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).
A continuous supply of fusion-competent synaptic vesicles is essential for sustainable neurotransmission. Mutations of the dicistronic stoned locus disrupt normal vesicle cycling and cause functional deficits in synaptic transmission. Although both Stoned A and B proteins putatively participate in reconstituting synaptic vesicles, their precise function is still unclear. This study investigated the effects of progressive depletion of Stoned B (STNB) on the release properties of neuromuscular synapses using a novel set of synthetic STNB hypomorphic alleles. Decreasing neuronal STNB expression to ~35% of wildtype level causes a strong reduction in EJC amplitude at low stimulation frequencies and a marked slowing in synaptic depression during high-frequency stimulation, suggesting vesicle depletion is attenuated by decreased release probability. Recovery from synaptic depression after prolonged stimulation is also decelerated in mutants, indicating a delayed recovery of fusion-ready vesicles. These phenotypes appear not to be due to a diminished vesicle population, since the docked vesicle pool is ultrastructurally unaffected, and the total number of vesicles is only slightly reduced in these hypomorphs, unlike lethal stoned mutants. Therefore, it is concluded that STNB not only functions as an essential component of the endocytic complex for vesicle reconstitution, as previously proposed, but also regulates the competence of recycled vesicles to undergo fusion. In support of such role of STNB, synaptic levels of the vesicular glutamate transporter (vGLUT) and synaptotagmin-1 are strongly reduced with diminishing STNB function, while other synaptic proteins are largely unaffected. It is concluded that STNB organizes the endocytic sorting of a subset of integral synaptic vesicle proteins thereby regulating the fusion-competence of the recycled vesicle (Mohrmann, 2008).
The Drosophila stoned locus was identified 35 years ago based on severe behavioral impairments. It is one of the few dicistronic loci characterized in Drosophila, encoding Stoned A and B proteins; however, the Stoned A protein appears totally dispensable for known functions. In contrast, the crucial importance of the Stoned B protein for synaptic transmission has been well established. Nevertheless, the exact mechanistic function of STNB remains enigmatic. This study has generated and characterized a graded set of STNB hypomorphic animals to provide evidence that STNB has a dose-dependent limiting function regulating neurotransmission strength as a potent sorting factor governing a specific set of integral vesicular proteins during synaptic vesicle reconstitution. The key findings supporting this interpretation of STNB function are (I) the occurrence of diminished and altered release in a progressive series of STNB hypomorphic alleles, in the absence of significant ultrastructural defects of the vesicle pools, thereby indicating a changed fusion competence of synaptic vesicles, and (II) the differential loss of integral synaptic vesicle proteins dependent on the level of STNB activity (Mohrmann, 2008).
In previous studies, severely compromised basal synaptic transmission has been reported as a prominent feature of the physiological phenotype of various classical stoned mutants. Simplistically, defective synaptic release might be entirely a secondary effect of a primary impairment in vesicle pool maintenance, due to disrupted vesicle endocytosis. However, recent studies of endophilin and synaptojanin endocytic mutants suggest that only a surprisingly small number of synaptic vesicles is actually required to support normal synaptic function at basal stimulation frequencies: In both endophilin and synaptojanin mutants, basal synaptic transmission is completely normal despite the near elimination of the presynaptic vesicle population, and the loss of synaptic uptake of FM1-43. This raises the question that STNB may be involved in other processes that affect exocytosis, apart from limiting the availability of vesicles. The observation that viable stnC mutants exhibit release defects without major alterations in vesicle pool size seems to support such notion. However, recent findings show that the stnC mutation induces the expression of a C-terminally truncated STNB variant, together with low levels of the full-length product, thereby possibly involving dominant-negative effects or a partial functionality of the truncated STNB variant. Thus, the physiological phenotype in stnC mutants cannot be clearly interpreted. To clarify whether a hypomorphic condition exists that would allow for a segregation of the putative release defect and vesicle pool depletion, a new collection of graded STNB hypomorphic alleles was generated by transgenic expression of STNB in the stn13-120 mutant background. In the hypomorphic condition, STNB levels should be sufficient to support vesicle resupply and to maintain normal vesicle pools, but the shortage would still compromise basal synaptic transmission (Mohrmann, 2008).
This study demonstrates that physiological defects first emerge when STNB expression is reduced to less than 40% of wildtype level. Below this threshold, the decrease in basal EJC amplitude correlates closely with the expression level of STNB. Hypomorphic stn-vl mutants expressing ~35% of the wildtype level are of particular interest for this analysis, because their expression is only slightly lower than this threshold, and yet this condition causes a large 40% drop in basal amplitude. Strikingly, the ultrastructural analysis of stn-vl synapses showed that clustered and docked vesicle pools at the presynaptic active zone were completely unaffected in this hypomorphic condition, although there is a slight reduction in overall vesicle density. At the Drosophila NMJ, an 'exo/endo cycling vesicle pool' (ECP) has been demonstrated at the bouton periphery, which probably corresponds to the electrophysiologically defined readily releasable pool (RRP), and a reserve pool (RP) at the bouton center. According to this study the ECP/RRP alone is sufficient to allow for full-scale basal synaptic transmission after pharmacological depletion of the RP, and a loss of RP vesicles mainly affects synaptic fatigue during high-frequency stimulation. Since ultrastructural data indicate the integrity of the ECP/RRP in stn-vl hypomorphs, it must be concluded that the reduction in average amplitude during low frequency stimulation cannot be simply due to a small vesicle depletion restricted to the RP. Rather, defective basal synaptic transmission must be caused by the reluctant fusion of existing vesicles, correlating with the loss of STNB (Mohrmann, 2008).
