The molecular pathways involved in retrograde signal transduction at synapses and the function of retrograde communication are poorly understood. This study demonstrates that postsynaptic calcium 2+ ion (Ca2+) influx through glutamate receptors and subsequent postsynaptic vesicle fusion trigger a robust induction of presynaptic miniature release after high-frequency stimulation at Drosophila neuromuscular junctions. An isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), serves as a postsynaptic Ca2+ sensor to release retrograde signals that stimulate enhanced presynaptic function through activation of the cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase pathway. Postsynaptic Ca2+ influx also stimulates local synaptic differentiation and growth through Syt 4-mediated retrograde signals in a synapse-specific manner (Yoshihara, 2005).

Neuronal development requires coordinated signaling to orchestrate pre- and postsynaptic maturation of synaptic connections. Synapse-specific enhancement of synaptic strength as occurs during long-term potentiation, as well as compensatory homeostatic synaptic changes, have been suggested to require retrograde signals for their induction. Although retrograde signaling has been implicated widely in synaptic plasticity, the molecular mechanisms that transduce postsynaptic Ca²⁺ signals during enhanced synaptic activity to alterations in presynaptic function are poorly characterized. Because postsynaptic Ca²⁺ is essential for synapse-specific potentiation, it is important to characterize how Ca²⁺ can regulate retrograde communication at synapses (Yoshihara, 2005).

To dissect the mechanisms underlying activity-dependent synaptic plasticity, test were performed to see whether newly formed Drosophila glutamatergic neuromuscular junctions (NMJs), which have ~30 active zones, show physiological changes after 100-Hz stimulation (5-1552+ chelator EGTA from the patch pipette caused a modest suppression of HFMR, whereas the fast Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) induced strong suppression by 2.5 min of perfusion. Longer perfusion with BAPTA for 5 min before stimulation abolished HFMR, indicating HFMR is induced after postsynaptic Ca²⁺ influx (Yoshihara, 2005).

Ca²⁺-induced vesicle fusion in presynaptic terminals provides a temporally controlled and spatially restricted signal essential for synaptic communication. Postsynaptic vesicles within dendrites have been visualized by transmission electron microscopy, and dendritic release of several neuromodulators has been reported. To test whether postsynaptic vesicle fusion might underlie the Ca²⁺-dependent release of retrograde signals, postsynaptic vesicle recycling was blocked by using the dominant negative shibire^ts1 mutation, which disrupts endocytosis at elevated temperatures. shibire^ts1 was expressed specifically in postsynaptic muscles by driving a UAS-shibire^ts1 transgene with muscle-specific myosin heavy chain (Mhc)-Gal4, keeping presynaptic activity intact. At the permissive temperature (23°C), high-frequency stimulation induced normal HFMR. However, raising the temperature to 31°C suppressed HFMR in the presence of postsynaptic shibire^ts1, whereas wild-type animals displayed normal HFMR at 31°C. Basic synaptic properties in Mhc-Gal4, UAS-shibire^ts1 animals were not affected at either the permissive or the restrictive temperature. The suppression of HFMR is not due to irreversible damage induced by postsynaptic UAS-shibire^ts1 expression, because a second high-frequency stimulation after recovery to the permissive temperature induced normal HFMR (Yoshihara, 2005).

The synaptic vesicle protein synaptotagmin 1 (Syt 1) is the major Ca²⁺ sensor for vesicle fusion at presynaptic terminals but is not localized postsynaptically. It has recently been shown that another isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), is present in the postsynaptic compartment (Adolfsen, 2004), suggesting Syt 4 might function as a postsynaptic Ca²⁺ sensor. Syt 4 immunoreactivity is observed in a punctate pattern surrounding presynaptic terminals, suggesting Syt 4 is present on postsynaptic vesicles. Postsynaptic vesicle recycling was blocked by using the UAS-shibire^ts1 transgene driven with Mhc-Gal4. Without a temperature shift, Syt 4-containing vesicles showed their normal postsynaptic distribution surrounding presynaptic terminals. When the temperature was shifted to 37°C for 10 min in the presence of high-K⁺ saline containing 1.5 mM Ca²⁺ to drive synaptic activity, Syt 4-containing vesicles translocated to the plasma membrane. After recovery at 18°C for 20 min, postsynaptic vesicles returned to their normal position. Removing extracellular Ca²⁺ during the high-K⁺ stimulation resulted in vesicles that did not translocate to the postsynaptic membrane (Yoshihara, 2005).

