Several members of the synaptotagmin (syt) family of vesicle proteins have been proposed to act as Ca2+ sensors on synaptic vesicles. The mechanism by which calcium activates this class of proteins has been the subject of controversy, yet relatively few detailed biophysical studies have been reported on how isoforms other than syt I respond to divalent metal ions. Here, we report a series of studies on the response of syt II to a wide range of metal ions. Analytical ultracentrifugation studies demonstrate that Ca2+ induces protein dimerization upon exposure to 5 mM Ca2+. Whereas Ba2+, Mg2+, or Sr2+ do not potentiate self-association as strongly as Ca2+, Pb2+ triggers self-association of syt II at concentrations as low as 10 microM. Partial proteolysis studies suggest that the various divalent metals cause different changes in the conformation of the protein. The high calcium concentrations required for self-association of syt II suggest that the oligomerized state of this protein is not a critical intermediate in vesicle fusion; however, low-affinity calcium sites on syt II may play a critical role in buffering calcium at the presynaptic active zone. In addition, the high propensity of lead to oligomerize syt II offers a possible molecular explanation for how lead interferes with calcium-evoked neurotransmitter release.

Synaptotagmins (Syts) I and II are believed to act as Ca2+ sensors in the control of neurotransmission. Here we demonstrate that mast cells express Syt II in their lysosomal fraction. We further show that activation of mast cells by either aggregation of FcepsilonRI or by Ca2+ ionophores results in exocytosis of lysosomes, in addition to the well documented exocytosis of their secretory granules. Syt II directly regulates lysosomal exocytosis, whereby overexpression of Syt II inhibited Ca2+-triggered release of the lysosomal processed form of cathepsin D, whereas suppression of Syt II expression markedly potentiated this release. These findings provide evidence for a novel function of Syt II in negatively regulating Ca2+-triggered exocytosis of lysosomes, and suggest that Syt II-regulated secretion from lysosomes may play an important role in mast cell biology.

Synaptotagmin 1 and 2 (syt 1, syt 2) are synaptic vesicle-associated membrane proteins that act as calcium sensors for fast neurotransmitter release from presynaptic nerve terminals. Here we show that widely used monoclonal antibodies, mab 48 and znp-1, stain nerve terminals in multiple species and, in mouse, recognize syt 1 and syt 2, respectively. With these antibodies, we examined the synaptic localization of these synaptotagmin isoforms in the mouse central nervous system. Syt 1 and syt 2 are localized predominantly to different subsets of synapses in retina, hippocampus, cerebellum, and median nucleus of the trapezoid body (MNTB). In the MNTB, syt 1 and syt 2 are present in different presynaptic terminals on the same postsynaptic principal neuron. In retina, horizontal and OFF-bipolar cell terminals contain syt 2, whereas most other terminals contain syt 1. Syt 1 localization in the immature retina resembles that seen in adult; however, syt 2 localization appears strikingly different at perinatal ages and continues to change dramatically prior to eye opening. For example, starburst amacrine cells, which lack syt 2 in adult retina, transiently express syt 2 during the first 2 postnatal weeks. In addition to differences in spatial and temporal distribution, species-specific differences in synaptotagmin localization were observed in retina and cerebellum. The cell-, temporal-, and species-specific expression of synaptotagmin isoforms suggests that each may have distinct functions in neurotransmitter release.

Synaptotagmin II is a type I signal-anchor protein, in which the NH(2)-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.

