InteractiveFly: GeneBrief

Synaptotagmin 4: Biological Overview | References


Gene name - Synaptotagmin 4

Synonyms -

Cytological map position - 84D3-84D3

Function - signaling transduction

Keywords - neuromuscular junction, mesoderm, a postsynaptic Ca2+ sensor, releases retrograde signals that stimulate enhanced presynaptic function, acts throught activation of the cAMP-dependent protein kinase pathway

Symbol - Syt4

FlyBase ID: FBgn0028400

Genetic map position - chr3R:3,091,783-3,096,531

Classification - C2 domain second repeat present in Synaptotagmin 4

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | NCBI Protein
BIOLOGICAL OVERVIEW

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 shibirets1 mutation, which disrupts endocytosis at elevated temperatures. shibirets1 was expressed specifically in postsynaptic muscles by driving a UAS-shibirets1 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 shibirets1, whereas wild-type animals displayed normal HFMR at 31°C. Basic synaptic properties in Mhc-Gal4, UAS-shibirets1 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-shibirets1 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-shibirets1 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 4BA1) 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 (DC0B3) 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).

As a general mechanism for memory storage, Hebb postulated that potentiated synapses maintain an activated state until structural changes occur to consolidate alterations in synaptic strength. The current results demonstrate that acute plasticity and synapse-specific growth require Syt 4-dependent retrograde signaling at Drosophila NMJs. The feedback mechanism described in this study could be a molecular basis for both input-specific postsynaptic tagging and an output-specific presynaptic mark or tag for long-lasting potentiation. The regenerative nature of a positive feedback signal allows individual synapses to be tagged in a discrete all-or-none manner until synaptic rewiring is completed. The synaptic tag is maintained as a large increase in miniature frequency at Drosophila NMJs, suggesting a previously unknown role for miniature release in neuronal function. The spatial resolution for input and output specificity would result from the accuracy insured by Ca2+-dependent vesicle fusion and subsequent diffusion, similar to the precision of presynaptic neurotransmitter release (Yoshihara, 2005).

The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling

Postsynaptic cells can induce synaptic plasticity through the release of activity-dependent retrograde signals. A Ca(2+)-dependent retrograde signaling pathway mediated by postsynaptic Synaptotagmin 4 (Syt4) has been previously described in this context. To identify proteins involved in postsynaptic exocytosis, this study conducted a screen for candidates that disrupt trafficking of a pHluorin-tagged Syt4 at Drosophila neuromuscular junctions (NMJs). The study further characterized one candidate, the postsynaptic t-SNARE Syntaxin 4 (Syx4). Analysis of Syx4 mutants reveals that Syx4 mediates retrograde signaling, modulating the membrane levels of Syt4 and the transsynaptic adhesion protein Neuroligin 1 (Nlg1). Syx4-dependent trafficking regulates synaptic development, including controlling synaptic bouton number and the ability to bud new varicosities in response to acute neuronal stimulation. Genetic interaction experiments demonstrate Syx4, Syt4, and Nlg1 regulate synaptic growth and plasticity through both shared and parallel signaling pathways. These findings suggest a conserved postsynaptic SNARE machinery controls multiple aspects of retrograde signaling and cargo trafficking within the postsynaptic compartment (Harris, 2016).

Synaptic connections form and mature through signaling events in both pre- and postsynaptic cells. The release of signaling molecules into the synaptic cleft depends on SNARE proteins that drive membrane fusion. This machinery is well understood for neurotransmitter release from the presynaptic cell: in response to an action potential, a v-SNARE in the synaptic vesicle membrane (Synpatobrevin/VAMP) engages t-SNARES in the presynaptic membrane (Syx1 and SNAP-25), forming a four-helix structure that brings the membranes into close proximity and initiates fusion. Although SNARE-dependent fusion drives membrane dynamics in all cell types, it is specialized in the presynaptic terminal to be Ca2+-dependent, employing Ca2+ sensors like Synaptotagmin 1 (Syt1) to link synaptic vesicle fusion to Ca2+ influx following an action potential (Harris, 2016).

The postsynaptic cell also exhibits activity-dependent exocytosis. Altering the composition of the postsynaptic membrane, including regulated trafficking of neurotransmitter receptors, is an important plastic response to neural activity (Chater, 2014). The postsynaptic cell also releases retrograde signals into the synaptic cleft to modulate synaptic growth and function. These retrograde messengers include lipid-derived molecules like endocannabinoids, gases like nitric oxide, neurotransmitters, neurotrophins, and other signaling factors like TGF-β and Wnt. Adhesion complexes that provide direct contacts across the synaptic cleft also participate in retrograde signaling (Harris, 2016).