To further test whether the Syt 4 vesicle population undergoes fusion with the postsynaptic membrane as opposed to mediating fusion between intracellular compartments, transgenic animals were constructed expressing a pH-sensitive green fluorescent protein (GFP) variant (ecliptic pHluorin) fused at the intravesicular N terminus of Syt 4. Ecliptic pHluorin increases its fluorescence 20-fold when exposed to the extracellular space from the acidic lumen of intracellular vesicles during fusion. Expression of Syt 4-pHluorin in postsynaptic muscles resulted in intense fluorescence at specific subdomains in the postsynaptic membrane, defining regions where Syt 4 vesicles undergo exocytosis. The fluorescence was not diffusely present over the postsynaptic membrane but directed to restricted compartments. Mhc-Gal4, UAS-Syt 4-pHluorin larvae were costained with antibodies against the postsynaptic density protein, DPAK, and nc82, a monoclonal antibody against a presynaptic active zone protein. Syt 4-pHluorin colocalized with DPAK and localized adjacent to nc82, demonstrating that Syt 4-pHluorin translocates from postsynaptic vesicles to the plasma membrane at postsynaptic densities opposite presynaptic active zones (Yoshihara, 2005).

To examine the function of Syt 4-dependent postsynaptic vesicle fusion, the phenotypes of a syt 4 null mutant (syt 4^BA1) and a syt 4 deficiency (rn¹⁶) were tested. Mutants lacking Syt 4 hatch from the egg case 21 hours after egg laying at 25°C, similar to wild type, and grow to fully mature larvae that pupate and eclose with a normal time course. To determine whether postsynaptic vesicle fusion triggered by Ca²⁺ influx is required for HFMR, the effects of high-frequency stimulation in syt 4 mutants were analyzed. In contrast to controls, the increase of miniature release was eliminated in syt 4 mutants. Postsynaptic expression of a UAS-syt 4 transgene completely restored HFMR in the null mutant, demonstrating that postsynaptic Syt 4 is required for triggering enhanced presynaptic function. Presynaptic expression of a UAS-syt 4 transgene did not restore HFMR. In addition, postsynaptic expression of a mutant Syt 4 with neutralized Ca²⁺-binding sites in both C2A and C2B domains did not rescue HFMR, indicating that retrograde signaling by Syt 4 requires Ca²⁺ binding (Yoshihara, 2005).

The large increase in miniature frequency observed during HFMR is similar to the enhancement of presynaptic release after activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) described in Aplysia and Drosophila. Bath application of forskolin, an activator of adenylyl cyclase, results in a robust enhancement of miniature frequency at Drosophila NMJs similar to that observed during HFMR, suggesting retrograde signals may function to increase presynaptic cAMP. To test the role of the cAMP-PKA pathway in HFMR, DC0 mutants were assayed for the presence of HFMR. DC0 encodes the major catalytic subunit of PKA in Drosophila and has been implicated in olfactory learning. Similar to the lack of forskolin-induced miniature induction, DC0 null mutants lacked HFMR. Bath application of forskolin in syt 4 mutants resulted in enhanced miniature frequency, suggesting activation of the cAMP pathway can bypass the requirement for Syt 4 in synaptic potentiation (Yoshihara, 2005).

To further explore the role of retrograde signaling at Drosophila synapses, the role of activity was tested in synapse differentiation and growth. During Drosophila embryonic development, presynaptic terminals undergo a stereotypical structural change from a flat path-finding growth cone into varicose synaptic terminals through dynamic reconstruction. Such developmental changes in synaptic structure may share common molecular mechanisms with morphological changes induced during activity-dependent plasticity. Synaptic transmission was eliminated by using a deletion mutation that removes the postsynaptic glutamate receptors, DGluRIIA and DGluRIIB (referred to as GluRs). Postsynaptic currents normally induced by nerve stimulation were completely absent in the mutants (gluR). Miniatures were also eliminated, even at elevated extracellular Ca²⁺ concentrations of 4 mM. In the absence of GluRs, the presynaptic morphology of motor terminals is abnormal, even though GluRs are only expressed in postsynaptic muscles. GluR-deficient terminals maintain a flattened growth cone-like structure and fail to constrict into normal synaptic varicosities. Synaptic development was also assayed in a null mutant of the presynaptic plasma membrane t-SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) receptor], syntaxin (syx), which eliminates neurotransmitter release, providing an inactive synapse similar to that in the gluR mutant. syx null mutants also have abnormal growth cone-like presynaptic terminals with less varicose structure (Yoshihara, 2005).