Botulinum neurotoxins (BoNTs) can cause paralysis at exceptionally low concentrations and include seven serotypes (BoNT/A-G). The chimeric BoNT/DC toxin has a receptor binding domain similar to the same region in BoNT/C. However, BoNT/DC does not share protein receptor with BoNT/C. Instead, it shares synaptotagmin (Syt) I and II as receptors with BoNT/B, despite their low sequence similarity. Here, we present the crystal structures of the binding domain of BoNT/DC in complex with the recognition domains of its protein receptors, Syt-I and Syt-II. The structures reveal that BoNT/DC possesses a Syt binding site, distinct from the established Syt-II binding site in BoNT/B. Structure-based mutagenesis further shows that hydrophobic interactions play a key role in Syt binding. The structures suggest that the BoNT/DC ganglioside binding sites are independent of the protein receptor binding site. Our results reveal the remarkable versatility in the receptor recognition of the BoNTs.

The lateral superior olive (LSO), a nucleus in the auditory brainstem, computes interaural intensity differences for sound localization by comparing converging excitatory and inhibitory inputs that carry tonotopically matched information from the two ears. Tonotopic refinement in the inhibitory projection pathway from the medial nucleus of the trapezoid body (MNTB) is known to be established during the first postnatal week in rats. During this period, immature MNTB terminals in the LSO contain vesicular transporters for both inhibitory and excitatory amino acids and release glutamate. The primary Ca(2+) sensors for vesicular release in the CNS are understood to be synaptotagmins, and in adult auditory brainstem synaptotagmin 2 is the predominant synaptotagmin. We asked here whether a different Ca(2+) sensor might be expressed in the immature auditory brainstem. We have found that synaptotagmin 1 is indeed expressed transiently in the immature auditory brainstem, most highly in those areas that receive glutamate-releasing immature inhibitory inputs from the MNTB, and that during the first postnatal week synaptotagmin 1 co-localizes with the vesicular glutamate transporter VGLUT3, a marker of glutamate-releasing immature inhibitory terminals from the MNTB. We suggest that immature MNTB terminals may contain two populations of synaptic vesicles, one expressing the vesicular inhibitory amino acid transporter together with synaptotagmin 2 and another expressing VGLUT3 together with synaptotagmin 1. Because Ca(2+) sensing is an important determinant of release properties for the presynaptic terminal, differential expression of the synaptotagmins might allow the differential release of excitatory and inhibitory neurotransmitters in response to differing patterns of neural activity.

Mature hair cells transduce information over a wide range of stimulus intensities and frequencies for prolonged periods of time. The efficiency of such a demanding task is reflected in the characteristics of exocytosis at their specialized presynaptic ribbons. Ribbons are electron-dense structures able to tether a large number of releasable vesicles allowing them to maintain high rates of vesicle release. Calcium entry through rapidly activating, non-inactivating CaV 1.3 (L-type) Ca2+ channels in response to cell depolarization causes a local increase in Ca2+ at the ribbon synapses, which is detected by the exocytotic Ca2+ sensors. The Ca2+ dependence of vesicle exocytosis at mammalian vestibular hair cell (VHC) ribbon synapses is believed to be linear, similar to that observed in mature cochlear inner hair cells (IHCs). The linear relation has been shown to correlate with the presence of the Ca2+ sensor synaptotagmin-4 (Syt-4). Therefore, we studied the exocytotic Ca2+ dependence, and the release kinetics of different vesicle pool populations, in Type II VHCs of control and Syt-4 knockout mice using patch-clamp capacitance measurements, under physiological recording conditions. We found that exocytosis in mature control and knockout Type II VHCs displayed a high-order dependence on Ca2+ entry, rather than the linear relation previously observed. Consistent with this finding, the Ca2+ dependence and release kinetics of the ready releasable pool (RRP) of vesicles were not affected by an absence of Syt-4. However, we did find that Syt-4 could play a role in regulating the release of the secondary releasable pool (SRP) in these cells. Our findings show that the coupling between Ca2+ influx and neurotransmitter release at mature Type II VHC ribbon synapses is faithfully described by a nonlinear relation that is likely to be more appropriate for the accurate encoding of low-frequency vestibular information, consistent with that observed at low-frequency mammalian auditory receptors.

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.