In order to fully characterize this potential adverse effect on exocytosis, synaptic response patterns evoked by different stimulation paradigms were analyzed. Strikingly, stn-vl mutants exhibited significantly less pronounced synaptic depression during short stimulus trains applied at different frequencies. Interestingly, altered depression was found only in those low STNB-expressing hypomorphs that also showed defective basal transmission, suggesting a linkage relationship between these phenotypes. A simplistic model of synaptic depression could be satisfied by a progressive depletion of fusion-ready synaptic vesicles during phases of increased activity. Using shibire mutants to study depression in the absence of compensating endocytosis, it has been demonstrated that short trains (5-20Hz) that selectively deplete the readily-releasable pool caused a depression profile whose shape is reminiscent of the de-staining kinetics of FM1-43 labeled RRP in presynaptic boutons. Hence, the initial phase of depression at NMJ synapses presumably reflects the depletion of the RRP. Based on this interpretation, the altered depression profile in STNB hypomorphs is most likely due to an alteration in mobilization and/or fusion-rate of RRP vesicles. Since abnormal response patterns are also observed for simple paired-pulse stimuli, a shortage of STNB might generally change release properties. It is widely accepted that basal release probability is a determinant factor for short-term plasticity. Indeed, the presence of a depletion-based mechanism of synaptic depression readily implies a dependence of depression kinetics on initial release probability. Therefore, the reduction in basal EJC amplitude and the slowing of depression kinetics represent concurring indicators of an underlying decrease of release probability caused by removal of STNB (Mohrmann, 2008).
Though STNB could in principle play a dual role by independently functioning in exocytosis and endocytosis, no evidence was found to support such hypothesis. In fact, stn-vl hypomorphs, which exhibited less pronounced synaptic depression, also demonstrated a delayed recovery after prolonged stimulation indicating an accompanying defect in vesicle recovery. More likely, STNB serves functions on two different levels during vesicle reconstitution: Apart from simply being an essential component constituting functional endocytic complexes, STNB potentially also acts on a governing stage ensuring proper recovery of fusion-competent vesicles. Since a compromised complement of synaptic proteins on recovered vesicles could readily account for the decreased release probability in stnB hypomorphs, assays were performed for possible alterations in presynaptic expression levels and localization of synaptic vesicle proteins. While the expression of all tested proteins was at least slightly reduced in stn-vl hypomorphs, a definite subset of integral vesicle proteins was clearly most affected by STNB depletion. Synaptobrevin and Synapsin expression levels were only slightly (≤30%) reduced, while the abundance of synaptotagmin and vGLUT were more severely decreased (≤50%). Synapsin adheres to available vesicular membranes in a dynamic fashion based on its phosphorylation state and presumably without obligatory endocytic sorting. Therefore, the reduction in synapsin levels likely represents a decrease in vesicle number within presynaptic terminals. Indeed, this conclusion is well supported by the comparable level of reduction in vesicle density observed at the ultrastructural level. However, the more pronounced effects on synaptotagmin and vGLUT cannot be attributed simply to a physical depletion of vesicles, suggesting a specifically reduced presence in vesicular membranes, consistent with trafficking defects (Mohrmann, 2008).
A mislocalization of synaptotagmin-1 was already reported in lethal stoned mutants, spawning discussions of a synaptotagmin-focused function of stoned proteins. New data shows that presynaptic expression of different vesicle proteins is differentially affected by reduced STNB levels, excluding the possibility that defective endocytosis causes an nonselective loss of vesicle proteins from synaptic boutons. The new data also suggest that STNB function might be important for the correct localization of a specified set of vesicle proteins, which questions whether the loss of synaptotagmin-1 alone is primarily responsible for the physiological phenotypes observed in stoned mutants. Based on the finding that stnB hypomorphs exhibit impaired neurotransmitter release, and accompanying alterations in vesicle protein configuration, it is considered most likely that STNB acts as a stabilizing and/or sorting factor for several synaptic vesicle proteins supporting this function. Similarly, a recent study (Diril, 2006) postulated that the mammalian STNB homolog stonin2 acts as an endocytic sorting adapter for synaptotagmin-1. Confusingly, however, STNB lacks several N-terminal WVxF motifs of its ortholog, which supposedly mediate a stonin2-AP2 interaction crucial for the reported stonin2-induced acceleration of synaptotagmin endocytosis. Nevertheless, association with other AP2-interacting proteins might also enable the recruitment of STNB to appropriate sites (Mohrmann, 2008).
Synaptotagmin sorting is predicted to critically depend on the ability to interact with the MHD of STNB. To examine the role of the MHD-synaptotagmin interaction, targeted mutations were generated to abolish this binding capability. Most interestingly, the complete removal of the MHD, and similarly the introduction of two point mutations within the putative binding interface for synaptotagmin, prevented the expressed STNB protein from restoring viability in lethal stoned mutants. Epitope-tagged mutant variant proteins could be neuronally expressed in wildtype animals, but completely failed to localize at synaptic sites. This suggests a crucial role for MHD-based interactions in presynaptic trafficking or anchorage of STNB. It is noteworthy that the STNB Y1125G, R1135A variant could still enter proximal parts of the axon, possibly indicating defective active transport of the protein. In contrast, the C-terminally truncated STNB variant is fully retained in discrete, punctate accumulations within the soma. Surprisingly, a similar, truncated variant of stonin2 seems to distribute uniformly in mammalian neurons (Walther, 2004). This might be due to higher expression levels, or different interactions of the remaining N-terminal portion of stonin2. A STNB-AP50 chimera, which contains corresponding sequences of AP50 instead of its original MHD, was also completely missing from synaptic sites, and exhibited punctate somatic localization similar to the truncated STNB variant. Thus, the MHD and the μ2-subunit are not functionally equivalent, even though the transplanted sequences contain the putative synaptotagmin-binding interface, and are predicted to confer the ability to bind synaptotagmin. Hence, STNB localization is not dependent on an association with synaptotagmin. This conclusion is independently confirmed by showing that synaptotagmin null mutants sytAD4 exhibit only relatively minor changes in STNB localization (Mohrmann, 2008).
