in autaptic hippocampal glutamatergic neurons from mice of either sex. We find that regulation of release probability is most sensitive to variation of expression levels, whereas its impact on vesicle priming is least sensitive. Also, loss of SYT1 phenotypes on spontaneous release and vesicle priming is compensated in less mature synaptic cultures by redundant support from SYT7. Overall, our data help in resolving some controversies in SYT1 functions in exocytosis and in our understanding of how SYT1 contributes to the pathophysiology underlying SYT1-related human neurologic disorders.SIGNIFICANCE STATEMENT Our work clarifies the functions of SYT1 protein in synaptic vesicle priming and spontaneous and calcium-evoked neurotransmitter release and analyzes whether these occur at different stages of synaptic responses by examining their relative sensitivity to protein concentration at the synaptic terminal. We demonstrate that these synaptic functions are unequally sensitive to both protein levels and neuronal stage, indicating that they operate under distinct molecular mechanisms. Furthermore, we analyze how these functions are modulated by another synaptotagmin isoform expression. We show that to understand the phenotype displayed by SYT1 knock-out neurons (Syt1^−/−) is necessary to consider the interplay between SYT1 and SYT7 molecules at the presynaptic terminal.     

Ca(2+)-independent syntaxin binding to the C(2)B effector region of synaptotagmin

Although synaptotagmin I, which is a calcium (Ca²⁺)-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remain controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca²⁺ is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca²⁺ to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca²⁺-independent manner, and the binding was abolished in the presence of 1 M NaCl. Synaptotagmin contains 2 Ca²⁺-binding domains (C₂A, C₂B). Mutating the positively charged lysine residues in the putative effector-binding region of the C₂B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C₂A domain had no effect on binding. Synaptotagmin-syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C₂B domain effector region in a Ca²⁺-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca²⁺ influx into presynaptic nerve terminals.     

Distinct developmental expression of synaptotagmin I and IX in the mouse brain

Synaptotagmin IX has been postulated to function as a major Ca2+ sensor for dense-core vesicle exocytosis in neuroendocrine cells. In this study, we investigated the subcellular localization and developmental expression profile of synaptotagmin IX in the mouse brain and found that it is mainly present in the dense-core vesicle fraction, which is devoid of synaptotagmin I and synaptophysin. We also found that the synaptotagmin IX expression level is constant throughout the postnatal development of the mouse brain, whereas the synaptotagmins I, II, III, VI, and XII are upregulated in parallel with synaptogenesis. These findings suggest that synaptotagmin IX regulates the transport of certain vesicles in the brain other than synaptic vesicles.     

Synaptic vesicle protein 2A: basic facts and role in synaptic function

In recent years, there has been considerable interest in determining the function of synaptic vesicle protein 2A and its role as a target for antiepileptic drugs. Although it is known that synaptic vesicle protein 2A is involved in normal synaptic vesicle function, its participation in synaptic vesicle cycling and neurotransmitter release in normal and pathological conditions is unclear. However, the experimental evidence suggests that synaptic vesicle protein 2A could be a vesicular transporter, regulate synaptic exocytosis as a gel matrix, or modulate synaptotagmin‐1 activity. This review describes and discusses the participation of synaptic vesicle protein 2A in synaptic modulation in normal and pathological conditions.     

The calcium sensor synaptotagmin 1 is expressed and regulated in hippocampal postsynaptic spines

Synaptotagmin 1 is a presynaptic calcium sensor, regulating SNARE‐mediated vesicle exocytosis of transmitter. Increasing evidence indicate roles of SNARE proteins in postsynaptic glutamate receptor trafficking. However, a possible postsynaptic expression of synaptotagmin 1 has not been demonstrated previously. Here, we used postembedding immunogold electron microscopy to determine the subsynaptic localization of synaptotagmin 1 in rat hippocampal CA1 Schaffer collateral synapses. We report for the first time that synaptotagmin 1 is present in rat hippocampal postsynaptic spines, both on cytoplasmic vesicles and at the postsynaptic density. We further investigated whether postsynaptic synaptotagmin 1 is regulated during synaptic plasticity. In a rat model of chronic temporal lobe epilepsy, we found that presynaptic and postsynaptic concentrations of the protein are reduced compared to control animals. This downregulation may possibly be an adaptive measure to decrease both presynaptic and postsynaptic calcium sensitivity in excitotoxic conditions.     

Reduced hippocampal LTP in mice lacking a presynaptic protein: complexin II.