Because activity is required for synapse development, whether Syt 4-dependent vesicle fusion may be required, similar to its role in acute retrograde signaling during HFMR, was tested. Physiological analysis revealed that the amplitude of evoked currents in mutants lacking Syt 4 was moderately reduced compared with wild type, suggesting weaker synaptic function or development. Similar to the morphological phenotype of the gluR mutant, syt 4 null mutant embryos showed defective presynaptic differentiation. Nerve terminals lacking Syt 4 displayed reduced varicose structure, whereas wild-type terminals have already formed individual varicosities at this stage of development. Postsynaptic expression with a UAS-syt 4 transgene rescued the physiological and morphological phenotypes. Syt 4 Ca²⁺-binding deficient mutant transgenes did not rescue either the morphological immaturity or the reduced amplitude of evoked currents, even though Syt 4 immunoreactivity at the postsynaptic compartment was restored by muscle-specific expression of the mutant syt 4 transgene, similar to the wild-type syt 4 transgene and endogenous Syt 4 immunoreactivity (Yoshihara, 2005).

Mammalian syt 4 was originally identified as an immediate-early gene that is transcriptionally up-regulated by nerve activity in certain brain regions. Therefore, this study analyzed gain-of-function phenotypes caused by postsynaptic Syt 4 overexpression specifically in muscle cells to increase the probability of postsynaptic vesicle fusion. Syt 4 overexpression induced overgrowth of presynaptic terminals in mature third instar larvae, in contrast to overexpression of Syt 1, which does not traffic to Syt 4-containing postsynaptic vesicles. In addition to synaptic overgrowth, Syt 4 overexpression occasionally induced the formation of abnormally large varicosities. Postsynaptic overexpression of the Syt 4 Ca²⁺-binding mutant did not induce synaptic overgrowth, indicating that retrograde signaling by Syt 4 also requires Ca²⁺ binding to promote synaptic growth (Yoshihara, 2005).

To determine whether the cAMP-PKA pathway is important in activity-dependent synaptic growth, the effects of PKA on synaptic morphology were assayed. Expression of constitutively active PKA presynaptically using a motor neuron-specific Gal4 driver induced not only synaptic overgrowth but also larger individual varicosities in mature third instar larvae, similar to those induced by postsynaptic overexpression of Syt 4. These observations are consistent with the presynaptic overgrowth observed in the learning mutant, dunce, which disrupts the enzyme that degrades cAMP, and with studies in Aplysia implicating PKA in synaptic varicosity formation. The loss-of-function phenotype of PKA mutants (DC0^B3) were characterized at the embryonic NMJ to compare with gluR and syt 4 mutants. Presynaptic terminals in the DC0 mutant were morphologically aberrant, with abnormal growth cone-like features and less varicose structure. Postsynaptic expression of a constitutively active PKA transgene in the DC0 or syt 4 mutant backgrounds rescued the immature morphology, suggesting activation of PKA is downstream of Syt 4-dependent release of retrograde signals (Yoshihara, 2005).

Similar to the role of Syt 1-dependent synaptic vesicle fusion in triggering synaptic transmission at individual synapses, Syt 4-dependent vesicle fusion might trigger synapse-specific plasticity and growth. To test synapse specificity, advantage was taken of the specific properties of the Drosophila NMJ at muscle fibers 6 and 7, where two motorneurons innervate both muscle fibers 6 and 7 during development. Syt 4 was expressed specifically in embryonic muscle fiber 6 but not muscle fiber 7 by using the H94-Gal4 driver. If Syt 4-dependent retrograde signals induce general growth of the motorneuron, one would expect to see a proliferation of synapses on both muscle fibers. Alternatively, if Syt 4 promoted local synaptic growth, one would expect specific activation of synapse proliferation only on target muscle 6, releasing the Syt 4-dependent signal. UAS-syt 4 driven by H94-Gal4 increased innervation on muscle fiber 6 compared with that on muscle fiber 7 in third instar larvae. Control experiments with Syt 4 Ca²⁺-binding deficient mutant transgenes, or a transgene encoding Syt 1, did not result in proliferation. Thus, synaptic growth can be preferentially directed to specific postsynaptic targets where Syt 4-dependent retrograde signals predominate, allowing differential strengthening of active synapses via local rewiring (Yoshihara, 2005).

On the basis of the results described in this study, a local feedback model is proposed for activity-dependent synaptic plasticity and growth at Drosophila NMJs. Synapse-specific Ca²⁺ influx triggers postsynaptic vesicle fusion through Syt 4. Fusion of Syt 4-containing vesicles with the postsynaptic membrane releases locally acting retrograde signals that activate the presynaptic terminal, likely through the cAMP pathway. Active PKA then triggers cytoskeletal changes by unknown effectors to induce presynaptic growth and differentiation. Moreover, PKA is well known to facilitate neurotransmitter release directly, triggering a local synaptic enhancement of presynaptic release as shown in HFMR. Therefore, postsynaptic vesicular fusion might initiate a positive feedback loop, providing a localized activated synaptic state that can be maintained beyond the initial trigger (Yoshihara, 2005).

Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. This study shows that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling (Korkut, 2013).

The Drosophila neuromuscular junction (NMJ) is a powerful system to investigate mechanisms underlying retrograde signaling. Spaced stimulation of Drosophila larval and embryonic NMJs results in potentiation of spontaneous (quantal) release, through a retrograde signaling mechanism requiring postsynaptic function of the vesicle protein, Synaptotagmin 4 (Syt4) (Barber, 2009; Yoshihara, 2005, Korkut, 2013 and references therein).

Synaptotagmins are a family of membrane trafficking proteins composed of an N-terminal transmembrane domain, a linker sequence, and two C-terminal C2 domains. The most abundant isoform in the nervous system, Synaptotagmin 1, is associated with synaptic vesicles and has been proposed to function as a Ca++ sensor for neurotransmitter release. Among Synaptotagmins, Syt4 occupies an interesting, yet poorly understood position. Its expression is regulated by electrical activity, it is present in vesicles containing regulators of synaptic plasticity and growth, such as BDNF, it regulates learning and memory , and in humans the syt4 gene is localized to a locus linked to schizophrenia and bipolar disorder (Korkut, 2013 and references therein).

At the fly NMJ, spaced stimulation not only results in potentiation of spontaneous neurotransmitter release (Ataman, 2008; Yoshihara, 2005), but also in structural changes at presynaptic arbors, the rapid formation of ghost boutons, nascent boutons which have still not developed postsynaptic specializations or recruited postsynaptic proteins . However, whether this activity-dependent bouton formation also required Syt4-dependent retrograde signaling was unknown (Korkut, 2013).

This study shows that Syt4 protein functions in postsynaptic muscles to mediate activity-dependent presynaptic growth and potentiation of quantal release. However, to mediate this function Syt4 needs to be transferred from presynaptic terminals to postsynaptic muscle sites. Evidence is presented that, most likely, the entire pool of postsynaptic Syt4 is derived from presynaptic cells. Like the Wnt binding protein, Evi, Syt4 is packaged in exosomes, which provides a mechanism for the unusual transfer of transmembrane proteins across cells. Taken together, these studies support a novel mechanism for the presynaptic control of a retrograde signal, through the presynaptic release of exosomes containing Syt4 (Korkut, 2013).

Larval NMJs continuously generate new synaptic boutons and their corresponding postsynaptic specializations, ensuring constant synaptic efficacy despite the continuous growth of muscle cells. This precise matching of pre- and postsynaptic compartments is regulated by electrical activity, which induces a retrograde signal in muscle to stimulate new presynaptic growth. This process is likely to fine-tune the magnitude of the retrograde signal in specific nerve terminal-muscle cell pairs, each with a characteristic size. Given that most larval muscle cells are innervated by multiple motorneurons, this mechanism may also enable spatial coincidence to ensure the synaptic specificity of plasticity, making certain that only those activated synapses within a cell become structurally regulated (Korkut, 2013).

Synaptotagmins (Syt) are a large family of proteins that regulate membrane traffic in neurons and other cell types. One isoform that has received considerable attention is SYT4, with apparently contradictory reports concerning the function of this isoform in fruit flies and mice. SYT4 was reported to function as a negative regulator of neurotrophin secretion in mouse neurons and as a positive regulator of secretion of a yet to be identified growth factor from muscle cells in flies. This study has directly compared the biochemical and functional properties of rat and fly SYT4. Rat SYT4 was found to inhibit SNARE-catalyzed membrane fusion in both the absence and presence of Ca(2+). In marked contrast, fly SYT4 stimulates SNARE-mediated membrane fusion in response to Ca(2+). Analysis of chimeric molecules, isolated C2 domains, and point mutants revealed that the C2B domain of the fly protein senses Ca(2+) and is sufficient to stimulate fusion. Rat SYT4 is able to stimulate fusion in response to Ca(2+) when the conserved Asp-to-Ser Ca(2+) ligand substitution in its C2A domain is reversed. In summary, rat SYT4 serves as an inhibitory isoform, whereas fly SYT4 is a bona fide Ca(2+) sensor capable of coupling Ca(2+) to membrane fusion (Wang, 2010).

Ca(2+) influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca(2+) in triggering vesicle fusion though the Ca(2+) sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca(2+) influx remain unclear. This study demonstrates that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca(2+)-binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca(2+)-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca(2+) sensor for presynaptic vesicle fusion (Barber, 2009).