Unlike the well-established SYT-STNB interaction, a direct binding activity between vGLUT and STNB has not been tested. Though the molecular mechanisms of STNB dependent vGLUT localization/stabilization are unclear, several recent studies in mammals report a direct interaction between its vertebrate homolog, VGLUT1, and endophilin, thereby establishing a connection to the endocytic protein network. It will be very interesting to examine the exact relationship between STNB and vGLUT in the future (Mohrmann, 2008).
The vesicle protein synaptotagmin I is the Ca(2+) sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca(2+) binding by the C(2)B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca(2+) binding results in vesicle fusion remains controversial. Ca(2+)-dependent interactions between synaptotagmin and SNARE complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C(2)B Ca(2+)-binding motif, suggesting that these interactions are facilitatory rather than essential. This study used Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca(2+)-binding pockets of synaptotagmin. Mutation of this residue in the C(2)A domain (F286) resulted in a ~50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C(2)B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in sytnull mutants, a response that more severe than the phenotype of C(2)B Ca(2+)-binding mutants. Thus, mutation of a single, C(2)B hydrophobic residue required for Ca(2+)-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. These results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C(2)B domain of synaptotagmin, is the critical effector interaction for coupling Ca(2+) binding with vesicle fusion (Paddock, 2011; full text of article).
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).
Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. This study, using adrenal chromaffin cells, identifies the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, led to a proposal that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes (de Wit, 2009).
These data identify two genes, Snap-25 and synaptotagmin-1, that, together with two previously characterized genes, munc18-1 and syntaxin-1, are required for docking of secretory vesicles. This study addressed the involvement of the syntaxin-1/SNAP-25 acceptor complex and found that two conditions that favor the formation of syntaxin-1/SNAP-25 acceptor complexes rescue the docking defects in munc18-1 null mutants: SNAP-25 overexpression and expression of truncated synaptobrevin. Furthermore, null mutations for SNAP-25 and the vesicular protein synaptotagmin-1 abolish docking, and SNAP-25 no longer rescues docking in synaptotagmin-1/munc18-1 double-null mutants. By using synaptotagmin-1 and SNAP-25 mutations that affect their interaction, both proteins were confirmed to act in concert for correct anchoring of secretory vesicles to fusion sites. Moreover, the rescue of docking, but not fusion, after expression of SNAP-25 or the synaptobrevin-2 C-terminal fragment on the munc18-1 null background indicates that Munc18-1 is not an essential constituent of the docking complex itself, but plays an essential downstream role. Together, the null mutation and (cross-) rescue experiments indicate that the corresponding four proteins work together to dock vesicles and at the same time suggest that Munc18-1 plays a unique, orchestrating role. While docking is established between syntaxin-1/SNAP-25 acceptor complexes at the target membrane and synaptotagmin-1 on the vesicle membrane, Munc18-1 promotes the formation or stability of the correct acceptor SNARE complexes (de Wit, 2009).
Munc18-1 can interact with both 'closed' and 'open' syntaxin-1, but it is unclear which binding mode is essential to perform its function in docking. Munc18-1 binding to 'open' syntaxin-1 involves an interaction with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. It has been shown that N-terminal interaction is not sufficient for docking, since a docking phenotype similar to syntaxin-1 and munc18-1 null was observed in chromaffin cells from knockin mice that express a mutant syntaxin-1 that only allows N-terminal interaction. In addition, when the well-characterized D34N/M38V double mutant of Munc18-1 that is known to perturb the interaction with 'closed' syntaxin was expressed, it was observed that docking was not restored in munc18-1 null chromaffin cells. Other studies have shown that Munc18-1 binding to 'open' syntaxin is essential to execute fusion. In the present study, docking and fusion phenotypes were experimentally separated in munc18-1 null chromaffin cells. The observations that SNAP-25 and SybCT overexpression, which both increase the number of syntaxin-1/SNAP-25 dimers, restore docking implies that Munc18-1 promotes the existence/stability of intermediate syntaxin-1/SNAP-25 dimers at the target membrane and therefore probably binds to these intermediate complexes. This increased number of acceptor complexes is not sufficient to restore fusion in the absence of Munc18-1, which firmly establishes a postdocking role for Munc18-1 in SNARE-dependent fusion. Currently, it is unclear whether Munc18-1's function downstream of docking requires either binding to intermediate syntaxin-1/SNAP-25 dimers alone or also binding to assembled SNARE complexes (containing synaptobrevin-2) to promote fusion as shown previously in vitro. In addition, these experiments with synaptotagmin-1 and SNAP-25 mutations, which have been shown to impair secretion, show that in the presence of Munc18-1 a correlation exists between mutations that impair secretion and those that impair docking. This is not the case in the absence of Munc18-1, emphasizing its postdocking role in SNARE-dependent fusion (de Wit, 2009).
This study identifies synaptotagmin-1 as a vesicular docking factor that binds to the assembled docking acceptor discussed above and has the capacity to anchor vesicles to the target membrane. This docking role of synaptotagmin-1 is consistent with previous findings in invertebrate synapses, which, however, have not been specifically interpreted in terms of docking because of additional phenotypes in these synapses: large effects on undocked vesicle populations near the active zone, which has been related to the increased mini rate observed in these mutant synapses, and/or impaired recruitment. Interestingly, a mutation used in the latter study is in an area of the molecule that was later identified to interact with SNAP-25(de Wit, 2009).
