Endocytosis of synaptic vesicles follows exocytosis, and both processes require external Ca2+. However, it is not known whether Ca2+ influx through one route initiates both processes. At larval Drosophila neuromuscular junctions, exocytosis and endocytosis were separately measured using the fluorescent dye FM1-43. In a temperature-sensitive Ca2+ channel mutant, cacophonyTS2, exocytosis induced by high K+ decreases at nonpermissive temperatures, while endocytosis remains unchanged. In wild-type larvae, a spider toxin Ca2+ blocker, PLTXII, preferentially inhibits exocytosis, whereas the T-type Ca2+ channel blocker flunarizine and the blocker La3+ selectively depresses endocytosis. None of these blockers affect exocytosis or endocytosis induced by a Ca2+ ionophore. Evoked synaptic potentials are depressed regardless of stimulus frequency in cacophonyTS2 at nonpermissive temperatures and in wild-type by PLTXII, whereas flunarizine or La3+ gradually depressed synaptic potentials only during high-frequency stimulation, suggesting depletion of synaptic vesicles due to blockade of endocytosis. In shibirets1, a dynamin mutant, flunarizine or La3+ inhibit assembly of clathrin at the plasma membrane during stimulation without affecting dynamin function (Kuromi, 2004).
To maintain synaptic transmission during intense neuronal activities, synaptic vesicles (SVs) are effectively recycled by endocytosis. Ca2+ influx through voltage-gated Ca2+ channels plays a crucial role in exocytosis. It has also been found, in early studies at frog neuromuscular junctions, that external Ca2+ is essential for SV recycling. Subsequent studies have confirmed that Ca2+ influx is also required for endocytosis in various types of central synapses and secretory cells. In contrast, endocytosis has been shown to occur even in the absence of external Ca2+ after cessation of stimulation in rat hippocampal neurons and in presynaptic boutons of a Drosophila temperature-sensitive mutant, shibirets1 (shits1), at room temperature after depletion of SVs at nonpermissive temperatures. These studies suggest that endocytosis can be triggered independently of the Ca2+ influx that initiates exocytosis. To reconcile these seemingly contradictory findings, it has been postulated that Ca2+ influx during stimulation that causes exocytosis also triggers the formation of intermediates for SV recycling, and once they are formed, the following steps of endocytosis proceed without Ca2+. However, because of difficulties in separating Ca2+ influx routes for exocytosis and endocytosis during stimulation, this hypothesis remains unproven (Kuromi, 2004 and references therein).
Immunostaining studies suggest that sites for endocytosis are distinct from those for exocytosis at nerve terminals. It is then possible that Ca2+ influx routes for these two processes are separate. Along the line of this idea, multiple subtypes of Ca2+ channels are demonstrated in nerve terminals. Those subtypes of Ca2+ channels are spatially segregated in presynaptic terminals, and their roles in transmitter release have been subject to speculation. Specific roles of these Ca2+ channel subtypes in exocytosis and endocytosis, however, have not been identified (Kuromi, 2004).
Does Ca2+ influx through one route trigger both exocytosis and endocytosis? To address this question, a temperature-sensitive Ca2+ channel mutant, cacophonyTS2 (cacTS2), and various Ca2+ channel blockers have been used at larval Drosophila neuromuscular junctions. A fluorescent dye, FM1-43, is incorporated into SVs in nerve terminals by endocytosis, and FM1-43 loaded in SVs is released by exocytosis. By measuring the amount of FM1-43 released from or taken up into nerve terminals, exocytosis and endocytosis were separately determined. Thus, it has been revealed that distinct Ca2+ influx routes separately regulate exocytosis and endocytosis. Taking advantage of drugs that selectively block endocytosis, it has been further shown in shits1 that selective blockade of the Ca2+ influx route linked to endocytosis inhibits clathrin assembly on the plasma membrane of nerve terminals. It is suggested that Ca2+ influx during stimulation through this route forms an intermediate complex, which leads to endocytosis (Kuromi, 2004).