Over the last decade, there has been a reversal in appreciation of retrograde signaling at synapses, as experimental evidence has challenged the well-established view that synaptic information flows unidirectionally from the presynaptic terminal to the postsynaptic side. Retrograde signaling can be mediated by cell–cell contact through surface-attached adhesion complexes, membrane-permeable factors, and conventional neurotransmitters. Recent experiments have also established the presence of membrane trafficking components in the postsynaptic compartment, including ER, Golgi, and postsynaptic vesicles. In contrast to the well-described mechanisms for presynaptic vesicle trafficking, how retrograde signals are released from the postsynaptic compartment is poorly understood. This study shows that Syt 4 plays a postsynaptic role in regulation of synaptic growth and plasticity in an activity-dependent manner at Drosophila synapses (Barber, 2009).

Phenotypic analysis of syt 4-null mutants reveals a decrease in the number of NMJ synaptic varicosities, a reduction in neurotransmission, and defective-enhanced spontaneous release after high frequency stimulation. The results at mature third instar synapses are qualitatively similar to those observed at newly formed synapses. However, although high frequency stimulation can induce an approximately two- to threefold increase in mini frequency at mature NMJs, similar stimulation of embryonic synapses results in an ~100-fold change, indicating that newly formed connections are exquisitely sensitive to Syt 4-dependent retrograde signaling. In contrast to the loss of Syt 4 function, overexpression of Syt 4 in the postsynaptic compartment has the opposite effect, inducing bouton proliferation and enhancing spontaneous release after high frequency stimulation. Whether Syt 4 overexpression increases the likelihood of individual postsynaptic vesicle fusion events by altering the Ca²⁺-dependent release machinery or results in an increased number of postsynaptic vesicles is currently unknown. Similar increases in spontaneous release frequency have been observed after repetitive pulses of high K⁺ saline at the NMJ. Although the link between increased mini frequency after strong stimulation and synaptic growth triggered by Syt 4 postsynaptic overexpression is unclear, it is tempting to speculate that increased Syt 4 levels enhance stimulation-dependent release of retrograde signals that act on presynaptic release and the synaptic growth machinery. It is hypothesized that enhanced spontaneous release may function to prolong postsynaptic Ca²⁺ transients that normally occur after high frequency neuronal firing, enhancing generation of second messengers that promote synaptic proliferation. Syt 4-dependent synaptic growth requires neuronal activity, as the effect can be blocked by decreased action potential firing in para^ts1 mutants. In addition, Syt 4 contributes to activity-induced synaptic growth triggered by rearing animals at higher temperatures or by seizure-inducing mutations. By imaging tagged versions of Syt 4 in CNS motor neurons, this study found that Syt 4 fusion-competent vesicles are also found in CNS dendrites, suggesting that the protein may play a general role in Ca²⁺ regulation of postsynaptic membrane traffic (Barber, 2009).

What is the role of Syt 4 in the postsynaptic compartment? It is hypothesized that Syt 4 functions as a postsynaptic Ca²⁺ sensor for regulating retrograde signaling by activating postsynaptic vesicle fusion. This would result in release of a diffusible ligand from the postsynaptic compartment or insertion of a transmembrane protein that would trigger bidirectional signaling. Given the role of Syt 1 in presynaptic neurotransmitter release and the acute effects on synaptic plasticity observed with manipulation of Syt 4 levels, release of a diffusible signal is the favored scenario. Alternatively, Syt 4 might be required for endocytosis of a transsynaptic signaling complex that would regulate short-term changes in bidirectional signaling. A role for Syt 4 solely in endocytosis is unlikely, as the protein is found on a vesicle compartment and not associated with the plasma membrane at rest, where it presumably would be required for endocytotic reactions. In addition, interactions between Syt 1 and the clathrin adapter AP-2 do not require Ca²⁺ binding by Syt 1. This study has demonstrated that Syt 4's ability to drive synaptic growth and enhance presynaptic release requires C2 domain Ca²⁺-binding residues. Finally, Syt 4 could have a unique postsynaptic function that is unrelated to vesicle trafficking. Given the established role of synaptotagmins in regulating membrane trafficking, this is viewed as a less likely option. The specific cargo contained in Syt 4 vesicles is currently unknown. Similar to the diverse set of cell type-specific neurotransmitters found in Syt 1-containing synaptic vesicles, a similar diversity of Syt 4 postsynaptic vesicle cargos is expected. Several target-derived retrograde factors function in synaptic growth, including FGF, Wingless/Wnt, TGF-β peptides, and the NGF family of neurotrophic factors. Recent work indicates that mammalian Syt 4 is localized to postsynaptic vesicles in hippocampal neurons that contain brain-derived neurotrophic factor where it regulates their release and subsequent effects on long-term potentiation, supporting an evolutionarily conserved role of Syt 4 as a regulator of postsynaptic fusion events (Barber, 2009).

