Drosophila Synaptotagmins

The synaptotagmin family has been implicated in calcium-dependent neurotransmitter release, although Synaptotagmin 1 is the only isoform demonstrated to control synaptic vesicle fusion. This study reports the characterization of the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologues of mammalian Synaptotagmins 4, 7, 12, and 14. Like Synaptotagmin 1, Synaptotagmin 4 is ubiquitously present at synapses, but localizes to the postsynaptic compartment. The remaining isoforms were not found at synapses (Synaptotagmin 7), expressed at very low levels (Synaptotagmins 12 and 14), or in subsets of putative neurosecretory cells (Synaptotagmins alpha and ß). Consistent with their distinct localizations, overexpression of Synaptotagmin 4 or 7 cannot functionally substitute for the loss of Synaptotagmin 1 in synaptic transmission. The results indicate that synaptotagmins are differentially distributed to unique subcellular compartments. In addition, the identification of a postsynaptic synaptotagmin suggests calcium-dependent membrane-trafficking functions on both sides of the synapse (Adolfsen, 2004).

Taking advantage of the recently completed Drosophila genome, putative synaptotagmin genes have been identified using BLAST analysis with known mammalian synaptotagmin isoforms. Seven synaptotagmin isoforms are present in the fly genome and show a conserved domain structure consisting of an NH2-terminal transmembrane domain followed by tandem C2 domains. A comparison of the amino acid sequence encompassing the negatively charged residues important for calcium coordination within each C2 domain is presented. Only the Syt 1 and Syt 7 isoforms encode all the coordination residues for both C2 domains. Three of the remaining isoforms (Syt 4, Syt alpha, and Syt ß) display at least 60% conservation of these charged residues, while two isoforms (Syt 12 and Syt 14) show significant divergence, suggesting that the function of some synaptotagmins may not require calcium binding (Adolfsen, 2004).

To determine the relationship between Drosophila and other metazoan synaptotagmin isoforms, a cluster analysis was performed of the predicted synaptotagmin proteins encoded in currently sequenced genomes. Synaptotagmin sequences were collected from the C. elegans, Mus musculus, and Homo sapiens genomes and aligned using ClustalW analysis software. This analysis suggests the synaptotagmin superfamily can be divided into eight subfamilies based on sequence relationships across species. The Syt 1, Syt 4, Syt 7, Syt 12, and Syt 14 subfamilies contain at least one Drosophila member and one or more mammalian homologues. Isoforms of the Syt 1 and Syt 4 families were identified in all vertebrate and invertebrate genomes, suggesting that these two synaptotagmin families mediate an evolutionarily conserved function required in all animals. The Syt 7, Syt 12, and Syt 14 subfamilies contain Drosophila and vertebrate members, but lack homologues in other invertebrate genomes. Similar to the Drosophila homologues, the mammalian 12 and 14 isoforms lack the majority of consensus calcium binding aspartate residues, whereas Syt 7 contains highly conserved calcium binding sites. The three remaining synaptotagmin subfamilies are not highly conserved across evolution. The Syt 3 family consists of only vertebrate members, including the mammalian 3, 5, 6, and 10 isoforms. In contrast to the Syt 3 family, the Syt alpha and Syt ß subfamiles do not contain any obvious vertebrate orthologues (Adolfsen, 2004).

Genetic analysis has demonstrated that Syt 1 is essential for calcium-dependent synchronous release, underlying the fourth order cooperativity of synaptic vesicle fusion, but does not abolish asynchronous calcium-dependent release. These observations are consistent with the current two calcium sensor model for synaptic transmission, with Syt 1 functioning as the calcium sensor regulating the fast synchronous component of release and an unidentified calcium sensor mediating the slow asynchronous component. Other synaptotagmin isoforms are obvious candidates for the asynchronous calcium sensor. In addition, synaptotagmins have unique calcium-binding properties and undergo heterooligomerization in vitro. Several plasticity models have been proposed, suggesting differential expression of synaptotagmin isoforms on synaptic vesicles might regulate presynaptic release probability or transitions from full fusion to kiss-and-run. These hypotheses require that synaptotagmins have a similar expression pattern to Syt 1 and localize presynaptically at synaptic terminals. These hypotheses have been addressed in vivo by performing an extensive expression and localization study of the entire synaptotagmin family in D. melanogaster. The localization data argue against the possibility that other synaptotagmin isoforms function with Syt 1 to regulate neurotransmitter release. Instead, the remaining synaptotagmin isoforms likely regulate distinct membrane trafficking steps in vivo (Adolfsen, 2004).

Syt 4 was found in the postsynaptic compartment, suggesting it regulates a postsynaptic membrane trafficking pathway. A small fraction of Syt 4 may also be present in some presynaptic compartments, though it does not localize to Syt 1–positive synaptic vesicles. The detection of the Syt 4 protein by Western analysis and immunocytochemistry with new antisera is abolished in syt 4 null mutants, confirming the antisera accurately reflects the subcellular localization of Syt 4. These results indicate that previous detection of Syt 4 on synaptic vesicles reflected cross-reactivity of the old antisera with Syt 1. Given that Syt 4 does not colocalize on Syt 1–positive synaptic vesicles, the reduction of neurotransmitter release by Syt 4 up-regulation observed in Drosophila is unlikely to be due to heteromultimerization of the two proteins on vesicles and may instead reflect competitive binding to Syt 1 effectors or altered presynaptic calcium buffering (Adolfsen, 2004).