A widely accepted model of endocytosis is that the clathrin coat assembles first on the presynaptic membrane, forming a shallow coated pit, which then invaginates to generate a bud with a constricted neck and eventually a free clathrin-coated vesicle by fission of the neck. A model has been proposed with two steps in SV recycling in which a Ca2+-dependent step (step I), which occurs during stimulation, is followed by a Ca2+-independent, shibire-dependent step (step II). In shits1 it has been shown that when FNZ or La3+ is added after high K+ stimulation, endocytosis at permissive temperatures occurs normally, indicating that FNZ or La3+ have no effect on step II. In contrast, when FNZ or La3+ is present during high K+ stimulation, endocytosis is not observed although exocytosis is unaffected. These observations strongly support the hypothesis that FNZ or La3+ selectively block Ca2+ influx through the route designated for endocytosis (step I) (Kuromi, 2004).
Immunostaining experiments with shits1 at nonpermissive temperatures reveal that synaptotagmin I is transferred to the plasma membrane during high K+ stimulation regardless of the presence of La3+, confirming that La3+ has no effect on exocytosis. However, it was noted that in the absence of La3+, clusters of clathrin immmunoreactivity are detected at the periphery of boutons after high K+ stimulation, while in the presence of La3+, clathrin remains in the cytosol of boutons after high K+ stimulation. These observations suggest that La3+ inhibits clathrin assembly at the plasma membrane. Dynamin plays an essential role in the fission of a clathrin-coated bud, and this process occurs in the absence of external Ca2+. It is suggested that the part of Ca2+ influx sensitive to FNZ or La3+ during stimulation (step I), plays a crucial role in clathrin assembly at the plasma membrane (Kuromi, 2004).
The morphological transition of growth cones to synaptic boutons characterizes synaptogenesis. This study isolated mutations in immaculate connections (imac; FlyBase name unc-104), encoding a member of the Kinesin-3 family. Whereas earlier studies in Drosophila has implicated Kinesin-1 in transporting synaptic vesicle precursors, this study found that Imac/Unc-104 is essential for this transport. An unexpected feature of imac mutants is the failure of synaptic boutons to form. Motor neurons lacking imac properly target to muscles but remain within target fields as thin processes, a structure that is distinct from either growth cones or mature terminals. Few active zones form at these endings. The arrest of synaptogenesis is not a secondary consequence of the absence of transmission. These data thus indicate that Imac transports components required for synaptic maturation and provide insight into presynaptic maturation as a process that can be differentiated from axon outgrowth and targeting (Pack-Chung, 2007).
Components of synaptic and dense-core vesicles were, however, markedly redistributed in imac mutants. Synaptotagmin-I immunoreactivity, for example, is normally intensely concentrated in the neuropil and scant in the cortex. In imac mutants this pattern was reversed: synaptotagmin-I accumulated in cell bodies and was low in the neuropil. Synaptotagamin-I-GFP, expressed in the imac mutant nervous system by an elav-GAL4 driver, showed the same redistribution. VGlut, normally found at a few CNS synapses that are glutamatergic, was similarly redistributed from neuropil to cell bodies in imac mutants, as was cysteine string protein and ANF-GFP. The concentration of the vesicular proteins in imac mutant cell bodies indicates that these proteins are synthesized in the mutants. The loss of these markers from axon tracts and synaptic regions indicates a defect in their transport from the cell bodies. Absence of immunoreactivity of these molecules in the peripheral axons of motor neurons likewise supports the idea that Imac has a direct role in the transport of synaptic materials. In addition, live-cell imaging of vesicle precursors in the segmental nerves of an imac mutant has revealed defects in the anterograde movement of these cargos (R. Barkus and W. Saxton, personal communication to Pack-Chung, 2007), further substantiating the idea that Imac has a motor function (Pack-Chung, 2007).