An interesting question raised by these experiments is how similar the biochemical activities of Syt 1 and 4 are in vivo. Syt 1 and 4 cannot functionally substitute for each other at synapses, but this reflects sorting to distinct vesicle populations. Syt 1 preferentially requires C2B Ca²⁺ binding for activity, with C2A playing a more minor role. Syt 4-dependent retrograde signaling at NMJs shows Ca²⁺ dependence in vivo, although in vitro methods have produced conflicting evidence about its potential to bind Ca²⁺. By examining the ability of mutant Syt 4 proteins to promote synaptic growth and to rescue growth and plasticity defects in syt 4 mutants, this study demonstrates that Ca²⁺-binding sites in both the C2A and C2B domains of Syt 4 are necessary. In particular, S284 of the C2A domain is required for Syt 4 activity in vivo. This was an unexpected finding, as the serine replaces an essential aspartic acid residue required for C2 domain Ca²⁺ binding in other synaptotagmin isoforms. This aspartic acid to serine substitution is conserved from Drosophila to humans, suggesting an evolutionarily important role for the residue, previously hypothesized to be inactivation of the C2A Ca²⁺-binding site. The finding that S284 is required for Syt 4 function in vivo raises the possibility that the serine may be phosphorylated, reintroducing a negative charge into the Ca²⁺-binding pocket and allowing it to functionally substitute for the aspartic acid present in Syt 1's C2A Ca²⁺-binding pocket. Indeed, substitution of an aspartic acid at S284 restores Ca²⁺ binding by Syt 4 in vitro and can promote synaptic growth when overexpressed in vivo. Phosphorylation of Syt 4 could potentially explain the conflicting in vivo and in vitro data concerning the protein's ability to bind Ca²⁺. There is precedent for phosphorylation of synaptotagmins regulating Ca²⁺ binding, as Syt 2 is phosphorylated by WNK1, reducing its Ca²⁺-dependent, phospholipid-binding affinity. Current studies are under way to determine whether S284 of Syt 4 is phosphorylated in vivo and which kinases are involved (Barber, 2009).

Two orthologues of Drosophila Syt 4 are found in mammalian genomes encoded by the Syt 4 and Syt 11 genes. Although mammalian Syt 4 has been implicated in retrograde signaling, little is known about Syt 11 other than it is abundantly expressed in the brain. However, Ca²⁺-dependent exocytosis of postsynaptic vesicles in mammalian neurons has been documented. Ca²⁺-dependent fusion of somatodendritic vesicles containing the VMAT2 monoamine transporter has also been demonstrated using immunocytochemistry and amperometric recording of dopamine release. Surprisingly, the kinetics of somatodendritic dopamine release is similar to quantal events recorded at presynaptic sites, with release occurring in <1 ms. The observation that Syt 4 mRNA and protein levels are modulated by neuronal activity, similar to findings in mammals and birds, suggests that not only has Syt 4's primary sequence been conserved but also its transcriptional modulation by neuronal activity. Given the profound effects that this study observed on synaptic growth when postsynaptic Syt 4 levels are altered, it is exciting to speculate that activity-dependent modulation of Syt 4 and Syt 11 may be a general mechanism for regulating synaptic growth and plasticity at activated synapses (Barber, 2009).

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 beta). 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).

At nerve terminals, a focal and transient increase in intracellular Ca(2+) triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca(2+)-sensing trigger is synaptotagmin I, whose Ca(2+)-dependent interactions with acidic phospholipids and syntaxin have largely been ascribed to its C(2)A domain, although the C(2)B domain also binds Ca(2+). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca(2+)-sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca(2+) in the C(2)A domain, might be an inhibitor of release. An essential aspartate of the Ca(2+)-binding site of the synaptotagmin I C(2)A domain was mutated and expressed in Drosophila lacking synaptotagmin I. Despite the disruption of the binding site, the Ca(2+)-dependent properties of transmission were not altered. Similarly, synaptotagmin IV could substitute for synaptotagmin I. It is concluded that the C(2)A domain of synaptotagmin is not required for Ca(2+)-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission (Robinson, 2002).

Drosophila Synaptotagmin is homologous and highly similar to the mammalian synaptotagmins, and possesses similar biochemical properties. Aspartate residues 223, 229, 282, 284 and 290 in Drosophila (hereafter D1-D5) correspond to those that coordinate Ca2+ binding in rat synaptotagmin I. D2 and D3 form the high-affinity Ca1 binding site, and mutations of D2 and D3 are the most deleterious to the Ca2+-dependent interactions of the C₂A domain. Mutating D2 or D3 to asparagine abolishes Ca2+-dependent binding to syntaxin and phospholipids. Furthermore, the synaptotagmin IV isoform has a serine in the D3 position and consequently does not bind either phospholipids or syntaxin in a Ca2+-dependent manner (Robinson, 2002).