The SNAP receptor (SNARE) complex is a core complex specialized for synaptic vesicle exocytosis, and the binding of SNAPs to the complex is an essential step for neurotransmitter release. Complexin I and II have been identified as SNARE-complex-associated proteins. Importantly, complexins compete with alpha-SNAP for binding to the complex, suggesting that complexins may modulate neurotransmitter release process. To examine this possibility and to understand the physiological function of complexins, we generated complexin II knockout mice. The complexin-II-deficient mice (-/-) were viable and fertile, and appeared normal. Electrophysiological recordings in the mutant hippocampus showed that ordinary synaptic transmission and paired-pulse facilitation, a form of short-term synaptic plasticity, were normal. However, long-term potentiation (LTP) in both CA1 and CA3 regions was impaired, suggesting that complexin II may not be essential for synaptic vesicle exocytosis, but it does have a role in the establishment of hippocampal LTP.     

An antibody to synaptotagmin I facilitates synaptic transmission

Proper functioning of the nervous system requires precise control of neurotransmitter release. Synaptotagmin, a synaptic vesicle protein, is crucial for the temporal control of neurotransmitter release. The mechanism of synaptotagmin function is still under debate. To investigate the mechanism by which synaptotagmin controls neurotransmitter release, we injected an antibody of rat synaptotagmin I into a crayfish motor axon. We found that the antibody enhanced synaptic transmission at crayfish neuromuscular junctions by increasing the amplitude of the evoked synaptic response. This effect was antibody-dose dependent. The antibody also reduced the rise time of the synaptic potentials. These effects were accompanied by a reduction in the Hill coefficient for Ca(2+)-dependence of synaptic transmission. Our findings support the hypothesis that synaptotagmin inhibits neurotransmitter release in the absence of Ca(2+).     

SV2A and SV2C contain a unique synaptotagmin-binding site

SV2 (Synaptic Vesicle Protein 2) is expressed in neurons and endocrine cells where it is required for normal calcium-evoked neurosecretion. In mammals, there are three SV2 genes, denoted SV2A, B and C. SV2A interacts with synaptotagmin, the prime candidate for the calcium sensor in exocytosis. Here, we report that all isoforms of native SV2 bind synaptotagmin and that binding is inhibited by calcium, indicating that all isoforms contain a common calcium-inhibited synaptotagmin-binding site. The isolated amino termini of SV2A and SV2C supported an additional interaction with synaptotagmin, and binding at this site was stimulated by calcium. The amino-terminal binding site was mapped to the first 57 amino acids of SV2A, and removal of this domain decreased calcium-mediated inhibition of binding to synaptotagmin, suggesting that it modulates calcium's effect on the SV2-synaptotagmin interaction. Introduction of the amino terminus of SV2A or SV2C into cultured superior cervical ganglion neurons inhibited neurotransmission, whereas the amino terminus of SV2B did not. These observations implicate the SV2-synaptotagmin interaction in regulated exocytosis and suggest that SV2A and SV2C, via their additional synaptotagmin binding site, function differently than SV2B.     

Synaptotagmin can cause an immune-mediated model of Lambert-Eaton myasthenic syndrome in rats

The possible antigenicity of synaptotagmin, a synaptic vesicle protein acting as a cooperative calcium (Ca²⁺) receptor in exocytosis, was tested to determine whether it is involved in the induction of Lambert-Eaton myasthenic syndrome in which antibodies against voltage-dependent Ca²⁺ channels or related molecules play a pathogenic role. Repeated injections to Lewis rats with peptides of synaptotagmin residues 20 through 53 or 1 through 30 that are presumably exposed at the nerve terminal surface during exocytosis induced corresponding antipeptide antibodies; on immunoblots, antibodies recognized synaptotagmin that was expressed in the clonal cells. Electrophysiologically, the peptide (residues 20–53)-immunized rats showed (1) reduced acetylcholine quantal content of end-plate potential, (2) an increase in quantal content at high extracellular Ca²⁺ concentration, and (3) early facilitation followed by less marked depression of end-plate potential amplitude at a tetanic rate of repetitive nerve stimulation. Findings are similar to those in human Lambert-Eaton myasthenic syndrome and passively transferred Lambert-Eaton myasthenic syndrome in mice, and thus suggest that antibody to a synaptotagmin–voltage-dependent Ca²⁺ channel complex may be involved in the pathogenesis of Lambert-Eaton myasthenic syndrome. The peptide (residues 1–30)-immunized rats showed no electrophysiological abnormality.     