Although retrograde signaling is a key modulator of synaptic function, little is known about how postsynaptic exocytosis is regulated and coordinated. Components of a postsynaptic SNARE complex have been recently identified in mammalian dendrites. The t-SNAREs Syntaxin 3 (Stx3) and SNAP-47 are required for regulated AMPA receptor exocytosis during long term potentiation, while the v-SNARE synaptobrevin-2 regulates both activity-dependent and constitutive AMPAR trafficking (Jurado, 2013). Stx4 has also been implicated in activity-dependent AMPAR exocytosis (Kennedy, 2010). In Drosophila, a Ca2+-dependent retrograde signaling pathway relies on the postsynaptic Ca2+ sensor Syt4. Syt4 vesicles fuse with the postsynaptic membrane in an activity-dependent fashion (Yoshihara, 2005), and loss of Syt4 leads to abnormal development and function of the NMJ. Syt4 null animals have smaller synaptic arbors, indicating a defect in synaptic growth, and also fail to exhibit several forms of synaptic plasticity seen in control animals, including robust enhancement of presynaptic release in response to high frequency stimulation, and rapid budding of synaptic boutons in response to strong neuronal stimulation (Barber, 2009; Korkut, 2013; Piccioli, 2014; Yoshihara, 2005). However, a detailed understanding of how the postsynaptic cell regulates constitutive and activity-dependent signaling of multiple retrograde pathways is lacking. In addition to exocytosis, it is likely that many cellular processes including vesicle trafficking and polarized transport of protein and transcript are specialized to facilitate postsynaptic signaling. Identifying such regulatory mechanisms is crucial for understanding synaptic development and function (Harris, 2016).

This study carried out a candidate-based transgenic RNAi screen to identify regulators of postsynaptic exocytosis at the Drosophila NMJ, a model for studying glutamatergic synapse growth and plasticit. Using a fluorescently tagged form of the postsynaptic Ca2+ sensor Syt4, candidate gene products were screened that disrupted the localization of Syt4 at the postsynaptic membrane. This study describes characterization of one candidate from this screen, Syntaxin 4 (Syx4). Drosophila Syx4 is the sole homolog of the mammalian Stx 3/4 family of plasma membrane t-SNAREs that also includes Syntaxin 1. The mammalian Stx3 and Stx4 homologs regulate activity-dependent AMPA receptor trafficking in mammalian neurons (Jurado, 2013; Kennedy, 2010), while Stx4 also participates in regulated secretory events in several other mammalian cell types, including insulin-stimulated delivery of the glucose transporter to the plasma membrane in adipocytes and glucose-stimulated insulin secretion from pancreatic beta cells (reviewed by Jewell, 2010). The results demonstrate that the Drosophila Syx4 homolog is essential for retrograde signaling, regulating the membrane delivery of both Syt4 and Neuroligin (Nlg1), a transsynaptic adhesion protein that plays important roles in synapse formation and function, and is linked to autism spectrum disorder (ASD). Through genetic interaction experiments, this study defined functions of the Syx4, Syt4, and Nlg1 pathway in regulating multiple aspects of synaptic growth and plasticity within the postsynaptic compartment (Harris, 2016).

To identify regulators of postsynaptic exocytosis, a screen was conducted for gene products regulating Syt4 plasma membrane accumulation, resulting in the identification of the plasma membrane t-SNARE Syx4. Analysis of a Syx4 null mutant indicates that Syx4 is essential for development of the Drosophila NMJ and regulates the membrane delivery of at least two proteins that are important for synaptic growth and plasticity: the postsynaptic Ca2+ sensor Syt4 and the transsynaptic adhesion protein Nlg1 (Harris, 2016).

The screen identified 15 candidate gene products that altered the localization of Syt4-pH. In addition to Syx4, several other candidates motivate interesting hypotheses about regulatory pathways for postsynaptic exocytosis. MyoV is a Ca2+-sensitive unconventional myosin that regulates polarized traffic. Thus, MyoV could play a role linking Ca2+ influx to vesicle delivery or release at the synapse. Indeed, MyoV homologs have been implicated in regulated AMPA trafficking in mammalian dendrites. Two Rab regulators (Gdi and Rabex) suggest that key vesicle trafficking steps en route to the synapse are modulated by Rab activation states. Also, two cell adhesion molecules (Neuroglian and Contactin) indicate potential transsynaptic mechanisms regulating retrograde signaling. Neuroglian has been shown to be required for synaptic stability and it is possible that Syt4-mediated retrograde signaling plays some role in this process (Harris, 2016).

Syt4 has also been shown to be transferred transsynaptically from the presynaptic terminal to the postsynaptic terminal on exosomes (Korkut, 2013). Thus, the approach of expressing Syt4-pH postsynaptically may not reveal components for the biosynthetic synthesis and transport of presynaptic Syt4. Nevertheless, the requirement for Syt4 in the postsynaptic cell for retrograde signaling is clear, and the results of the screen highlight regulators of Syt4 trafficking to and from the postsynaptic membrane where Syt4 vesicles fuse in an activity-dependent manner. The observation that endogenously expressed Syt4-GFP (Syt4GFP-2M) shows a similar distribution to Syt4-pH supports the biological relevance of the screen data for identifying regulators of Syt4 trafficking in the postsynaptic cell (Harris, 2016).