To test the functional significance of Ca2+ binding to the C₂A domain, an asparagine was substituted for the D2 residue and the Ca2+ dependence of syntaxin binding was assayed. In the absence of Ca2+, little binding was observed of syntaxin to a fusion protein of glutathione S-transferase (GST) and the C₂A domain; in 1 mM Ca2+, binding was greatly enhanced. The D2N mutation prevents this binding even in the presence of Ca2+; the syntaxin bound by the mutated C₂A is similar to that of GST alone. When the synaptotagmin construct includes C₂A and C₂B, the syntaxin-synaptotagmin I interaction is independent of Ca2+ and persists in synaptotagmin I D2N. Binding of phospholipid to the wild-type Drosophila C₂A domain is dependent on Ca2+ and is abrogated by the D2N mutation. The D2N mutation reduces, but does not entirely block, Ca2+-dependent binding of phospholipids to a synaptotagmin I construct containing both C2 domains. Thus, mutating this aspartate drastically alters the Ca2+-dependent interactions of the C₂A domain of synaptotagmin I, and reveals a residual Ca2+ dependence on some interactions that is likely to reside in the C₂B domain (Robinson, 2002).

Although the equivalence of the Drosophila and mammalian synaptotagmins can not be presumed from sequence alone, the D2 residue of the Drosophila protein is necessary for conferring Ca2+ sensitivity on protein-protein and protein-phospholipid interactions of the C₂A domain of synaptotagmin I. The use of this mutation in this study does not depend on the total ablation of all Ca2+ binding to this domain. It rests instead on the biochemical and structural evidence that the mutation causes substantial changes to the site. If the C₂A domain binds the Ca2+ ions that trigger membrane fusion, and if these protein-protein or protein-lipid interactions are related to the initiation of fusion, a dramatic alteration in Ca2+ affinity should be mirrored in a profound alteration in the ability of Ca2+ to stimulate transmitter release (Robinson, 2002).

To test the functional competence of synaptotagmin I^D2N, it and wild-type Synaptotagmin I were epitope tagged, placed under the control of an upstream activating sequence (UAS) promoter, and reintroduced into the fly (P[UAS-HA-syt] and P[UAS-D2N]). The expression of each construct was driven by the heat-shock-dependent driver hsp70-Gal4. This expression system approximately doubled the amount of Synaptotagmin I protein present in the adult head compared with synaptotagmin I (sytI) heterozygotes, although some of this protein may reside in non-neuronal tissues. Each synaptotagmin transgene and driver was crossed into a sytI-null background. The sytI gene encodes the major synaptotagmin isoform at neuromuscular junctions and its absence greatly reduces transmitter release. Thus these null genotypes provide a suitable background for judging the efficacy of a synaptotagmin in which Ca2+ binding has been altered (Robinson, 2002).

Both control and D2N transgenes rescue the synaptic phenotype in a heat-shock-dependent manner. The amplitude of excitatory junctional potentials (EJPs) in the P[UAS-HA-syt] larvae fell within the range observed in the syt+ control, although they were on average smaller, suggesting that the heat-shock-driven wild-type transgene is not a perfect substitute for the normal gene. The P[UAS-D2N] transgene also confers robust EJPs. In a second set of experiments, an elav-Gal4 driver was used to induce neuronal expression of P[UAS-D2N]; under this circumstance the response was fast, tightly coupled to nerve stimulation, and very similar in time course to those of the wild type. Thus, despite mutation of the essential aspartate, the transgene can alleviate most of the disruption of synaptic transmission caused by removing synaptotagmin I (Robinson, 2002).

The Ca2+ dependence of transmission mediated by the synaptotagmin I^D2N construct was examined by looking at the apparent cooperativity (N) of release from the slope of double-log plots of quantal content versus extracellular Ca2+ concentration. This study shows that a major alteration of the C₂A Ca2+-binding site has a negligible effect on the Ca2+-dependent properties of the synapse. This result stands in contrast to a previous study that looked at minor perturbations of Ca2+ binding in the C₂A domain and observed a decrease in release probability that was interpreted as a correlation of those changes (Robinson, 2002).