In terms of Syt 4's postsynaptic localization, there is evidence in several experimental systems for a regulated form of postsynaptic vesicular trafficking. Studies in hippocampal culture neurons indicate that long-term labeling with FM1–43 loads dendritic organelles that undergo rapid calcium-triggered exocytosis that is blocked by tetanus toxin. In addition, pharmacological blockage of postsynaptic membrane fusion reduces LTP, suggesting postsynaptic vesicle trafficking contributes to synaptic plasticity. Mammalian Syt 4 has been localized within dendrites and soma, suggesting Syt 4 and the related homologue Syt 11 may also function postsynaptically. Although the exact role for regulated postsynaptic fusion remains unclear, possibilities include the release of retrograde signals, trafficking of postsynaptic receptors, and/or trafficking of synaptic cell adhesion proteins (Adolfsen, 2004).

The remaining synaptotagmins were not ubiquitously localized to synapses. Unlike Syt 1 or Syt 4, Syt 7 could not be detected at synapses; it was expressed in both neuronal and nonneuronal tissues. Mammalian Syt 7 has been found in secretory lysosomes and in synaptic active zones where it has been postulated to function as a plasma membrane calcium sensor. Genetic studies of Syt 7 will be required to determine if it also functions at Drosophila active zones. Peripheral Syt ß staining was restricted to muscle fiber 8 synapses that are known to release the neuropeptide leukokinin. In the CNS, Syt ß was observed in a pair of bilateral neurons that may be the DPM neurosecretory neurons known to secrete the amnesiac neuropeptide. The only staining outside the nervous system is detected at tracheal branch points, where a group of myomodulin-releasing neurosecretory cells are located. These localization studies suggest Syt ß is a candidate calcium sensor for mediating dense core vesicle fusion and release of neuropeptides. Similar to Syt ß, Syt alpha showed specific expression in another set of putative CNS neuropeptide-releasing neurons, as well as within the mushroom bodies. In the periphery, staining was restricted to the LBD neurosecretory neuron, which is consistent with a role in neuropeptide release. In addition, the localization of Syt alpha in mushroom bodies and the possible localization of Syt ß in DPM neurons makes these isoforms attractive candidates for potential roles in vesicular trafficking pathways contributing to neuronal plasticity. The two remaining synaptotagmins, Syt 12 and Syt 14, were not localized. It is likely that the proteins are below the detection level of the antisera, which is consistent with the microarray and in situ experiments, indicating that these isoforms are expressed at low levels in embryos and adults. Unlike the other synaptotagmins, these two isoforms lack most of the calcium coordination residues in C2A and C2B in both vertebrates and flies, indicating that they may function in trafficking pathways not regulated by calcium (Adolfsen, 2004).

In summary, Drosophila synaptotagmin isoforms identify unique membrane-trafficking compartments. The data indicate that only the Syt 1 isoform is found on synaptic vesicles and so argue against heterooligomerization models. In addition, Syt 4 and Syt 7 cannot rescue the behavioral or physiological defects in syt 1 mutants, suggesting that synaptotagmins define unique membrane trafficking pathways within neurons. It is possible synaptotagmins function in an analogous manner to control vesicle fusion, but do so in distinct compartments. Given that Syt 4 localizes to the postsynaptic compartment, the findings indicate that calcium-dependent membrane trafficking occurs on both sides of the synapse (Adolfsen, 2004).

Interaction of Synaptotagmin with Syntaxin and SNAP-25

Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. NMR spectroscopy was used to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release (Fernandez, 1998).

A remarkable feature of neurotransmitter release is that it occurs very fast after Ca2+ influx. Thus, the synaptic vesicles that are 'ready to fuse' appear to be in a metastable state that is hindered to proceed toward fusion in the absence of Ca2+. Based on previous structural analyses of the C2A domain, it was predicted that the region of syntaxin involved in Ca2+-dependent binding to the C2A domain is highly negatively charged, and it was proposed that the switch in electrostatic potential caused by Ca2+ binding to the C2A domain causes the change in affinity for syntaxin. The results described in this paper demonstrate that the region of the N-terminal sequence of syntaxin that binds to the C2A domain is indeed highly acidic. Given the high density of negative charge in the Ca2+-binding region of the C2A domain before Ca2+ binding, it is most likely that the N-terminal sequence of syntaxin and the C2A domain repel each other in the absence of Ca2+. Such repulsion may be the force that prevents synaptic vesicle exocytosis to proceed before Ca2+ influx into a presynaptic terminal. Upon nerve stimulation, Ca2+ binding to the C2A domain could attract syntaxin, initiating fusion. Whether the C2A domain interaction with the N-terminal sequence of syntaxin is physiologically relevant remains to be demonstrated, since the C2A domain has also been shown to bind in a Ca2+-dependent manner to the C-terminal region of syntaxin and to negatively charged phospholipid vesicles. The picture that emerges shows that the N-terminal sequence of syntaxin is a highly conserved, independently folded domain of syntaxin with the following features: (1) it is covalently linked to the C-terminal region of syntaxin, which may be directly involved in membrane fusion; (2) it contains a synaptotagmin-binding site, which may be a part of the Ca2+ trigger; (3) it forms a highly conserved groove between two helices that may bind munc13, munc18, and/or the C-terminal region of syntaxin, with potential regulatory roles in exocytosis. These characteristics suggest that the N-terminal sequence of syntaxin may act as a multifunctional domain in neurotransmitter release. It will be interesting to study which protein(s) directly interacts with the groove and what the consequences are in vivo of mutations introduced in this groove (Fernandez, 1998).