The Syx4 null allele phenocopies the Syx4-RNAi knockdown, reducing the delivery of Syt4-pH to the postsynaptic membrane. Consistent with this finding, loss of Syx4 produces similar phenotypes to loss of Syt4. Both null mutants exhibit a reduction in the total number of boutons at the NMJ, indicating a defect in synaptic growth. Moreover, genetic interaction experiments clearly indicate that Syx4 and Syt4 interact with respect to synaptic growth. A strong genetic interaction between Syx4 and Syt4 is also evident at the level of lethality, as double mutant animals are lethal at a much earlier stage than either single mutant alone. Thus, even though Syx4 affects the localization of Syt4, suggesting they act in the same pathway, the genetic interaction data do not support a simple epistatic relationship. The difference in phenotypic severity, with the Syx4 bouton number defect being significantly stronger than the Syt4 defect, also points to Syt4 not being absolutely required for Syx4 signaling. A similar phenomenon is observed presynaptically where the t-SNARE Syx1 is indispensible for synaptic vesicle fusion, while fusion is only reduced in the absence of the synaptic vesicle Ca2+ sensor Syt1. Taken together, it is hypothesized that (1) Syx4 and Syt4 act together in a single pathway where Syx4 regulates the exocytosis of vesicles containing Syt4, and (2) Syx4 and Syt4 also act in divergent pathways, where Syt4 cooperates with other t-SNARES, and Syx4 mediates the exocytosis of vesicles in a Syt4-independent manner. This model allows for multiple possible postsynaptic SNARE complexes, regulating distinct release events. Dissecting the other components of these fusion machineries, and distinguishing activity-dependent from constitutive release events, will be important to build understanding of how retrograde signaling is regulated (Harris, 2016).

In addition to affecting the localization of Syt4, Syx4 mutants also exhibit a decrease in the amount of Nlg1 at the postsynaptic membrane. Nlg1 has several functions at the synapse, along with its presynaptic binding partner Nrx-1. Together they regulate bouton number as well as the size and spacing of active zones and glutamate receptors, though some aspects of Nlg1 signaling appear to be independent of Nrx-1. Mutations in Nrx and Nlg family genes are also linked to autism spectrum disorder (ASD), highlighting the importance of Nrx-Nlg signaling in neuronal development. Consistent with a reduction of Nlg1 levels at the synapse, strong genetic interactions were observed between Syx4, Nlg1 and Nrx-1 with respect to bouton number. However, the prominent AZ/GluR defects seen in Nlg1 and Nrx-1 mutants were not observed in Syx4 mutants, and heterozygous combinations did not produce these defects. It is likely that Syx4 mutants exhibit a partial loss of function of Nlg1, and that bouton number is sensitive to this loss while AZ/GluR organization can be maintained with low levels of Nlg1 (Harris, 2016).

A dramatic change in distribution of Nlg1Δcyto is observed in the Syx4 mutant background, providing further evidence that Syx4 regulates the localization of Nlg1. The redistribution of Nlg1Δcyto to large accumulations is striking compared to full-length Nlg1, which is simply reduced at the synapse in the Syx4 mutant background. This observation points to complex Syx4-dependent regulation of Nlg1 localization. One model is that trafficking of Nlg1 involves both a Syx4-dependent pathway and a second pathway that depends on an interaction with the Nlg1 C-terminus, which includes a PDZ-domain-binding motif. In this scenario, a severe Nlg1 trafficking defect is revealed only when both pathways are compromised. A second possibility is that in the absence of Syx4, a portion of the Nlg1 content in the cell is degraded, but that this degradation step depends on the presence of the Nlg1 cytoplasmic tail, leading to the observed aggregation of Nlg1Δcyto in Syx4 mutants (Harris, 2016).

Analysis of Nlg1 trafficking in live animals reveals that Nlg1 is strikingly stable, in both control and Syx4 mutant backgrounds. A motivation in performing these experiments was to test possible mechanisms underlying the decrease in Nlg1 levels in Syx4 mutants. It is possible that some Nlg1 mobility would be observed over a longer time course. Mammalian Nlg has been shown to undergo significant turnover at postsynaptic sites under LTP conditions in neuronal cell culture. Also, synaptic activity has been shown to induce cleavage of Nlg and the subsequent destabilization of the Nrx-Nlg complex. Thus, it remains a possibility that Nlg1 would be mobilized in response to activity in the preparation; however, no increased mobility was observed in response to high K+ incubations in preliminary tests. The data are most consistent with Syx4 regulating Nlg1 over a developmental time course. A detailed examination of the relationship between Syx4 and Nlg1 dynamics will be crucial to understand how Syx4 contributes to this important pathway in synaptic development (Harris, 2016).