These observations prompted a reinvestigation of the function of synaptotagmin IV, the isoform whose sequence contains a serine substitution for the D3 aspartate and whose inability to bind Ca2+ in the C₂A domain causes it to act as an inhibitor of release. To this end, the UAS-sytIV transgene, with elav-Gal4 to activate neuronal expression, was crossed into the sytI-null background. Larvae lacking synaptotagmin I typically die at early stages, but, if separated from their heterozygous siblings, they can survive to adulthood. Nonetheless, transmission at this synapse is nearly absent. When sytIV was expressed in the sytI^AD4/AD4 background, transmission was restored to near-control levels. Consistent with previous studies, a modest increase in the frequency, but not the amplitude, of spontaneous miniature EJPs was observed in sytI-null larvae. This phenomenon was reversed by the expression of synaptotagmin IV. Thus, despite the inability of the C₂A domain of synaptotagmin IV to bind Ca2+ and the reported interference of this protein with Ca2+-dependent biochemical processes, synaptotagmin IV can substitute efficiently for synaptotagmin I in synaptic transmission (Robinson, 2002).

The synaptotagmin I^D2N mutation afforded a stringent test of the hypothesis that the C₂A domain of synaptotagmin I is the Ca2+ sensor that transduces the change in cytosolic Ca2+ into a signal for exocytosis. This transgene restored fast, Ca2+-dependent transmission with properties extremely similar to those of wild-type animals and sytI-null animals that had been rescued with a wild-type transgene. Similarly, although synaptotagmin IV does not bind Ca2+ in its C₂A domain, it efficiently supports transmission in a sytI-null background. These findings are not easily reconciled with the hypothesis that the C₂A domain of synaptotagmin is the Ca2+-sensing trigger. The release observed was dependent on the induction of the transgenes, so transmission in these animals should predominantly reflect the properties of the transgene and not those of any minor second synaptotagmin gene that might be present. If the mutated Ca2+-binding site was indeed the trigger for fusion, alterations in the Ca2+ dependence of transmission should have been observed. It cannot be ruled out that some residual Ca2+ binding takes place in the D2N mutation, although biochemical assays indicate it must be very slight, but the profound reduction in Ca2+ binding that occurred should have been mirrored by a sharp decrease in synaptic function (Robinson, 2002).

The apparent cooperativity of Ca2+ action in the nerve terminal is often used to characterize the sensor. This cooperativity has been inferred from the non-linearity of secretion to changes in extracellular Ca2+, which should, at low Ca2+ concentrations, be proportional to local change in cytosolic Ca2+. The ability of synaptotagmin to bind multiple Ca2+ ions has been put forward as a potential correlate for the multiple predicted Ca2+ ions that trigger exocytosis. This parameter should be a sensitive reporter of changes in the triggering site, including changes in ions bound per site or the removal of one of several Ca2+-binding steps. Thus, the observation that this property is indifferent to the D2N mutation is a further indication that the C₂A domain is not involved in the triggering of exocytosis. Similarly, the range of Ca2+ concentrations that evoke release is unaffected, suggesting that the Michaelis constant (Km) for the sensor had not been greatly altered (Robinson, 2002).

The finding that Ca2+ binding by the C₂A domain is not essential for release raises the possibility that the C₂B domain fulfils this function or that the two domains share the task. Yet, if Ca2+ sensing for fusion were accomplished by Ca2+ binding to both C₂A and C₂B, then the mutations in this study should have affected transmission significantly, and the apparent cooperativity of fusion should have been reduced by the impairment of C₂A function. Functions of C₂B that are independent of C₂A should be addressed directly with further mutations (Robinson, 2002).

In Drosophila, C. elegans and mice, some synaptic transmission persists in the absence of synaptotagmin I, and this transmission is dependent on Ca2+ concentration, similar to what has been observed in wild type. The present observation that synaptotagmin IV can participate in fast, Ca2+-dependent transmission suggests that synaptotagmin IV or another isoform could mediate the residual release in sytI-null mutants. In contrast, the fact that Ca2+-dependent properties of transmission are constant in the face of the different Ca2+-binding properties of either synaptotagmin IV or I^D2N suggests that Ca2+ binding, at least by the C₂A domain, is not the primary means by which synaptotagmin promotes fusion. Decreased release, which was consistently observed in synaptotagmin I mutations, could instead be accounted for by a defect in any of several steps in nerve terminal function, including a decrease in the pool of releasable docked vesicles or a decrease in the probability of fusion of a docked vesicle. This model would be consistent with the interactions of synaptotagmin with plasma membrane proteins and the AP-2 complex and with ultrastructural studies of mutants. The Ca2+-binding site in the C2 domains of synaptotagmins may permit Ca2+ to modulate vesicle docking or endocytosis, and thereby have subtler synaptic effects that were not assayed in this study (Robinson, 2002).

This study reopens the question of the nature of the Ca2+ sensor that triggers release. In addition to the C₂B domain of synaptotagmin, many other candidates have been identified, and multiple sensors may exist to account for the steep dependence of fusion on Ca2+ concentration. These alternatives now merit closer examination (Robinson, 2002).