Parkin is an E3 ubiquitin ligase whose mutations cause autosomal recessive juvenile Parkinson's disease (PD). Unlike the human phenotype, parkin knockout (KO) mice show no apparent dopamine neuron degeneration, although they demonstrate reduced expression and activity of striatal mitochondrial proteins believed to be necessary for neuronal survival. Instead, parkin-KO mice show reduced striatal evoked dopamine release, abnormal synaptic plasticity, and non-motor symptoms, all of which appear to mimic the preclinical features of Parkinson's disease. Extensive studies have screened candidate synaptic proteins responsible for reduced evoked dopamine release, and synaptotagmin XI (Syt XI), an isoform of Syt family regulating membrane trafficking, has been identified as a substrate of parkin in humans. However, its expression level is unaltered in the striatum of parkin-KO mice. Thus, the target(s) of parkin and the molecular mechanisms underlying the impaired dopamine release in parkin-KO mice remain unknown. In this study, we focused on Syt IV because of its highly homology to Syt XI, and because they share an evolutionarily conserved lack of Ca2+-binding capacity; thus, Syt IV plays an inhibitory role in Ca2+-dependent neurotransmitter release in PC12 cells and neurons in various brain regions. We found that a proteasome inhibitor increased Syt IV protein, but not Syt XI protein, in neuron-like, differentiated PC12 cells, and that parkin interacted with and polyubiquitinated Syt IV, thereby accelerating its protein turnover. Parkin overexpression selectively degraded Syt IV protein, but not Syt I protein (indispensable for Ca2+-dependent exocytosis), thus enhancing depolarization-dependent exocytosis. Furthermore, in parkin-KO mice, the level of striatal Syt IV protein was increased. Our data indicate a crucial role for parkin in the proteasomal degradation of Syt IV, and provide a potential mechanism of parkin-regulated, evoked neurotransmitter release.

Ca(2+)-dependent regulation of fusion pore dilation and closure is a key mechanism determining the output of cellular secretion. We have recently described 'fusion-activated' Ca(2+) entry (FACE) following exocytosis of lamellar bodies in alveolar type II cells. FACE regulates fusion pore expansion and facilitates secretion. However, the mechanisms linking this locally restricted Ca(2+) signal and fusion pore expansion were still elusive. Here, we demonstrate that synaptotagmin-7 (Syt7) is expressed on lamellar bodies and links FACE and fusion pore dilation. We directly assessed dynamic changes in fusion pore diameters by analysing diffusion of fluorophores across fusion pores. Expressing wild-type Syt7 or a mutant Syt7 with impaired Ca(2+)-binding to the C2 domains revealed that binding of Ca(2+) to the C2A domain facilitates FACE-induced pore dilation, probably by inhibiting translocation of complexin-2 to fused vesicles. However, the C2A domain hampered Ca(2+)-dependent exocytosis of lamellar bodies. These findings support the hypothesis that Syt7 modulates fusion pore expansion in large secretory organelles and extend our picture that lamellar bodies contain the necessary molecular inventory to facilitate secretion during the exocytic post-fusion phase. Moreover, regulating Syt7 levels on lamellar bodies appears to be essential in order that exocytosis is not impeded during the pre-fusion phase.