Redistribution of presynaptic proteins during alpha-latrotoxin-induced release of neurotransmitter and membrane retrieval at the frog neuromuscular junction

Calcium-dependent exocytosis at the nerve terminal involves the synaptic core (SNARE) complex composed of the t-SNAREs syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), and the v-SNARE vesicle-associated membrane protein (VAMP/synaptobrevin), a stable heterotrimer which can associate with the putative calcium sensor protein, synaptotagmin. The distribution of these proteins at the frog neuromuscular junction was examined by immunofluorescent staining and confocal microscopy following exocytosis induced by alpha-latrotoxin. Experiments were performed under conditions in which synaptic vesicle recycling was either maintained in balance with exocytosis, or completely blocked, or during recovery from block of endocytosis. When endocytosis was maintained, protein distribution was essentially identical to that of unstimulated nerve terminals, in which syntaxin 1 and SNAP-25 are localized to the presynaptic active zones coincident with the postsynaptic folds that contain a high density of acetylcholine receptors (AChRs). Block of endocytosis led to complete incorporation of vesicle proteins into the plasmalemma, and t-SNARE distribution was no longer restricted to active zones. Five minutes after the onset of recovery, both synaptic vesicle proteins and t-SNARE proteins were concentrated into small spots, in a similar pattern to that obtained following endocytosis of the vital styryl dye FM1-43. These findings are consistent with a model in which following sustained exocytosis, t-SNARE trafficking involves internalization and transit via a vesicular compartment before recycling to the presynaptic plasma membrane.     

Synaptotagmin,a synaptic vesicle protein,is present in human cerebrospinal fluid

Using a novel approach, including affinity chromatography, reversed-phase chromatography, and chemiluminescence immunoblotting, we have for the first time been able to demonstrate one of the small synaptic vesicle proteins, synaptotagmin I, in cerebrospinal fluid (CSF). Two other small synaptic vesicle proteins, rab3a and synaptophysin, were not detectable. The approximate molecular weight of CSF-synaptotagmin was 65 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Further characterization of CSF synaptotagmin by high-performance capillary electrophoresis (HPCE) showed a single peak. These findings support that the whole synaptotagmin molecule is present in CSF, without significant proteolytic degradation. After high-speed centrifugation of CSF, synaptotagmin was exclusively found in the supernatant, suggesting that synaptotagmin is present in CSF as a free protein, and not as a constituent of synaptic vesicles. In a preliminary study, we found a marked reduction of CSF synaptotagmin in patients with early onset Alzheimer disease (EAD) as compared with age-matched healthy individuals. To elucidate the biological relevance of this finding, we also quantified synaptotagmin in brain tissue. A marked reduction in synaptotagmin was found both in the hippocampus and frontal cortex of EAD, suggesting that a decrease in synaptotagmin in the brain is followed by a concomitant decrease in the CSF. Analysis of CSF synaptotagmin might provide a tool to study synaptic function and pathology in the human brain.     

Synaptic vesicle reuse and its implications.

Presynaptic nerve terminals are exquisite vesicle trafficking machines. Neurotransmission is sustained by constant recycling of a handful of vesicles. Therefore, the rate and the pathway of vesicle trafficking can critically determine synaptic efficacy during activity. However, it is yet unclear whether synaptic vesicle recycling becomes rate limiting on a rapid time scale during physiologically relevant forms of activity in the brain. Several forms of synaptic plasticity arise from persistent alterations in the dynamics of vesicle trafficking in presynaptic terminals. What makes presynaptic forms of plasticity particularly interesting is that they not only increase or decrease the amplitude of synaptic responses but also cause frequency-dependent changes in neurotransmission. In this manner, plasticity can alter the information coding in neural circuits beyond simple scaling of synaptic responses. However, studying the synaptic vesicle cycle beyond exocytosis and endocytosis has been difficult. In the past decade, several methods have been developed to infer vesicles' trajectory during their cycle in the synapse. Nevertheless, several questions remain. A better understanding of the role of synaptic vesicle trafficking in neurotransmission will require novel approaches that either combine existing methods or the development of new methods to trace vesicles during their cycle. Recent evidence suggests that various presynaptic proteins involved in the synaptic function and homeostasis are either mutated or altered in their expression in several neurological and psychiatric disorders. Therefore, elucidation of the mechanisms that underlie the synaptic vesicle cycle may reveal novel therapeutic targets for brain disorders.     

Crosstalk between huntingtin and syntaxin 1A regulates N-type calcium channels

We have identified a novel interaction between huntingtin (htt) and N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis. Htt is a widely expressed 350-kDa cytosolic protein bearing an N-terminal polyglutamine tract. Htt is proteolytically cleaved by calpains and caspases and the resultant htt N-terminal fragments have been proposed to be biologically active; however, the cellular function of htt and/or the htt fragments remains enigmatic. We show that N-terminal fragments of htt (consisting of exon1) and full-length htt associate with the synaptic protein interaction (synprint) region of the N-type calcium channel. Given that synprint has previously been shown to bind syntaxin 1A and that this association elicits inhibition of N-type calcium channels, we tested whether htt(exon1) affects the modulation of these channels. Our data indicate that htt(exon1) enhances calcium influx by blocking syntaxin 1A inhibition of N-type calcium channels and attributes a key role for htt N-terminal fragments in the fine tuning of neurotransmission.     