To determine whether Imac is involved in the transport of other proteins and organelles, the distribution of various intracellular components was examined. The normal axonal growth and guidance observed in imac mutants implies that the transport of post-Golgi vesicles with new membrane and cell-surface proteins persists. The neuronal membrane marker, HRP, did not reveal any significant differences between wild type and imac mutants. Post-Golgi membrane trafficking was examined in imac mutants by using a fusion of the extracellular and transmembrane domains of CD8 to a cytoplasmic GFP, a construct that serves as a nonspecific reporter of the constitutive transport of membrane proteins to the cell surface, including axons and terminals. The distribution of CD8-GFP was not affected in imac mutants. Similarly, Fasciclin II (FasII), a Drosophila homolog of vertebrate neural cell-adhesion molecules and an essential regulator of motor neuron growth and guidance, remained concentrated in the axon tracts and synaptic regions in imac mutants. Another plasma membrane protein, syntaxin, is needed for exocytosis and addition of membrane to the cell surface. No change in the distribution of syntaxin, visualized with the monoclonal antibody 8C3, was detected in imac-null embryos. These data are consistent with the observation that axon outgrowth and targeting proceeds normally in imac mutants, and they demonstrate that the vesicles required for membrane extension and axon targeting are not conveyed by Imac. In addition, the data support the current model wherein mechanisms regulating membrane outgrowth are distinct from mature synaptic vesicle exocytosis (Pack-Chung, 2007).
Synaptotagmin serves as the major Ca2+ sensor for regulated exocytosis from neurons. While the mechanism by which synaptotagmin regulates membrane fusion remains unknown, studies using Drosophila indicate that the molecule functions as a multimeric complex and that its second C2 domain is essential for efficient excitation-secretion coupling. Biochemical data is described that may account for these phenomena. Ca2+ causes synaptotagmin to oligomerize, primarily forming dimers, via its second C2 domain. This effect is specific for divalent cations that can stimulate exocytosis of synaptic vesicles (Ca2+ >> Ba2+, Sr2+ >> Mg2+) and occurs with an EC50 value of 3-10 microM Ca2+. In contrast, a separate Ca2+-dependent interaction between synaptotagmin and syntaxin, a component of the fusion apparatus, occurs with an EC50 value of approximately 100 microM Ca2+ and involves the synergistic action of both C2 domains of synaptotagmin. It is proposed that Ca2+ triggers two consecutive protein-protein interactions: the formation of synaptotagmin dimers at low Ca2+ concentrations followed by the association of synaptotagmin dimers with syntaxin at higher Ca2+-concentrations. These findings, in conjunction with physiological studies, indicate that the Ca2+-induced dimerization of synaptotagmin is important for the efficient regulation of exocytosis by Ca2+ (Chapman, 1996).
While recent studies have focused on the proteins involved in exocytosis, it is not clear whether (or to what extent) the vesicle membrane recycles via clathrin-coated vesicles, or whether the membrane is directly retrieved by a fast endocytotic process. Biochemical studies have led to the proposal that the AP2 complex, a heterotetramer containing alpah-adaptin (See Drosophila alpah-Adaptin), plays an essential role in orchestrating different steps of endocytosis at the synapse. AP2 is able to bind to the cytoplasmic tail of a number of membrane receptors including synaptotagmin, a transmembrane protein that controls the Ca2+-dependent membrane fusion during exocytosis. Synaptotagmin interaction with AP2 is consistent with its proposed function during endocytosis, which is based on a synaptotagmin endocytotic mutant phenotype. This would argue that synaptotagmin plays a dual role in the vesicle cycle by acting in the final step of exocytosis and the initial step of endocytosis, thereby coupling the two processes at the plasma membrane. Once recruited to the inner surface of the plasma membrane, AP2 is likely to initiate the formation of clathrin-coated pits by triggering the assembly of clathrin triskelion subunits into a polygonal lattice that causes a bending of the membrane into the coated pit structure. Clathrin-coated pits detach from the plasmalemma by a GTP-dependent fission reaction that is mediated by the GTPase dynamin, and the resulting coated membrane vesicles become internalized. Dynamin has been shown to bind the AP2 complex in vitro, and to be functionally required for the detachment of the clathrin-coated vesicles from the membrane. After internalization, the clathrin-coated vesicles shed their coats, a process that involves a number of proteins, including auxilin, Hsp-70, and the cysteine string protein (CSP), which may function in a chaperone-like manner to unfold the clathrin lattice at the outer surface (González-Gaitán, 1997 and references therein).