The docking role of synaptotagmin-1 proposed in this study does not conflict with its well-established role in fusion. However, while its role in fusion is strictly Ca2+ dependent, its role in docking is probably Ca2+ independent, since resting chromaffin cells have a strong docking phenotype in the absence of synaptotagmin-1 and its Ca2+ affinity is insufficient to be activated by resting Ca2+ levels in the cytosol. This is in line with a Ca2+-independent, upstream role previously suggested in rescue experiments in fly neuromuscular junction (Loewen, 2006). It is tempting to speculate that on top of this principally Ca2+-independent docking role, synaptotagmins may also contribute to the well-known but incompletely understood Ca2+-dependent acceleration of vesicle recruitment/docking/priming (de Wit, 2009).
Secretory systems typically express multiple synaptotagmins. In chromaffin cells, synaptotagmin-7 can partially compensate for the loss of synaptotagmin-1, but the secretion phenotype of the synaptotagmin-1 null cells is still drastic. In analogy, the docking phenotype in synaptotagmin-1 null cells is also drastic, but still slightly less severe than the munc18-1 null phenotype. This may be explained by a partial compensation by other synaptotagmins. The presence of multiple synaptotagmins, with different Ca2+ sensitivities and the new evidence that they are not only involved in fusion (and endocytosis), but also in docking, may require reinterpretation of previous studies on these proteins. Most studies assess upstream processes by measuring the final one (fusion) and thereby sample a composite measure of the combined effects of experimental manipulations on all upstream steps. For these combined effects to be dissected, new methodologies may be required to directly assess these upstream steps and to go beyond what current secretion assays have revealed about the complexity of the secretory pathway (de Wit, 2009).
Invertebrate synapses, docking phenotypes for Munc18-1, syntaxin-1, SNAP-25, and synaptotagmin-1 have not been described or are at least less evident. It is possible that these proteins are dispensable for synaptic vesicle docking and that distinct mechanisms dock vesicles in synapses. However, it seems more likely that docking principles are conserved among secretory systems. This idea is strongly supported by the fact that docking phenotypes have been observed in invertebrate synapses upon mutations in three of the four genes. However, these phenotypes are generally subtle and sometimes require advanced methodology and new docking definitions to become evident. In the case of synaptotagmin, invertebrate phenotypes are robust, but additional phenotypes were observed that prevented a specific interpretation in terms of docking. It is likely that docking phenotypes are less evident in vertebrate synapses either because of redundancy arising from the expression of multiple isoforms for some of the docking genes identified here or because structurally unrelated proteins that are not expressed in chromaffin cells restrict undocking of synaptic vesicles even when essential docking factors are not expressed. Finally, it is plausible that undocking and docking phenotypes are simply not as evident in the densely packed nerve terminal (de Wit, 2009).
With the currently identified four genes for docking and the link to SNARE complex assembly, a consistent (minimal) working model for the exocytotic pathway from the initial docking step until the final fusion reaction can now be synthesized for the first time, proposing the following four steps: First, Munc18-1 binds the closed conformation of syntaxin-1. Munc18-1 interacts with two epitopes in syntaxin-1, the Habc domain, and the N-terminal domain. Second, SNAP-25 binds the syntaxin-1/Munc18-1 heterodimer. Third, secretory vesicles reach the target membrane area and associate via synaptotagmin-1 to this trimeric syntaxin-1/Munc18-1/SNAP-25 complex, which effectuates docking. This binding requires the C2B domain of synaptotagmin-1, and recent studies suggest that Munc18-1's function here is to further help stabilize the syntaxin-1/SNAP-25 (1:1) acceptor complex for subsequent binding of synaptobrevin-2. In addition, since only vesicles docked in the presence of Munc18-1 are able to fuse, Munc18-1 might help restrict fusion to specific sites on the plasma membrane. By attaching the vesicle to the plasma membrane, the calcium sensor for exocytosis (synaptotagmin-1) has the additional function of localizing vesicles close to calcium channels. Fourth, synaptobrevin-2 then binds to the synaptotagmin-1/syntaxin-1/Munc18-1/SNAP-25 complex and the four helical SNARE bundle forms, which subsequently allows complexins to associate with the four helical SNARE bundle, and ultimately the vesicle fuses upon Ca2+ entry. It has been proposed that synaptobrevin-2 replaces Munc18-1, but, given the proposed fusion-promoting actions of Munc18-1 while associated to SNARE complexes, Munc18-1 may also continue to associate with the ternary SNARE complex until fusion is triggered (de Wit, 2009).
SNARE-mediated synaptic exocytosis is orchestrated by facilitatory and inhibitory mechanisms. Genetic ablations of Complexins (see Drosophila complexin), a family of SNARE-complex-binding proteins, in mice and Drosophila cause apparently opposite effects on neurotransmitter release, leading to contradictory hypotheses of Complexin function. Reconstitution experiments with different fusion assays and Complexins also yield conflicting results. Cross-species rescue experiments were therefore performed to compare the functions of murine and Drosophila Complexins in both mouse and fly synapses. It was found that murine and Drosophila Complexins employ conserved mechanisms to regulate exocytosis despite their strikingly different overall effects on neurotransmitter release. Both mouse and fly Complexins contain distinct domains that facilitate or inhibit synaptic vesicle fusion, and the strength of each facilitatory or inhibitory function differs significantly between them. These results show that a relative shift in the balance of facilitatory and inhibitory functions results in differential regulation of neurotransmitter release by murine and Drosophila Complexins in vivo, reconciling previous incompatible findings (Xue, 2009).
SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor)-mediated synaptic vesicle exocytosis is tightly controlled by a large number of regulatory proteins to ensure the exquisite temporal and spatial precision of neurotransmitter release at synapses. Complexins constitute a family of small and highly charged proteins that bind to the assembled SNARE complex. They generally contain a central α helix and an accessory α helix in the middle portion of the protein, and the N- and C-terminal sequences that are probably largely unstructured. Complexins bind to the SNARE complex with high affinity. The central α helix of Complexins interacts with the SNARE motifs of Syntaxin-1 and Synaptobrevin-2 within the SNARE complex in an antiparallel fashion. Complexins can also bind to the target-SNAREs' (Syntaxin-1 and SNAP-25) heterodimer with a lower affinity (Xue, 2009 and references therein).
Biophysical and physiological studies have indicated diverse functions for Complexins in vesicle fusion, some of which are incompatible. Complexins have been shown to inhibit SNARE-mediated cell fusion and proteoliposome fusion in bulk ensemble assay, and this inhibition is released by the Ca2+ sensor Synaptotagmin-1 and Ca2+. Biochemically, Synaptotagmin-1 competes with Complexins for the SNARE complex binding and displaces Complexins from the SNARE complex in a Ca2+-dependent manner. These studies suggest a fusion clamp model for Complexin function, in which Complexins inhibit the transfer of the force generated by the SNARE complex assembly onto the fusing membranes and arrest synaptic vesicle fusion before Ca2+ influx. Upon Ca2+ binding, Synaptotagmin-1 displaces Complexins from the SNARE complex to release this inhibition and triggers exocytosis. However, Complexins have also been shown to stimulate proteoliposome fusion in both single-vesicle fusion assay and bulk ensemble assay, indicating a facilitatory role. These in vitro results are further confounded by in vivo genetic studies. Genetic knockout of Complexins in mice leads to a reduction in both evoked and spontaneous release at multiple glutamatergic and GABAergic synapses in cultures and in acute brain slicesand a decrease in Ca2+-triggered exocytosis in adrenal chromaffin cells, supporting a stimulatory function for Complexins. In contrast, genetic deletion of Complexin in fruit fly Drosophila melanogaster greatly enhances spontaneous release but decreases Ca2+-evoked release, favoring the fusion clamp model. Moreover, knockdown of Complexins by RNA interference in mass-cultured mouse cortical neurons decreases evoked release and increases spontaneous release at glutamatergic synapses. To explain the discrepancy between this result and those obtained previously from Complexin knockout mice, the authors (Maximov, 2009) suggest that this is due to the different preparations used (autaptic cultures for knockout studies versus mass cultures for knockdown study, disavowing the fact that the knockout studies also employed mass cultures and acute brain slices, and similar results were found to those obtained from autaptic cultures. Hence, many studies seem at odds with each other and the precise in vivo role of Complexins in exocytosis is still unclear (Xue, 2009 and references therein).
An in vivo structure-function analysis of murine Complexin I (CplxI) in Complexin I/II double knockout mouse neurons indicates that the SNARE complex binding is essential for CplxI function, and that the N terminus of CplxI facilitates release, whereas an accessory α helix between the N terminus and the central α helix inhibits release. A biophysical study reveals that CplxI inhibits SNARE complex formation, but strongly stimulates membrane fusion after the assembly of the SNARE complex in vitro. These studies indicate that Complexins play both facilitatory and inhibitory roles in exocytosis, but they still do not explain why genetic deletions of Complexins in two model organisms, mouse and fly, have such different effects on neurotransmitter release. Furthermore, the amino acid sequence homology is low between murine and Drosophila Complexins except for the central α helix that is essential for the binding to the SNARE complex, and part of the N terminus. Thus, the dramatic difference in loss-of-function phenotypes of Complexin-deficient mice and flies leads to the conclusion that Complexin function must differ between mice and flies (Xue, 2009).
To test whether the functions of murine and Drosophila Complexins are conserved in synaptic vesicle exocytosis, and to gain insight into their functional and structural differences, it is essential to compare murine and Drosophila Complexins in the same experimental in vivo systems. A detailed structure-function analysis is also necessary because a complete removal of Complexins is unlikely to reveal all aspects of their function. Therefore a systematic cross-species rescue approach was undertaken to compare the functions of murine and Drosophila Complexins at both mouse and fly synapses. It was found that both murine and Drosophila Complexins contain distinct functional domains and play dual roles in neurotransmitter release. They facilitate and inhibit release via similar domains, but the facilitatory or inhibitory strength of a given domain varies between murine and Drosophila Complexins. Thus, both murine and Drosophila Complexins utilize conserved mechanisms in release process, but the integration of facilitation and inhibition differs substantially between them, leading to an apparently opposite overall effect on exocytosis. These results reveal conserved functions of Complexins between species and indicate that the interplay of dual functions orchestrates neurotransmitter release (Xue, 2009).
Synaptic exocytosis is exquisitely controlled by a set of facilitatory and inhibitory mechanisms, some of which are often executed by the very same protein. As a key regulator of the release machinery, Complexins play both facilitatory and inhibitory roles in vesicle fusion through distinct mechanisms. However, the remarkable phenotypic difference between mouse and fly Complexin null animals remained unexplained. This work compared the functions of murine and Drosophila Complexins in cross-species rescue experiments. The data establish that murine and Drosophila Complexins share a set of conserved mechanisms in synaptic vesicle fusion (Xue, 2009).
First, the SNARE complex binding mediated by the central α helix (residues 48-70 for CplxI and 54-76 for dmCplx) is essential for Complexin function. Mutations that diminish the interaction between the central α helix and the SNARE complex abolish the functions of both CplxI and dmCplx, indicating that the actions of other domains all depend on this high-affinity interaction. The binding of the central α helix not only can stabilize the assembled SNARE complex, but perhaps more importantly, can strategically position the accessory α helix and the N terminus for their actions (Xue, 2009 and references therein).