A strong suppression of acute structural plasticity was observed in null mutants of Syx4, Syt4 and Nlg1. Double heterozygous combinations also indicated strong genetic interactions between all three of these genes with respect to plasticity. GB budding is regulated by both acute and developmental signaling. Because Syt4 postsynaptic vesicles fuse in an activity-dependent manner, it is possible that Syt4-dependent signaling releases an acute instructive cue for GB budding. Thus, one attractive model is that Nlg1 is delivered to the membrane in response to stimulation, depending on the Ca2+ sensitivity of Syt4 and the presence of the t-SNARE Syx4 at the membrane. It is also possible that Syx4-Syt4-Nlg1 signaling is required throughout development to potentiate the synapse to respond to strong neuronal stimulation. In conclusion, Syx4, Syt4, and Nlg1 interact to regulate several aspects of synaptic biology. The data support multiple overlapping signaling pathways regulated by these proteins, reflecting a complex modulation of retrograde signaling to control synaptic growth and plasticity at the Drosophila NMJ (Harris, 2016).

Regulation of postsynaptic retrograde signaling by presynaptic exosome release

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).

Rat and Drosophila synaptotagmin 4 have opposite effects during SNARE-catalyzed membrane fusion

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).

Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains

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 parats1 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).

Synaptotagmins are trafficked to distinct subcellular domains including the postsynaptic compartment

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).

Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain

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 ID2N, 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 ID2N 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 sytIAD4/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 ID2N 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 ID2N 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).


REFERENCES

Search PubMed for articles about Drosophila Syt4

Adolfsen, B., Saraswati, S., Yoshihara, M., Littleton, J. T. (2004). Synaptotagmins are trafficked to distinct subcellular domains including the postsynaptic compartment. J Cell Biol 166: 249-260. PubMed ID: 15263020

Ataman, B., Ashley, J., Gorczyca, M., Ramachandran, P., Fouquet, W., Sigrist, S. J. and Budnik, V. (2008). Rapid activity-dependent modifications in synaptic structure and function require bidirectional Wnt signaling. Neuron 57: 705-718. PubMed ID: 18341991

Barber, C. F., Jorquera, R. A., Melom, J. E. and Littleton, J. T. (2009). Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains. J Cell Biol 187: 295-310. PubMed ID: 19822673

Chater, T. E. and Goda, Y. (2014). The role of AMPA receptors in postsynaptic mechanisms of synaptic plasticity. Front Cell Neurosci 8: 401. PubMed ID: 25505875

Harris, K.P., Zhang, Y.V., Piccioli, Z.D., Perrimon, N. and Littleton, J.T. (2016). The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling. Elife 5. PubMed ID: 27223326

Jewell, J. L., Oh, E. and Thurmond, D. C. (2010). Exocytosis mechanisms underlying insulin release and glucose uptake: conserved roles for Munc18c and syntaxin 4. Am J Physiol Regul Integr Comp Physiol 298: R517-531. PubMed ID: 20053958

Jurado, S., Goswami, D., Zhang, Y., Molina, A. J., Sudhof, T. C. and Malenka, R. C. (2013). LTP requires a unique postsynaptic SNARE fusion machinery. Neuron 77: 542-558. PubMed ID: 23395379

Kennedy, M. J., Davison, I. G., Robinson, C. G. and Ehlers, M. D. (2010). Syntaxin-4 defines a domain for activity-dependent exocytosis in dendritic spines. Cell 141: 524-535. PubMed ID: 20434989

Korkut, C., Li, Y., Koles, K., Brewer, C., Ashley, J., Yoshihara, M., Budnik, V. (2013). Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron 77: 1039-1046. PubMed ID: 23522040

Piccioli, Z. D. and Littleton, J. T. (2014). Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM kinase regulation of cofilin. J Neurosci 34: 4371-4381. PubMed ID: 24647957

Robinson, I. M., Ranjan, R. and Schwarz, T. L. (2002). Synaptotagmins I and IV promote transmitter release independently of Ca(2+) binding in the C(2)A domain. Nature 418: 336-340. PubMed ID: 12110845

Wang, Z. and Chapman, E. R. (2010). Rat and Drosophila synaptotagmin 4 have opposite effects during SNARE-catalyzed membrane fusion. J Biol Chem 285: 30759-30766. PubMed ID: 20688915

Yoshihara, M., Adolfsen, B., Galle, K. T. and Littleton, J. T. (2005). Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth. Science 310: 858-863. PubMed ID: 16272123


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date revised: 10 July 2014

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