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a ß sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, the C2B-domain binds phospholipids in a Ca2+-dependent manner similar to the C2A-domain. Evolutionary analysis has revealed that the C-terminal helix is fully conserved in the synaptotagmin 1 homologs reported from Drosophila and C. elegans, but is absent in those from squid and Aplysia, suggesting that either synaptotagmin 1 is different in molluscs or the synaptotagmins described for molluscs are not true synaptotagmin 1 homologs. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release (Fernandez, 2001).

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but any understanding of the mechanism by which Ca2+ triggers membrane fusion is uncertain. SNAP-25 plays a role in the Ca2+ regulation of secretion. Synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) have been identified that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations leads to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion (Zhang, 2002).

Synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: syntaxin and SNAP-25 on the plasma membrane (t-SNAREs) and synaptobrevin/VAMP on the synaptic vesicles (v-SNARE). Vesicular synaptotagmin 1 is essential for fast synchronous SNARE-mediated exocytosis and interacts with the SNAREs in brain material. To uncover the step at which synaptotagmin becomes linked to the three SNAREs, all four proteins were purified from brain membranes and their interactions were analyzed. In the absence of calcium, native synaptotagmin 1 binds the t-SNARE heterodimer, formed from syntaxin and SNAP-25. This interaction is both stoichiometric and of high affinity. Synaptotagmin contains two divergent but conserved C2 domains that can act independently in calcium-triggered phospholipid binding. Both C2 domains are strictly required for the calcium-independent interaction with the t-SNARE heterodimer, indicating that the double C2 domain structure of synaptotagmin may have evolved to acquire a function beyond calcium/phospholipid binding (Rickman, 2003).

Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca(2+), but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. This study, directly addresses this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion (Hui, 2009).

Ca2+ binding to Synaptotagmin

C2-domains are widespread protein modules with diverse Ca2+-regulatory functions. Although multiple Ca2+ ions are known to bind at the tip of several C2-domains, the exact number of Ca2+-binding sites and their functional relevance are unknown. The first C2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca2+-dependent interactions with syntaxin and phospholipids. The Ca2+-binding mode of this C2-domain has been studied as a prototypical C2-domain using NMR spectroscopy and site-directed mutagenesis. The C2-domain is an elliptical module composed of a beta-sandwich with a long axis of 50 A. The C2-domain is found to bind three Ca2+ ions in a tight cluster spanning only 6 A at the tip of the module. The Ca2+-binding region is formed by two loops whose conformation is stabilized by Ca2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C2-domains. All three Ca2+ ions are required for the interactions of the C2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C2-domain function whereby the beta-sheets of the domain provide a fixed scaffold for the Ca2+-binding loops, and whereby interactions with target molecules are triggered by a Ca2+-induced switch in electrostatic potential (Ubach, 1998).

Synaptotagmins constitute a large family of membrane proteins implicated in Ca2+-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca2+ affinities of synaptotagmin C2-domains in a complex with phospholipids, it has been shown that different synaptotagmins exhibit distinct Ca2+ affinities, with plasma membrane synaptotagmins binding Ca2+ with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca2+ affinities are functionally important, the effects of synaptotagmin C2-domains on Ca2+-triggered exocytosis were examined in permeabilized PC12 cells. A precise correlation is observed between the apparent Ca2+ affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This is extended to PC12 cell exocytosis triggered by Sr2+, which is also selectively affected by high-affinity C2-domains of synaptotagmins. Together, these results suggest that Ca2+ triggering of exocytosis involves tandem Ca2+ sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca2+ sensors involved in slow Ca2+-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca2+ sensors specialized for fast Ca2+-dependent exocytosis (Sugita, 2002).

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, two C2A domain mutations, D232N and D238N, were studied using recombinant proteins and knock-in mice. Both mutations severely decrease intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain. Neither mutation, however, alters the apparent Ca2+ affinity of the double C2 domain fragment, although both decrease the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations have no apparent effect on morbidity and mortality and cause no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice reveal that neither mutation induces major changes in synaptic transmission. The D232N mutation, however, causes increased synaptic depression during repetitive stimulation, whereas the D238N mutation does not exhibit this phenotype. These data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+-dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. These data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses (Fernández-Chacón, 2002).

Sr2+ triggers neurotransmitter release similar to Ca2+, but less efficiently. In synaptotagmin 1 knockout mice, the fast component of both Ca2+- and Sr2+-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C2A and the C2B domain of synaptotagmin 1 bind Ca2+ in phospholipid complexes, but only the C2B domain forms Sr2+/phospholipid complexes; therefore, Sr2+ binding to the C2B domain is sufficient to trigger fast release, although with decreased efficacy. Ca2+ induces binding of the synaptotagmin C2 domains to SNARE proteins, whereas Sr2+ even at high concentrations does not. Thus, triggering of the fast component of release by Sr2+ as a Ca2+ agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding (Shin, 2003).