Second, the accessory α helix (approximately residues 29-47 for CplxI and 33-53 for dmCplx) located between the N terminus and the central α helix inhibits vesicle fusion. It was proposed that the inhibitory action of the accessory α helix might arise from its interference with the binding of Synaptobrevin-2 to Syntaxin-1 and SNAP-25 heterodimer, which would consequently prevent the complete zippering of the SNARE complex. This model has recently been supported by the findings that Complexins can bind to Syntaxin-1 and SNAP-25 heterodimer in vitro and may form an alternative four-helix bundle with target-SNAREs to inhibit fusion in a reconstituted fusion system (Xue, 2009 and references therein).
Third, the N termini (residues 1-16) of both CplxI and dmCplx promote release. It has been speculated that the CplxI N terminus may interact with lipid membranes, but so far, there are no supporting biochemical data. Instead, this facilitatory effect is likely mediated by a direct interaction of the Complexin N terminus with the SNARE complex C terminus. Mutations of methionine 5 and lysine 6 of CplxI disrupt the binding of the CplxI N terminus to the SNARE complex C terminus and abolish the facilitatory activity of the N terminus. Interestingly, methionine 5 is not conserved in dmCplx and an alanine residue is at position 6 (corresponding to residue 5 of CplxI). It is possible that a methionine is not absolutely required for dmCplx and other residues may compensate for the interaction with the SNARE complex C terminus (Xue, 2009).
Furthermore, at fly neuromuscular junctions, both murine and Drosophila Complexins promote Ca2+-triggered release and suppress spontaneous release, but to very different degrees. Neuronal expression of murine or Drosophila Complexins rescues the lethality and sterility of Complexin null mutant flies, showing again that murine and Drosophila Complexins share conserved functions (Xue, 2009).
Therefore, these cross-species rescue experiments show that murine and Drosophila Complexins have both facilitatory and inhibitory functions associated with similar protein domains in synaptic vesicle exocytosis. It is proposed that the Complexin central α helix binds to the middle portion of the SNARE complex, stabilizing the SNARE complex and positioning the accessory α helix and the N terminus. The accessory α helix replaces the C terminus of the Synaptobrevin-2 SNARE motif in the four-helix bundle, preventing the full assembly of the SNARE complex to suppress fusion. The N terminus directly interacts with the C-terminal portion of the SNARE complex, likely stabilizing this unstable region of the SNARE complex to promote membrane fusion. However, the relative strengths of these functions are remarkably different between murine and Drosophila Complexins. It is proposed that the integration of facilitation and inhibition, which are associated with distinct domains, determines the overall effect of murine and Drosophila Complexins on neurotransmitter release in a given synapse. The overall action of murine and Drosophila Complexins is unlikely to be a linearly additive effect of all facilitatory and inhibitory actions. However, it is clear that the facilitatory function is preponderant in murine Complexins, whereas the inhibitory functions of the accessory α helix and the C terminus predominate in Drosophila Complexin. Thus, a relative shift in the balance of facilitatory and inhibitory functions results in differential roles of murine and Drosophila Complexins in neurotransmitter release, and leads to apparently very different loss-of-function phenotypes in flies and mice. These results emphasize the functional similarities and differences between murine and Drosophila Complexins, and reconcile previous contradictory hypotheses of Complexin in vivo function. Moreover, the data illustrate the complexity of Complexin function and strongly support the notion that Complexins play dual roles in vesicle fusion (Xue, 2009).
This model is clearly different from the previous models of Complexins based on the fusion clamp hypothesis. These models propose that Complexins arrest primed synaptic vesicles at a hemifused and metastable state, which provides the substrate for Ca2+-bound Synaptotagmin-1 to release the clamping function of Complexins, allowing the fast and synchronous fusion. The lack of Complexins and therefore the lack of metastable vesicles for Synaptotagmin-1 action causes excessive spontaneous release and deficient Ca2+-triggered fast release. However, the current in vivo results speak against this model because murine Complexins do not completely clamp the excessive spontaneous release in Drosophila Complexin null mutants, yet they actually enhance Ca2+-evoked fast release even better than Drosophila Complexin. This observation indicates that the decreased Ca2+-evoked fast release in Drosophila Complexin null mutants is not functionally coupled to the increased spontaneous release frequency. Could it be that the reduced evoked release in Drosophila Complexin null mutants is due to a partial depletion of readily releasable vesicles by the high-frequency spontaneous release? This is unlikely because the vesicle recruitment rate is usually at least 100-fold higher than the spontaneous release rate at resting intracellular Ca2+ level, and therefore a 20- to 30-fold increase in spontaneous release rate should not significantly change the vesicle pool size in Drosophila Complexin null mutants. In addition, murine-Complexin-rescued Drosophila Complexin null synapses still exhibit strongly increased spontaneous release, yet the evoked release is even larger than that of WT synapses, arguing that high-frequency spontaneous release in null mutants is unlikely to exhaust vesicles, causing a decreased evoked release (Xue, 2009).
A recent fusion clamp model proposes that Complexins control the force transfer from the SNARE complex to the membranes and assist the SNAREs in exerting force on the membranes (Maximov, 2009). This model assumes that Complexins are released from the SNARE complex by Synaptotagmin-1 and Ca2+, but it is physically unclear how Complexins can help SNAREs exert force on the membranes if they are dissociated upon Ca2+ influx. In contrast, the current model requires Complexins to remain bound to the SNARE complex upon Ca2+ influx and is consistent with the notion that Complexins could function independently from Synaptotagmin-1 (Xue, 2009).