Numerous studies have verified that electroacupuncture (EA) can relieve neuropathic pain through a variety of mechanisms. Synaptotagmin 1 (Syt-1), a synaptic vesicle protein for regulating exocytosis of neurotransmitters, was found to be affected by EA stimulation. However, the roles of Syt-1 in neuropathic pain and EA-induced analgesic effect remain unclear. Here, the effect of Syt-1 on nociception was assessed through an antibody blockade, siRNA silencing, and lentivirus-mediated overexpression of spinal Syt-1 in rats with spared nerve injury (SNI). EA was used for stimulating bilateral "Sanjinjiao" and "Zusanli" acupoints of the SNI rats to evaluate its effect on nociceptive thresholds and spinal Syt-1 expression. The mechanically and thermally nociceptive behaviors were assessed with paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different temperatures, respectively, at day 0, 7, 8, 14, and 20. Syt-1 mRNA and protein levels were determined with qRT-PCR and Western blot, respectively, and its distribution was observed with the immunohistochemistry method. The results demonstrated Syt-1 antibody blockade and siRNA silencing increased ipsilateral PWTs and PWLs of SNI rats, while Syt-1 overexpression decreased ipsilateral PWTs and PWLs of rats. EA significantly attenuated nociceptive behaviors and down-regulated spinal Syt-1 protein levels (especially in laminae I-II), which were reversed by Syt-1 overexpression. Our findings firstly indicate that Syt-1 is involved in the development of neuropathic pain and that EA attenuates neuropathic pain, probably through suppressing Syt-1 protein expression in the spinal cord.

Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA-mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.

GABAergic synapses in brain circuits generate inhibitory output signals with submillisecond latency and temporal precision. Whether the molecular identity of the release sensor contributes to these signaling properties remains unclear. Here, we examined the Ca2+ sensor of exocytosis at GABAergic basket cell (BC) to Purkinje cell (PC) synapses in cerebellum. Immunolabeling suggested that BC terminals selectively expressed synaptotagmin 2 (Syt2), whereas synaptotagmin 1 (Syt1) was enriched in excitatory terminals. Genetic elimination of Syt2 reduced action potential-evoked release to ∼10%, identifying Syt2 as the major Ca2+ sensor at BC-PC synapses. Differential adenovirus-mediated rescue revealed that Syt2 triggered release with shorter latency and higher temporal precision and mediated faster vesicle pool replenishment than Syt1. Furthermore, deletion of Syt2 severely reduced and delayed disynaptic inhibition following parallel fiber stimulation. Thus, the selective use of Syt2 as release sensor at BC-PC synapses ensures fast and efficient feedforward inhibition in cerebellar microcircuits.

Plant viruses cross the barrier of the plant cell wall by moving through intercellular channels, termed plasmodesmata, to invade their hosts. They accomplish this by encoding movement proteins (MPs), which act to alter plasmodesmal gating. How MPs target to plasmodesmata is not well understood. Our recent characterization of the first plasmodesmal localization signal (PLS) identified in a viral MP, namely, the MP encoded by the Tobamovirus Tobacco mosaic virus (TMV), now provides the opportunity to identify host proteins that recognize this PLS and may be important for its plasmodesmal targeting. One such candidate protein is Arabidopsis synaptotagmin A (SYTA), which is required to form endoplasmic reticulum (ER)-plasma membrane contact sites and regulates the MP-mediated trafficking of begomoviruses, tobamoviruses, and potyviruses. In particular, SYTA interacts with, and regulates the cell-to-cell transport of, both TMV MP and the MP encoded by the Tobamovirus Turnip vein clearing virus (TVCV). Using in planta bimolecular fluorescence complementation (BiFC) and yeast two-hybrid assays, we show here that the TMV PLS interacted with SYTA. This PLS sequence was both necessary and sufficient for interaction with SYTA, and the plasmodesmal targeting activity of the TMV PLS was substantially reduced in an Arabidopsis syta knockdown line. Our findings show that SYTA is one host factor that can recognize the TMV PLS and suggest that this interaction may stabilize the association of TMV MP with plasmodesmata.IMPORTANCE Plant viruses use their movement proteins (MPs) to move through host intercellular connections, plasmodesmata. Perhaps one of the most intriguing, yet least studied, aspects of this transport is the MP signal sequences and their host recognition factors. Recently, we have described the plasmodesmal localization signal (PLS) of the Tobacco mosaic virus (TMV) MP. Here, we identified the Arabidopsis synaptotagmin A (SYTA) as a host factor that recognizes TMV MP PLS and promotes its association with the plasmodesmal membrane. The significance of these findings is two-fold: (i) we identified the TMV MP association with the cell membrane at plasmodesmata as an important PLS-dependent step in plasmodesmal targeting, and (ii) we identified the plant SYTA protein that specifically recognizes PLS as a host factor involved in this step.