When an action potential invades a nerve terminal, Ca2+ influx induces neurotransmitter release with an exquisite temporal specificity. Release exhibits at least two components, a fast synchronous component that dominates at low-frequency stimulation, and a slower asynchronous component that dominates at high-frequency stimulation. Compared to other Ca2+-regulated biological processes, both components of release are rapid since Ca2+ triggers synchronous release in as little as 100 µs, and asynchronous release in 10-50 ms. Both release components are Ca2+ dependent with similar apparent Ca2+ cooperativities but different apparent Ca2+ affinities, suggesting that multiple Ca2+ sensors control release (Shin, 2003).

Sr2+ is a divalent cation that can substitute for Ca2+ in triggering neurotransmitter release. Although Sr2+, similar to Ca2+, induces both synchronous and asynchronous release, the properties of Sr2+- and Ca2+-evoked release are different. Synchronous and asynchronous release triggered by Sr2+ exhibit reduced peak amplitudes and longer time constants, suggesting that Sr2+ is a Ca2+ agonist for vesicle exocytosis that exhibits a lower efficiency than Ca2+. In addition, Sr2+ causes a relative increase in asynchronous release, probably because Sr2+ buffering and clearance is less effective than Ca2+ buffering and clearance. Furthermore, a relatively higher Sr2+ affinity of the Ca2+ sensor for asynchronous release may contribute to the Sr2+-induced increase in asynchronous release (Shin, 2003).

Synaptotagmins constitute a large family of membrane proteins that are candidate Ca2+ sensors for exocytosis. Synaptotagmins contain a single N-terminal transmembrane region, a linker sequence, and two conserved C2 domains. In most synaptotagmins, both C2 domains bind multiple Ca2+ ions in a complex with phospholipids, and additionally form Ca2+-dependent complexes with SNARE proteins and other molecules implicated in exocytosis. Synaptotagmins 1 and 2 are the most abundant synaptotagmins that are enriched on synaptic vesicles and differentially expressed in brain. Studies in mutant mice have demonstrated that synaptotagmin 1 functions as the Ca2+ sensor for the fast component of release in hippocampal neurons. Direct evidence for this conclusion was derived from a point mutation (R233Q) that was introduced by homologous recombination into the murine synaptotagmin 1 gene. The R233Q mutation, although localized to the C2A domain, decreases approximately 2-fold the apparent Ca2+ affinity of the double C2 domain fragment of synaptotagmin 1 and the apparent Ca2+ affinity of neurotransmitter release. In contrast, point mutations in the Ca2+ binding sites of the C2A domain that partly abolish Ca2+ binding have no effect in vivo, probably because they impair only Ca2+-dependent phospholipid binding to the isolated C2A domain but not to the double C2 domain fragment. Mutations in Ca2+ binding sites of the C2B domain, conversely, abolish Ca2+-triggered release. Together these results suggest that Ca2+ binding to the C2B domain of synaptotagmin 1 is essential for Ca2+ triggering of fast exocytosis. However, these results do not clarify the precise contribution of the C2A domain to Ca2+ triggering of release and do not reveal whether Ca2+ binding to the C2B domain is sufficient for inducing exocytosis (Shin, 2003).

Mouse mutants of synaptotagmin 1 exhibit no change in the slow, asynchronous component of release or in Ca2+-independent forms of exocytosis, suggesting that synaptotagmin 1 function is selective for the fast component. Other synaptotagmins, possibly synaptotagmins 3 and 7 (the most abundant after synaptotagmins 1 and 2), may act as Ca2+ sensors for asynchronous release. This suggestion was raised because these synaptotagmins bind Sr2+ comparatively better than synaptotagmins 1 and 2, and because they exhibit a higher apparent Ca2+ affinity than synaptotagmins 1 and 2. In addition, the C2 domains of synaptotagmins 3 and 7 are effective inhibitors of Ca2+-dependent exocytosis in neuroendocrine PC12 cells where synaptotagmin 1 is not required for release, although additional synaptotagmins (such as synaptotagmin 9) may also be involved (Shin, 2003).

A key question in understanding neurotransmitter release is how Ca2+ binding to synaptotagmin 1 triggers fast release. Two major interactions mediated by synaptotagmin 1, Ca2+-dependent binding to phospholipids and to SNARE proteins, have been suggested as the triggering mechanism. However, the nature and importance of these interactions continue to be disputed. Based on indirect data in cracked PC12 cells, it was proposed in one study that Ca2+-induced SNARE binding is essential for exocytosis. In contrast, a second study using a similar approach observed a better correlation of exocytosis with phospholipid binding, although SNARE binding was not excluded as a mechanism. At the synapse (where exocytosis is quite different from PC12 cells), the importance of phospholipid binding is supported by the fact that changes in the apparent Ca2+ affinity of synaptotagmin 1/phospholipid complexes correlate with release, but are not accompanied by equivalent changes in SNARE binding. However, no direct test of the importance of Ca2+-induced SNARE binding to synaptotagmin 1 has been reported. Attempts have been made to address this question at the synapse under conditions that do not require overexpression or dominant-negative effects, but make use of the insights gained in electrophysiological studies on Sr2+ as a low-affinity Ca2+ agonist in neurotransmitter release. The data show that Sr2+ binding to the synaptotagmin 1 C2B domain is sufficient to trigger fast exocytosis. This action involves the formation of Sr2+-induced complexes of synaptotagmin with phospholipids but not with SNAREs, demonstrating that synaptotagmin/SNARE complexes induced by divalent cations are not essential for triggering fast neurotransmitter release. This conclusion rules out an essential role for the Ca2+-dependent synaptotagmin 1/SNARE complex in stimulating fast release, but does not rule out a role for the Ca2+-dependent synaptotagmin 1/SNARE complex in other stages of exocytosis, e.g., vesicle recruitment (Shin, 2003).