One can envision a scenario where the recruitment of the AP2 complex to the plasma membrane is a rate-limiting step, which in turn could be controlled by membrane-associated AP2 receptors that are released from exocytotic vesicles. Such a molecular link between exocytosis and endocytosis events would guarantee exocytosis-dependent membrane retrieval, as suggested by the temporal link of exocytosis and endocytosis and by the in vitro interaction between AP2 and synaptotagmin. The results of this study show, however, that synaptotagmin does not colocalize with alpha-Adaptin in shi mutants. This suggests that the role of synaptotagmin and AP2 association is more likely to serve the recycling of synaptotagmin from the membrane, returning it to the cytoplasmic pool of synaptic vesicles to take part in the subsequent exocytosis event. Alternatively, synaptotagmin could be one of several functionally redundant receptors to anchor the AP2 complex to the membrane to initiate a new vesicle cycle (González-Gaitán, 1997).
Communication within the nervous system is mediated by Ca2+-triggered fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic and biochemical evidence indicates that synaptotagmin I may function as a Ca2+ sensor in neuronal exocytosis because it can bind Ca2+ and penetrate into lipid bilayers. Chronic depolarization or seizure activity results in the upregulation of a distinct and unusual isoform of the synaptotagmin family, synaptotagmin IV. A Drosophila homolog of synaptotagmin IV has been identified that is enriched on synaptic vesicles and contains an evolutionarily conserved substitution of aspartate to serine that abolishes its ability to bind membranes in response to Ca2+ influx. Synaptotagmin IV forms hetero-oligomers with synaptotagmin I, resulting in synaptotagmin clusters that cannot effectively penetrate lipid bilayers and are less efficient at coupling Ca2+ to secretion in vivo: upregulation of synaptotagmin IV, but not synaptotagmin I, decreases evoked neurotransmission. These findings indicate that modulating the expression of synaptotagmins with different Ca2+-binding affinities can lead to heteromultimers that can regulate the efficiency of excitation-secretion coupling in vivo and represent a new molecular mechanism for synaptic plasticity (Littleton, 1999).
The Drosophila stoned locus encodes two novel gene products, termed stonedA and stonedB, which possess sequence motifs shared by proteins involved in intracellular vesicle traffic. A specific requirement for stoned in the synaptic vesicle cycle has been suggested by synthetic genetic interactions between stoned and shibire, a gene essential for synaptic vesicle recycling. A synaptic role for stoned gene products is also suggested by altered synaptic transients in electroretinograms recorded from stoned mutant eyes. The StonedA protein is highly enriched at Drosophila nerve terminals. Mutant alleles that affect stonedA disrupt the normal regulation of synaptic vesicle exocytosis at neuromuscular synapses in Drosophila. Spontaneous neurotransmitter release is enhanced dramatically, and evoked release is reduced substantially in such stoned mutants. Ultrastructural studies reveal no evidence of major disorganization at stoned mutant nerve terminals. Thus, these data indicate a direct role for stonedA in regulating synaptic vesicle exocytosis. However, genetic and morphological observations suggest additional, subtle effects of stoned mutations on synaptic vesicle recycling. Remarkably, almost all phenotypes for stoned mutants are similar to those for mutants of synaptotagmin, a protein postulated to regulate both exocytosis and the recycling of synaptic vesicles. A model is proposed in which stonedA functions together with synaptotagmin to regulate synaptic vesicle cycling (Stimson, 1998).