Drosophila Complexin in Cplx-TKO neurons abolishes both evoked and spontaneous release without altering the number of fusion-competent vesicles measured by hypertonic sucrose solution. This effect is intriguing, because very few molecular manipulations specifically block the synaptic vesicle cycle at the final fusion step. Drosophila Complexin does not change the number of primed vesicles, indicating that the initial formation of the SNARE complex is not affected by Drosophila Complexin. The inhibitory effect of Drosophila Complexin requires its binding to the SNARE complex. Hence, it is hypothesized that when the Drosophila Complexin central α helix binds to the partially assembled SNARE complex, the accessory α helix together with the C terminus prevents the further assembly of the SNARE complex C terminus, thereby arresting vesicles at the primed state. It is currently unknown how, mechanistically, the C terminus of Drosophila Complexin inhibits release. One possibility is that the C terminus may fold back toward the N-terminal direction and cooperate with the accessory α helix to inhibit vesicle fusion (Xue, 2009).
The phenotypic differences between fly and mouse knockouts seem dramatic, but it is worth noting that an increase of just 1.4 kcal/mol in the strength of a protein-protein interaction, which can arise simply from the formation of one hydrogen bond or salt bridge, leads to a 10-fold increase in affinity according to the Boltzmann equation. Hence, subtle changes in the molecular interactions of murine and Drosophila Complexins can suffice to tip the balance between facilitatory and inhibitory strengths. For example, protein sequence alignments show that the lengths and the amino acid compositions of the accessory α helices differ among different Complexins, which may cause different interactions of the accessory α helix with Syntaxin-1 and SNAP-25 heterodimer, thus changing its inhibitory strength (Xue, 2009).
The effects of murine Complexins in murine and fly synapses are not identical, as murine Complexins promote evoked release and inhibit spontaneous release in fly neuromuscular junctions, and promotes both types of release in mouse central synapses. Likewise, the effects of Drosophila Complexin in murine and fly synapses are not identical either, as it strongly inhibits spontaneous release and mildly promotes evoked release in fly neuromuscular junctions, and strongly inhibits both types of release in mouse synapses. These observations indicate that in addition to the Complexin-intrinsic properties, the molecular differences between species or synapses could differentially affect the facilitatory and inhibitory functions of murine and Drosophila Complexins, thereby tilting the facilitation and inhibition balance and contributing to the phenotypic differences (Xue, 2009).
Complexins represent a family of proteins that maintain a highly conserved core of sequences and at the same time display great diversity across paralogs and orthologs. This is likely reflected in their functions, namely conserved facilitatory and inhibitory mechanisms with varying strengths in neurotransmitter release. It will be interesting to test Complexin function in some other model organisms along the phylogenetic tree, such as worm and fish, to determine if and how the balance between facilitatory and inhibitory functions of Complexins has changed during evolution. At different synapses, the strengths of facilitation and inhibition of Complexins may be differentially regulated in a paralog- and ortholog-dependent fashion, thereby regulating release in a synapse-specific manner, and contributing to synaptic diversity and specificity. Furthermore, the ability of Drosophila Complexin to inhibit neurotransmitter release in mammalian neurons potentially provides a powerful tool to manipulate synaptic function to study neural circuits, as one should be able to express Drosophila Complexin to inhibit or even abolish synaptic transmission in a spatially and temporally specific manner (Xue, 2009).
Neurotransmitter release following synaptic vesicle (SV) fusion is the fundamental mechanism for neuronal communication. Synaptic exocytosis is a specialized form of intercellular communication that shares a common SNARE-mediated fusion mechanism with other membrane trafficking pathways. The regulation of synaptic vesicle fusion kinetics and short-term plasticity is critical for rapid encoding and transmission of signals across synapses. Several families of SNARE-binding proteins have evolved to regulate synaptic exocytosis, including Synaptotagmin (Syt) and Complexin (Cpx). This study demonstrates that Drosophila Cpx controls evoked fusion occurring via the synchronous and asynchronous pathways. cpx-/- mutants show increased asynchronous release, while Cpx overexpression largely eliminates the asynchronous component of fusion. It was also found that Syt and Cpx coregulate the kinetics and Ca(2+) co-operativity of neurotransmitter release. Cpx functions as a positive regulator of release in part by coupling the Ca(2+) sensor Syt to the fusion machinery and synchronizing its activity to speed fusion. In contrast, syt-/-; cpx-/- double mutants completely abolish the enhanced spontaneous release observe in cpx-/- mutants alone, indicating Cpx acts as a fusion clamp to block premature exocytosis in part by preventing inappropriate activation of the SNARE machinery by Syt. Cpx levels also control the size of synaptic vesicle pools, including the immediate releasable pool and the ready releasable pool-key elements of short-term plasticity that define the ability of synapses to sustain responses during burst firing. These observations indicate Cpx regulates both spontaneous and evoked fusion by modulating the timing and properties of Syt activation during the synaptic vesicle cycle (Jorquera, 2012).
The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the micro-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. This study report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the micro-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles (Soekmadji, 2012).
This study has investigated the effect of Ca2+ upon Stoned proteins binding to SYT-1 C2B domain. Previous study has show the Drosophila μHD of STNB binds to C2B domain of SYT-1, which is in agreement with a study that showed the μHD of the stonin-2, the mammalian homologue of the Drosophila STNB protein, could also bind SYT-1. It has also been reported that stonin-2 is able to bind the C2A domain, even though the role of Ca2+ in this binding was not explored. This study also observed that STNB is able to bind to the C2A domain in vitro, albeit at a much lower level as compared to the binding to the C2B domain. The data show that the binding of Drosophila STNB to Drosophila SYT-1 is different from that of the murine μ2 subunit of AP-2. While μ2 binding to C2B domain requires phosphatidylserine (PS) and Ca2+, the binding of the STNA and STNB proteins did not require PS. In fact PS-containing phospholipids nearly abolished the STNB binding to SYT-1 C2B. Deletion of the polyK region of the C2B domain also did not abolished STNB binding to SYT-1, indicating that this region cannot be the binding site for either STNA or STNB. The data support a recent publication that showed that, in Drosophila, μ2 cannot replace the function of the μHD of STNB in vivo, and suggest that STNB and AP-2 might represent alternative mechanisms for synaptic vesicle recycling (Soekmadji, 2012).