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. 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. It is concluded 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 (Stevens, 2003).

These observations are consistent with coupling occurring through synaptotagmin's calcium-dependent interactions with SNARE proteins, with itself, and/or with phospholipids at the C2B domain, but do not support the idea that calcium-dependent phospholipid binding by C2A is an essential part of the coupling mechanism. It is concluded that C2A is part of the calcium sensor for fast synchronous neurotransmitter release and the existence of some other component of the sensor at mammalian central synapses has been establised. Furthermore, some properties of this other component have been established. If this analysis is correct, the second part of the calcium sensor must be present in two copies, each copy must bind a single dominant calcium to trigger release, and the calcium affinity for this sensor must be the same as that of the C2A domain. The C2B domain appears to have these properties, and this structure is the obvious leading candidate for the rest of the calcium sensor. Determining the precise role of the C2B domain in calcium sensing awaits further evaluation by the sort of analysis presented here (Stevens, 2003).

Linker mutations reveal the complexity of synaptotagmin 1 action during synaptic transmission

The Ca(2+) sensor for rapid synaptic vesicle exocytosis, synaptotagmin 1 (syt), is largely composed of two Ca(2+)-sensing C2 domains, C2A and C2B. This study investigated the apparent synergy between the tandem C2 domains by altering the length and rigidity of the linker that connects them. The behavior of the linker mutants revealed a correlation between the ability of the C2 domains to penetrate membranes in response to Ca(2+) and to drive evoked neurotransmitter release in cultured mouse neurons, uncovering a step in excitation-secretion coupling. Using atomic force microscopy, this study found that the synergy between these C2 domains involved intra-molecular interactions between them. Thus, syt function is markedly affected by changes in the physical nature of the linker that connects its tandem C2 domains. Moreover, the linker mutations uncoupled syt-mediated regulation of evoked and spontaneous release, revealing that syt also acts as a fusion clamp before the Ca(2+) trigger (Lium 2014).

Linker mutations reveal the complexity of synaptotagmin 1 action during synaptic transmission

The Ca(2+) sensor for rapid synaptic vesicle exocytosis, synaptotagmin 1 (syt), is largely composed of two Ca(2+)-sensing C2 domains, C2A and C2B. This study investigated the apparent synergy between the tandem C2 domains by altering the length and rigidity of the linker that connects them. The behavior of the linker mutants revealed a correlation between the ability of the C2 domains to penetrate membranes in response to Ca(2+) and to drive evoked neurotransmitter release in cultured mouse neurons, uncovering a step in excitation-secretion coupling. Using atomic force microscopy, this study found that the synergy between these C2 domains involved intra-molecular interactions between them. Thus, syt function is markedly affected by changes in the physical nature of the linker that connects its tandem C2 domains. Moreover, the linker mutations uncoupled syt-mediated regulation of evoked and spontaneous release, revealing that syt also acts as a fusion clamp before the Ca(2+) trigger (Lium 2014).

Miscellaneous protein interactions of Synaptotagmin

Synaptotagmin, a major intrinsic membrane protein of synaptic vesicles that binds Ca2+, was purified from bovine brain and immobilized onto Sepharose 4B. Affinity chromatography of brain membrane proteins on immobilized synaptotagmin reveals binding of alpha- and beta-neurexins to synaptotagmin in a calcium ion-independent manner. Synaptotagmin specifically interacts with the cytoplasmic domains of neurexins (see Neurexin) but not of control proteins. This interaction is dependent on a highly conserved, 40 amino acid sequence that makes up most of the cytoplasmic tails of the neurexins. These data suggest a direct interaction between the cytoplasmic domains of a plasma membrane protein (neurexin) and a protein specific for a subcellular organelle (synaptotagmin). Such an interaction could have an important role in the docking and targeting of synaptic vesicles in the nerve terminal (Hata, 1993).

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein-protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 shows a biphasic calcium dependence with maximal binding at 10-20 microM. The synprint sites of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels have different patterns of interactions with synaptic proteins. The BI isoform of alpha1A interacts specifically with syntaxin, SNAP-25, and synaptotagmin, all independent of Ca2+ concentrations and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of alpha1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of alpha1A is Ca2+-dependent, with maximum affinity at 10-20 microM Ca2+. Although the rbA isoform of alpha1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca2+-dependent. These differential, Ca2+-dependent interactions of Ca2+ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca2+ influx through these channels (Kim, 1997).