StonedA is not the first protein for which dual roles in synaptic vesicle exocytosis and endocytosis have been proposed. Both biochemical and genetic studies suggest that, in addition to its role in regulating synaptic vesicle fusion, synaptotagmin may regulate synaptic vesicle recycling. Although the possible role of synaptotagmin in synaptic vesicle recycling has not been addressed in studies of Drosophila synaptotagmin mutants, Caenorhabditis elegans synaptotagmin (snt-1) mutants exhibit phenotypes consistent with impaired synaptic vesicle recycling. For example, the synaptic vesicle protein synaptobrevin exhibits an abnormally diffuse distribution in the snt-1 nerve cord, suggesting an accumulation and lateral spreading of this protein within neuronal plasma membrane. This altered distribution of synaptobrevin in snt-1 nerve cord is qualitatively similar to the altered distributions of synaptotagmin and csp in stoned boutons. Thus, parallels between stoned mutants and synaptotagmin mutants suggest that stonedA and synaptotagmin may share functions in synaptic vesicle recycling as well as in synaptic vesicle fusion. If stonedA does in fact regulate general synaptic vesicle endocytosis, could defects in transmitter release at stonedts2 and stnc synapses be secondary to a primary defect in synaptic vesicle recycling? This is thought to be unlikely for the following reasons: (1) EM studies show that any recycling defects at stnts2 and stnc mutant synapses must be very subtle; (2) partial depletion of synaptic vesicles, which may be achieved by stimulating shabirets1 mutants at nonpermissive temperature, does not result in the specific phenotypes observed at stoned mutant synapses. The elevated mini frequency and the reduced evoked release are unique phenotypes of stoned mutants and thus probably reflect a specific function of StonedA in regulating Ca2+-dependent neurotransmitter release. The simplest interpretation of the data is that stonedts2 and stonednc mutants have independent defects in synaptic vesicle fusion and synaptic vesicle recycling (Stimson, 1998 and references).
The stoned locus of Drosophila encodes two novel proteins, stonedA (STNA) and stonedB (STNB), both of which are expressed in the nervous system. Flies with defects at the stoned locus have abnormal behavior and altered synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicate a presynaptic function for stoned and suggest an involvement in vesicle cycling. Immunological studies revealed colocalization of the Stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein Synaptotagmin (SYT). stoned interacts genetically with synaptotagmin to produce a lethal phenotype. The STNB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the µ2-homology domain of STNB and the C2B domain of the SYTI isoform. The STNA protein is also found in association with vesicles, and it too exhibits an in vitro association with SYTI. However, the bulk of STNA is in a nonmembranous fraction. By using the shibire mutant to block endocytosis, STNB has been shown to be present on some synaptic vesicles before exocytosis. However, STNB is not associated with all synaptic vesicles. It is hypothesized that STNB specifies a subset of synaptic vesicles with a role in the synaptic vesicle cycle that is yet to be determined (Phillips, 2000).
The Drosophila dicistronic stoned locus encodes two distinctive presynaptic proteins, Stoned A (StnA) and Stoned B (StnB); StnA is a novel protein without homology to known synaptic proteins, and StnB contains a domain with homology to the endocytotic protein AP50. Both Stoned proteins colocalize precisely with endocytotic proteins including the clathrin-associated coated pit adaptor protein complex AP2 and Dynamin in the 'lattice network' characteristic of endocytotic domains in Drosophila presynaptic terminals. FM1-43 dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the endo-exo-cycling synaptic vesicle pool and loss of spatial regulation of the vesicular recycling intermediates. Mutant synapses display a significant delay in vesicular membrane retrieval after depolarization and neurotransmitter release. These studies suggest that the Stoned proteins play a role in mediating synaptic vesicle endocytosis. A highly specific synaptic mislocalization and degradation of Synaptotagmin I has been documented in stoned mutants. Transgenic overexpression of Synaptotagmin I rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Furthermore, overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin I directly regulates the endo-exo-cycling synaptic vesicle pool size. In parallel with recent biochemical studies, this genetic analysis strongly suggests that Stoned proteins regulate the AP2-Synaptotagmin I interaction during synaptic vesicle endocytosis. It is concluded that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin I from the plasma membrane (Fergestad, 2001).
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).
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