Synaptotagmin is a synaptic vesicle protein that has been proposed to be the calcium sensor responsible for fast neurotransmitter release at synapses. Synaptotagmin's two C2 domains, C2A and C2B, each provide a calcium binding pocket lined with negative charges contributed by five conserved aspartates. We find that even when all of C2A's conserved aspartates are neutralized by replacement with asparagines, neurotransmitter release still occurs at hippocampal synapses in culture. Because exocytosis continues to be dependent on extracellular calcium concentration, the C2A domain cannot represent the entire calcium sensor. C2A does appear to be part of the calcium sensor, however, because substitution of D232 alters the calcium dependence of release, perhaps by reducing the number of calcium ions that must bind to trigger exocytosis. We conclude that neutralization of the negative charge at D232 by coordination of a calcium ion is necessary--but not sufficient--for fast neurotransmission at mammalian CNS synapses.

The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P(2)-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.

Synaptotagmin 7 (Syt7) is thought to be a Ca2+ sensor that mediates asynchronous transmitter release and facilitation at synapses. However, Syt7 is strongly expressed in fast-spiking, parvalbumin-expressing GABAergic interneurons, and the output synapses of these neurons produce only minimal asynchronous release and show depression rather than facilitation. To resolve this apparent contradiction, we examined the effects of genetic elimination of Syt7 on synaptic transmission at the GABAergic basket cell (BC)-Purkinje cell (PC) synapse in cerebellum. Our results indicate that at the BC-PC synapse, Syt7 contributes to asynchronous release, pool replenishment, and facilitation. In combination, these three effects ensure efficient transmitter release during high-frequency activity and guarantee frequency independence of inhibition. Our results identify a distinct function of Syt7: ensuring the efficiency of high-frequency inhibitory synaptic transmission.

Synaptotagmins are a large family of synaptic vesicle membrane proteins, that appear to be involved in neurotransmitter secretion from small secretory vesicles. We have quantitatively analysed the messenger RNA levels of synaptotagmin I-IV isoforms in adult hypothalamic and pituitary tissues in order to determine which of these isoforms dominate in these tissues which mainly secrete peptides from large dense core vesicles. We also studied the expression of these isoforms during prenatal (E15, and E17) and postnatal (P1, P7, P14 and P21) rat hypothalamic development. In order to assay small individual samples (e.g., pituitary and embryonic tissues), we employed quantitative reverse transcription-polymerase chain reaction methods. Our results show that synaptotagmin I messenger RNA is the most abundant isoform in all tissues, and is about 5.4- or 38-fold higher in hypothalamus than in neurointermediate and anterior pituitary lobe, respectively. Synaptotagmin II, which is very abundant in cerebellum, is relatively low in hypothalamus (5% of cerebellum) and virtually absent from the pituitary. Synaptotagmin III is about 10 times greater in the neural tissues versus the pituitary, and synaptotagmin IV was the least abundant isoform in all the tissues. Developmental analyses of the synaptotagmin isoforms in rat hypothalamus shows that all isoforms are at low levels during embryonic stages and increase postnatally. Synaptotagmin I and II have similar patterns and rise to maximum (adult) levels around P14, whereas synaptotagmin III and IV reach their maximum levels considerably earlier, at P1. These data show that synaptotagmin I is the dominant isoform in both predominantly peptide secreting systems (e.g., in pituitary tissues) and in neurotransmitter secreting systems (e.g., in cerebellum). While the developmental expression patterns of synaptotagmin I and II parallels the temporal development of synaptogenesis in the nervous system, the early maximal expression of synaptotagmin III and IV suggests that these isoforms may have other functions during early postnatal development.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.