AP-2 is ubiquitously expressed and implicated in general mechanisms of endocytosis from the plasma membrane, while in Drosophila, STNB is expressed and functions only in the nervous system. Differences in the μHD of STNB and the μ2 domain of AP-2 may reflect the need for flexibility of μ2 subunit of AP-2 to act in a number of endocytic pathways, while the function of STNB may be specific for synaptic vesicle retrieval. The data, in conjunction with a report that STNB may specifically regulate the sorting of a subset of SV, suggest the Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound SYT-1 associated SV (Soekmadji, 2012).
Upon Ca2+ binding, SYT-1 was reported to undergo a conformational change that protects SYT-1 against trypsin and chymotrypsin digestion in vitro. Ca2+ has also promoted homo-oligomerization of SYT-1 and/or hetero-oligomerization with other synaptotagmins. Studies using mutations that affect Ca2+ dependent oligomerization, such as Y311N, showed a partial inhibition for internalization of SYT-1 in PC12 cells while the corresponding mutation in Drosophila (AD3) alters the rate of exocytosis. The AD3 SYT-1 is still capable of binding to SNARE complexes, synprint and AP-2, which implies that the mutation does not cause a complete loss of function. Hence the docked vesicles in AD3 flies (a mutation that affects Ca2+ dependent oligomerization) require higher Ca2+ concentration to undergo exocytosis, suggesting that defects in Ca2+ dependent oligomerization renders C2B SYT-1 inefficient for exocytosis. It was reported that Ca2+-triggered SYT-1 clustering is via the C2B domain and is required for exocytosis. In a potential endocytic model that includes STNB, the oligomerized SYT-1 that has led to exocytosis, could then be a target for STNB binding, and STNB, in turn, could recruit dynamin via the intersectin DAP-160. This would create what amounts to a Ca2+-dependent endocytic complex. However, a previous study has shown that STNB can be found bound to synaptic vesicles, via SYT-1, prior to exocytosis, that is, prior to the Ca2+ influx that might trigger SYT-1 oligomerization and the coupling of excitation and vesicle fusion. Either this bound STNB reflects the low level of oligomerization of SYT-1 in the absence of Ca2+, or that other factors, such as those that might alter the structure of the C-terminal region of SYT-I C2B, are playing a role in potentiating the binding of STNB to SYT-1 even in the absence of Ca2+. Certainly there is no STNB protein in the soluble fraction from fly head extracts, suggesting that all STNB is in a bound form, and although some is certainly is, perhaps not all is bound to SYT-1 (Soekmadji, 2012).
The D3,4N mutation in transgenic flies results in a reduction in the rate of endocytosis of synaptic vesicles, this may be due to a failure of this mutant SYT-I to interact with Stoned proteins. Another mutation which gives constitutive dimerization, the D3,4N mutation, was incapable to restore internalization in CHO cells. The D3,4N mutation did not only show a reduction in the rate of endocytosis in Drosophila, but also exocytosis defect by decrease evoked transmitter release and reduce in apparent Ca2+ affinity for synaptic transmission. Indeed, similar to these synaptotagmin mutant flies, the stoned mutants showed alterations in both spontaneous and evoked release at larval NMJ and severe neurotransmission defect that may lead to embryonic lethality, as well as depletion of synaptic vesicle and increase of membrane recycling intermediate that might be due to mislocalization of synaptotagmin during endocytosis. In contrast with μ2 and SYT-1 interaction, the binding of Stoned proteins do not require phospholipids. Studies using Folch liposome has shown that D3,4N mutant are not able to induce a close proximity membrane curvature, which may be a prerequisite for SNARE mediated membrane fusion. A synthesized peptide consist of the 3rd loop of C2B SYT-1 can outcompete the μ-homology domain of STNB, suggesting the interaction of STNB to SYT-1 is mediated by a region in loop 3 of C2B domain. Thus, it is an attractive hypothesis that STNB may act as an inhibitor for membrane fusion. In a recent paper, it is shown that SYT-1 bound to PtdIns at the same level as PS and that this binding is also required Ca2+; while the binding of PIP2 with SYT-1 is less affected by Ca2+. It would be interesting to investigate whether these lipids will have similar effect as PS in affecting Stoned and C2B SYT-1 binding (Soekmadji, 2012).
The role of STNA in the synaptic vesicle cycle remains elusive. The presence of STNA at the larval NMJ appears nonessential. However STNA is certainly associated with synaptic vesicles and it is clear that STNA has a strong affinity for SYT-1. This study has shown that the oligomerization of SYT-1 dramatically increases the binding of STNA and presumably STNA will bind under similar in vivo conditions as STNB. The presence of putative AP-2 binding motifs in STNA, may make vesicles bound by STNA targets for AP-2 mediated endocytosis. This is in contrast to STNB that lacks such AP-2 binding motifs. Whatever the specific mechanism of action of the STNA protein, it is clear from this study that the action of Ca2+ has a marked effect on STNA and STNB association with SYT-1 and confirms them as important proteins in the mechanism(s) of synaptic vesicle recycling in Drosophila (Soekmadji, 2012).
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