Synaptic vesicle docking and fusion are mediated by the assembly of a stable SNARE core complex of proteins, which includes the synaptic vesicle membrane protein VAMP/synaptobrevin and the plasmalemmal proteins syntaxin and SNAP-25. Another SNAP-25-binding protein, called Snapin, has been identified. Snapin is enriched in neurons and exclusively located on synaptic vesicle membranes. It is associated with the SNARE complex through direct interaction with SNAP-25. Binding of recombinant Snapin-CT to SNAP-25 blocks the association of the SNARE complex with synaptotagmin. Introduction of Snapin-CT and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons in culture reversibly inhibits synaptic transmission. These results suggest that Snapin is an important component of the neurotransmitter release process through its modulation of the sequential interactions between the SNAREs and synaptotagmin (Ilardi, 1999).

Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery, have been identified. The human stonins are homologous to the Drosophila melanogaster Stoned B protein and exhibit a modular structure consisting of an NH(2)-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the mu subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP-stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery (Martina, 2001).

Synaptic vesicle recycling is in part mediated by clathrin-mediated endocytosis. This process involves the coordinated assembly of clathrin and adaptor proteins and the concomitant selection of cargo proteins. The endocytotic protein stonin 2 has been shown to localize to axonal vesicle clusters through its micro-homology domain. Interaction of this domain with synaptotagmin I is sufficient to recruit stonin 2 to the plasmalemma. The N-terminal domain of stonin 2 harbors multiple AP-2-interaction motifs that bind to the clathrin adaptor complex AP-2. These motifs with the consensus sequence WVxF are capable of binding to the alpha-adaptin ear domain and to micro2. Mutation of the tyrosine motif-binding pocket of micro2 abolishes recognition of the WVxF peptide, suggesting that association with stonin 2 renders AP-2 incompetent to sort tyrosine motif-containing cargo proteins. It is hypothesized that stonin 2 may function as an AP-2-dependent sorting adaptor for synaptic vesicle recycling (Walther, 2001).

N-Glycosylation is essential for vesicular targeting of Synaptotagmin 1

Synaptotagmins 1 and 7 are candidate Ca2+ sensors for exocytosis localized to synaptic vesicles and plasma membranes, respectively. The N-terminal intraluminal sequence of synaptotagmin 1, when transplanted onto synaptotagmin 7, redirects synaptotagmin 7 from the plasma membrane to secretory vesicles. Conversely, mutation of the N-terminal N-glycosylation site of synaptotagmin 1 redirects synaptotagmin 1 from vesicles to the plasma membrane. In cultured hippocampal neurons, the plasma membrane-localized mutant of synaptotagmin 1 suppresses the readily releasable pool of synaptic vesicles, whereas wild-type synaptotagmin 1 does not. In addition to the intraluminal N-glycosylation site, the cytoplasmic C2 domains of synaptotagmin 1 are required for correct targeting but can be functionally replaced by the C2 domains of synaptotagmin 7. These data suggest that the intravesicular N-glycosylation site of synaptotagmin 1 collaborates with its cytoplasmic C2 domains in directing synaptotagmin 1 to synaptic vesicles via a novel N-glycosylation-dependent mechanism (Han, 2004).

Why is intravesicular N-glycosylation required for the correct localization and function of synaptotagmin 1? The mechanistic basis for this requirement may be a neuron-specific factor that couples the N-glycosylated N terminus to other vesicle components. The dependence of the localization of synaptotagmin 1 on its intravesicular N-glycosylation is an unexpected result, since protein targeting normally involves cytoplasmic sequences and since protein glycosylation generally mediates protein folding or protein-protein interactions. However, at least in polarized epithelia, N-glycosylation has been associated with targeting reactions. Apical sorting of some membrane proteins in polarized epithelial cells appears to be mediated by N-glycosylation, whereas other membrane proteins are targeted to apical membranes independent of N-glycosylation. N-glycosylation is probably also not generally responsible for the localization of synaptic vesicle proteins, since many synaptic vesicle proteins are not N-glycosylated, and some vesicle proteins do not even have an intravesicular sequence, suggesting that diverse mechanisms are involved in building a synaptic vesicle (Han, 2004).

Mutation of Synaptotagmin

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors--which are the gatekeepers to neurotransmission--remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. A point mutation in synaptotagmin I was studied that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse (Fernández-Chacón, 2001)

Signaling upstream of Synaptotagmin

cAMP-dependent protein kinase A (PKA) can modulate synaptic transmission by acting directly on unknown targets in the neurotransmitter secretory machinery. Snapin, a protein of relative molecular mass 15,000 has been identified that is implicated in neurotransmission by binding to SNAP-25, as a possible target. Deletion mutation and site-directed mutagenetic experiments pinpoint the phosphorylation site to serine 50. PKA-phosphorylation of Snapin significantly increases its binding to synaptosomal-associated protein-25 (SNAP-25). Mutation of Snapin serine 50 to aspartic acid (S50D) mimics this effect of PKA phosphorylation and enhances the association of synaptotagmin with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Furthermore, treatment of rat hippocampal slices with nonhydrolyzable cAMP analog induces in vivo phosphorylation of Snapin and enhances the interaction of both Snapin and synaptotagmin with the SNARE complex. In adrenal chromaffin cells, overexpression of the Snapin S50D mutant leads to an increase in the number of release-competent vesicles. These results indicate that Snapin may be a PKA target for modulating transmitter release through the cAMP-dependent signal-transduction pathway (Chheda, 2001).