Interaction of synaptotagmin with voltage gated calcium channels: A role in Lambert-Eaton myasthenic syndrome?

Plasma from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, contains antibodies that bind to the synaptic vesicle protein synaptotagmin. Synaptotagmin associates with calcium channels and appears to regulate synaptic vesicle docking at the plasma membrane prior to rapid neurotransmitter release. Autoantibodies directed against a synaptotagmin-calcium channel complex may be involved in the etiology of LEMS. In the majority of patients LEMS is associated with small cell lung cancer (SCLC). We have detected the expression of proteins of the secretory pathway, including synaptotagmin, syntaxin and N-type calcium channels, in a panel of SCLC tumor lines. These observations are compatible with the hypothesis that the initial autoimmune response in LEMS is triggered by the tumor.     

Synaptotagmin I and IV are differentially regulated in the brain by the recreational drug 3,4-methylenedioxymethamphetamine (MDMA)

3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy) is a widely abused drug. In brains of mice exposed to MDMA, we recently detected altered expression of several cDNAs and genes by using the differential display polymerase chain reaction (PCR) method. Expression of one such cDNA, which exhibited 98% sequence homology with the synaptic vesicle protein synaptotagmin IV, decreased 2 h after MDMA treatment. Herein, the effect of MDMA on expression of both synaptotagmin I and IV was studied in detail, since the two proteins are functionally interrelated. PCR analyses (semi-quantitative and real-time) confirmed that upon treatment with MDMA, expression of synaptotagmin IV decreased both in the midbrain and frontal cortex of mice. Decreases in the protein levels of synaptotagmin IV were confirmed by Western immunoblotting with anti-synaptotagmin IV antibodies. In contrast, the same exposure to MDMA increased expression of synaptotagmin I in the midbrain, a region rich in serotonergic neurons, but not in the frontal cortex. This differential expression was confirmed at the protein level with anti-synaptotagmin I antibodies. MDMA did not induce down- or up-regulation of synaptotagmin IV and I, respectively, in serotonin transporter knockout mice (-/-) that are not sensitive to MDMA. Therefore, psychoactive drugs, such as MDMA, appear to modulate expression of synaptic vesicle proteins, and possibly vesicle trafficking, in the brain.     

The synaptotagmins: calcium sensors for vesicular trafficking.

The synaptotagmin family of vesicle proteins is believed to mediate calcium-dependent regulation of membrane trafficking. Detailed biochemical and in vivo studies of the most characterized isoform, synaptotagmin 1 (syt 1), have provided compelling evidence that it functions as a calcium sensor for fast neurotransmitter release at synapses. However, the function of the remaining isoforms is unclear, and multiple roles have been hypothesized for several of these. Recent evidence in Drosophila has given insight into the function of some of the remaining synaptotagmin family members. Of the five evolutionarily conserved isoforms in Drosophila, only two, syt 1 and syt 4, localize to most, if not all, synapses. The former is localized to presynaptic terminals, whereas the latter is predominantly postsynaptic. This suggests an intriguing possibility that syt 4 may mediate a postsynaptic vesicle trafficking pathway, providing a molecular basis for an evolutionarily conserved bidirectional vesicular trafficking communication system at synapses.     

The synaptic ribbon is a site of phosphatidic acid generation in ribbon synapses

Ribbon synapses continuously transmit graded membrane potential changes into changes of synaptic vesicle exocytosis and rely on intense synaptic membrane trafficking. The synaptic ribbon is considered central to this process. In the present study we asked whether tonically active ribbon synapses are associated with the generation of certain lipids, specifically the highly active signaling phospholipid phosphatidic acid (PA). Using PA-sensor proteins, we demonstrate that PA is enriched at mouse retinal ribbon synapses in close vicinity to the synaptic ribbon in situ. As shown by heterologous expression, RIBEYE, a main component of synaptic ribbons, is responsible for PA binding at synaptic ribbons. Furthermore, RIBEYE is directly involved in the synthesis of PA. Using various independent substrate binding and enzyme assays, we demonstrate that the B domain of RIBEYE possesses lysophosphatidic acid (LPA) acyltransferase (LPAAT) activity, which leads to the generation of PA from LPA. Since an LPAAT-deficient RIBEYE mutant does not recruit PA-binding proteins to artificial synaptic ribbons, whereas wild-type RIBEYE supports PA binding, we conclude that the LPAAT activity of the RIBEYE(B) domain is a physiologically relevant source of PA generation at the synaptic ribbon. We propose that PA generated at synaptic ribbons likely facilitates synaptic vesicle trafficking.