The results presented here indicate that Snapin can serve as a target for PKA at the synapse to modulate transmitter release and neuronal plasticity through a second-messenger pathway. Phosphorylation of Snapin at serine 50, both in vitro and in vivo, leads to an increase in binding of Snapin to SNAP-25. Furthermore, this phosphorylation apparently strengthens the association of synaptotagmin, a proposed Ca2+ sensor for exocytosis, with the assembled SNARE complex. In physiological experiments, overexpression of constitutively phosphorylated Snapin (S50D Snapin) leads to an increase in the number of release-ready vesicles in adrenal chromaffin cells, whereas overexpression of the unphosphorylated form (S50A Snapin) reduces this number. In the context of the current model of exocytosis in chromaffin cells, these findings lead to the following conclusions: (1) both the S50D and S50A mutations increase the magnitude of the sustained component of exocytosis, indicating that Snapin acts as a positive modulator of the priming step by increasing the forward rate constant of priming; (2) S50D Snapin increases the size of the exocytotic burst, wheresa S50A Snapin reduces it. Thus, phosphorylation of Snapin leads to stabilization of release-ready vesicles, most probably by reducing the backward rate constant for the priming-unpriming reaction (Chheda, 2001).

Synaptotagmin-1, -2, and -9: Ca2+ sensors for fast release that specify distinct presynaptic properties in subsets of neurons

Synaptotagmin-1 and -2 are known Ca2+ sensors for fast synchronous neurotransmitter release, but the potential Ca2+-sensor functions of other synaptotagmins in release remain uncharacterized. This study shows that besides synaptotagmin-1 and -2, only synaptotagmin-9 (also called synaptotagmin-5) mediates fast Ca2+ triggering of release. Release induced by the three different synaptotagmin Ca2+ sensors exhibits distinct kinetics and apparent Ca2+ sensitivities, suggesting that the synaptotagmin isoform expressed by a neuron determines the release properties of its synapses. Conditional knockout mice producing GFP-tagged synaptotagmin-9 revealed that synaptotagmin-9 is primarily expressed in the limbic system and striatum. Acute deletion of synaptotagmin-9 in striatal neurons severely impairs fast synchronous release without changing the size of the readily-releasable vesicle pool. These data show that in mammalian brain, only synaptotagmin-1, -2, and -9 function as Ca2+ sensors for fast release, and that these synaptotagmins are differentially expressed to confer distinct release properties onto synapses formed by defined subsets of neurons (Xu, 2007).

Synaptotagmin and fusion pores

In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolongs the time from fusion pore opening to dilation, whereas synaptotagmin IV shortens this time. Both synaptotagmin isoforms reduce norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid (Wang, 2001).

Neuronal exocytosis is mediated by Ca(2+)-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca(2+)-sensing protein proposed to regulate fusion pore dynamics via Ca(2+)-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca(2+)-triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Ca(2+) does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca(2+).C2A-C2B bound to lipid monolayers, using electron microscopy, has revealed the formation of ring-like heptameric oligomers that are approximately 11 nm long and approximately 11 nm in diameter. In some cases, C2A-C2B also assembles into long filaments. Oligomerization, but not membrane binding, is disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca(2+) first drives C2A-C2B interactions with the membrane, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca(2+)-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion (Wu, 2003).

Synaptotagmin and release of neurotransmitter

Ca2+ triggers neurotransmitter release in at least two principal modes, synchronous and asynchronous release. Synaptotagmin 1 functions as a Ca2+ sensor for synchronous release, but its role in asynchronous release remains unclear. In cultured cortical neurons stimulated at low frequency (~;0.1 Hz), deletion of synaptotagmin 1 blocks synchronous GABA and glutamate release without significantly increasing asynchronous release. At higher stimulation frequencies (~4 Hz), deletion of synaptotagmin 1 also alters only synchronous, not asynchronous, release during the stimulus train, but dramatically enhances 'delayed asynchronous release' following the stimulus train. Thus synaptotagmin 1 functions as an autonomous Ca2+ sensor independent of asynchronous release during isolated action potentials and action potential trains, but restricts asynchronous release induced by residual Ca2+ after action potential trains. It is proposed that synaptotagmin 1 occupies release 'slots' at the active zone, possibly in a Ca2+-independent complex with SNARE proteins that are freed when action potential-induced Ca2+ influx activates synaptotagmin 1 (Maximov, 2005).

Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. It is now shown that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. These results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release (Rhee, 2005).

Synaptotagmin-1 and synaptotagmin-7 trigger synchronous and asynchronous phases of neurotransmitter release

In forebrain neurons, knockout of synaptotagmin-1 blocks fast Ca2+-triggered synchronous neurotransmitter release but enables manifestation of slow Ca2+-triggered asynchronous release. This study shows, using single-cell PCR, that individual hippocampal neurons abundantly coexpress two Ca2+-binding synaptotagmin isoforms, synaptotagmin-1 and synaptotagmin-7. In synaptotagmin-1-deficient synapses of excitatory and inhibitory neurons, loss of function of synaptotagmin-7 suppresses asynchronous release. This phenotype is rescued by wild-type but not mutant synaptotagmin-7 lacking functional Ca2+-binding sites. Even in synaptotagmin-1-containing neurons, synaptotagmin-7 ablation partly impairs asynchronous release induced by extended high-frequency stimulus trains. Synaptotagmins bind Ca2+ via two C2 domains, the C2A and C2B domains. Surprisingly, synaptotagmin-7 function selectively requires its C2A domain Ca2+-binding sites, whereas synaptotagmin-1 function requirs its C2B domain Ca2+-binding sites. These data show that nearly all Ca2+-triggered release at a synapse is due to synaptotagmins, with synaptotagmin-7 mediating a slower form of Ca2+-triggered release that is normally occluded by faster synaptotagmin-1-induced release but becomes manifest upon synaptotagmin-1 deletion (Bacaj, 2013).

Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling

Synaptic vesicle recycling is in part mediated by clathrin-mediated endocytosis. This process involves the coordinated assembly of clathrin and adaptor proteins and the concomitant selection of cargo proteins. The endocytotic protein stonin 2 (see Drosophila stoned) localizes to axonal vesicle clusters through its mu-homology domain. Interaction of this domain with synaptotagmin I is sufficient to recruit stonin 2 to the plasmalemma. The N-terminal domain of stonin 2 harbors multiple AP-2-interaction motifs that bind to the clathrin adaptor complex AP-2. These motifs with the consensus sequence WVxF are capable of binding to the alpha-adaptin ear domain and to mu2. Mutation of the tyrosine motif-binding pocket of mu2 abolishes recognition of the WVxF peptide, suggesting that association with stonin 2 renders AP-2 incompetent to sort tyrosine motif-containing cargo proteins. It is hypothesized that stonin 2 may function as an AP-2-dependent sorting adaptor for synaptic vesicle recycling (Walther, 2004).

Clathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. This study shows that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact mu-homology domain, and functional AP-2 heterotetramers. These data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling (Diril, 2006).

Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo

Neurotransmission requires a balance of synaptic vesicle exocytosis and endocytosis. Synaptotagmin I (Syt I) is widely regarded as the primary calcium sensor for synaptic vesicle exocytosis. Previous biochemical data suggest that Syt I may also function during synaptic vesicle endocytosis; however, ultrastructural analyses at synapses with impaired Syt I function have provided an indirect and conflicting view of the role of Syt I during synaptic vesicle endocytosis. Until now it has not been possible experimentally to separate the exocytic and endocytic functions of Syt I in vivo. This study directly tested the role of Syt I during endocytosis in vivo. Quantitative live imaging of a pH-sensitive green fluorescent protein fused to a synaptic vesicle protein (synapto-pHluorin) was used to measure the kinetics of endocytosis in sytI-null Drosophila. Live imaging of the synapto-pHluorins were combineed with photoinactivation of Syt I, through fluorescein-assisted light inactivation, after normal Syt I-mediated vesicle exocytosis. By inactivating Syt I only during endocytosis, it was demonstrate that Syt I is necessary for the endocytosis of synaptic vesicles that have undergone exocytosis using a functional Syt I protein (Poskanzer, 2004).

Kinetic efficiency of endocytosis at mammalian CNS synapses requires synaptotagmin I

At nerve terminals, synaptic vesicle components are retrieved from the cell surface and recycled for local reuse soon after exocytosis. The kinetics of this coupling is critical for the proper functioning of synapses during repetitive action potential firing, because deficiencies in this process lead to abnormal depletion of the releasable vesicle pool. Although the molecular basis of this coupling is poorly understood, numerous biochemical data point to a role for synaptotagmin I (SytI), an essential synaptic vesicle protein required for fast calcium-dependent exocytosis. Using synapto-pHluorin in an approach that allows the dissection of endocytosis and exocytosis into separate components during periods of stimulation, this study examined exocytic-endocytic coupling in synapses from SytI knockout mice and their WT littermates. It is shown that endocytosis is significantly impaired in the absence of SytI with the relative rates of endocytosis compared with exocytosis reduced approximately 3-fold with respect to WT. Thus, in addition to regulating exocytosis, SytI also controls the kinetic efficiency of endocytosis at nerve terminals (Nicholson-Tomishima, 2004).

Developmental expression of Synaptotagmin

The ascidian embryo, a model for the primitive mode of chordate development, rapidly forms a dorsal nervous system that consists of a small number of neurons. The transcriptional regulation of an ascidian synaptotagmin (syt) gene has been characterized to explore the molecular mechanisms underlying development of synaptic transmission. In situ hybridization has shown that syt is expressed in all neurons transiently in the embryonic epidermis. Neuronal expression of syt requires induction from the vegetal side of the embryo, whereas epidermal expression occurs autonomously in isolated ectodermal blastomeres. Introduction of green fluorescent protein reporter gene constructs into the ascidian embryos indicates that a genomic fragment of the 3.4-kb 5' upstream region contains promoter elements of syt gene. Deletion analysis of the promoter suggests that syt expression in neurons and in the embryonic epidermis depends on distinct cis-regulatory regions. The region between -1680 and -824 contains the ability to enhance neuronal expression. The construct lacking sequence between -2223 and -824 is capable of inducing neuronal gene expression in all injected larvae, indicating that the region distal to -2223 has enhancing activity of neuronal expression that can substitute for the region between -2223 and -824 (Katsuyma, 2002).

Complexin activates exocytosis of distinct secretory vesicles controlled by different synaptotagmins

Complexins are SNARE-complex binding proteins essential for the Ca(2+)-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. This study shows that, in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a cofactor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, the data indicate that complexins are essential for activation of multiple types of Ca(2+)-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is inv

synaptotagmin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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