InteractiveFly: GeneBrief

complexin: Biological Overview | References


Gene name - complexin

Synonyms -

Cytological map position - 82A1-82A3

Function - signaling

Keywords - CNS, SNARE-mediated synaptic exocytosis

Symbol - cpx

FlyBase ID: FBgn0041605

Genetic map position - 3R:105,906..128,309 [+]

Classification - Synaphin superfamily

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Mahoney, R. E., Azpurua, J. and Eaton, B. A. (2016). Insulin signaling controls neurotransmission via the 4eBP-dependent modification of the exocytotic machinery. Elife 5. PubMed ID: 27525480
Summary:
Altered insulin signaling has been linked to widespread nervous system dysfunction including cognitive dysfunction, neuropathy and susceptibility to neurodegenerative disease. However, knowledge of the cellular mechanisms underlying the effects of insulin on neuronal function is incomplete. This study shows that cell autonomous insulin signaling within the Drosophila CM9 motor neuron regulates the release of neurotransmitter via alteration of the synaptic vesicle fusion machinery. This effect of insulin utilizes the FOXO-dependent regulation of the thor gene, which encodes the Drosophila homologue of the eif-4e binding protein (4eBP). A critical target of this regulatory mechanism is Complexin, a synaptic protein known to regulate synaptic vesicle exocytosis. The amounts of Complexin protein observed at the synapse was found to be regulated by insulin, and genetic manipulations of Complexin levels support the model that increased synaptic Complexin reduces neurotransmission in response to insulin signaling.
Vasin, A., Volfson, D., Littleton, J. T. and Bykhovskaia, M. (2016). Interaction of the Complexin accessory helix with Synaptobrevin regulates spontaneous fusion. Biophys J 111: 1954-1964. PubMed ID: 27806277
Summary:
Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the plasma membrane. Vesicles attach to membranes via a specialized protein machinery composed of membrane-attached (t-SNARE; Syntaxin 1A) and vesicle-attached (v-SNARE; n-synaptobrevin) proteins that zipper together to form a coiled-coil SNARE bundle that brings the two fusing membranes into close proximity. Neurotransmitter release may occur either in response to an action potential or through spontaneous fusion. A cytosolic protein, Complexin (Cpx), binds the SNARE complex and restricts spontaneous exocytosis by acting as a fusion clamp. A model has been proposed in which the interaction between Cpx and the v-SNARE serves as a spring to prevent premature zippering of the SNARE complex, thereby reducing the likelihood of fusion. To test this model, molecular-dynamics (MD) simulations and site-directed mutagenesis of Cpx and SNAREs were combined in Drosophila. MD simulations of the Drosophila Cpx-SNARE complex demonstrated that Cpx's interaction with the v-SNARE promotes unraveling of the v-SNARE off the core SNARE bundle. Clamping properties were investigated in the syx3-69 paralytic mutant, which has a single-point mutation in the t-SNARE and displays enhanced spontaneous release. MD simulations demonstrated an altered interaction of Cpx with the SNARE bundle that hindered v-SNARE unraveling by Cpx, thus compromising clamping. This mode was used to predict mutations that should enhance the ability of Cpx to prevent full assembly of the SNARE complex. Transgenic Drosophila were generated with mutations in Cpx and the v-SNARE that disrupted a salt bridge between these two proteins. As predicted, both lines demonstrated a selective inhibition in spontaneous release, suggesting that Cpx acts as a fusion clamp that restricts full SNARE zippering.
Sabeva, N., Cho, R. W., Vasin, A., Gonzalez, A., Littleton, J. T. and Bykhovskaia, M. (2016). Complexin mutants reveal partial segregation between recycling pathways that drive evoked and spontaneous neurotransmission. J Neurosci [Epub ahead of print]. PubMed ID: 27913592
Summary:
Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. Synaptic vesicle recycling pathways were examined at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. Loading of the lipophilic dye FM1-43 was combined with photoconversion, electron microscopy (EM), and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. The total number of recycling vesicles was found to be equal to those retrieved through spontaneous and evoked pools, suggesting retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with EM analysis indicated spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, while vesicles recycled following evoked release involve a larger intra-terminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion.
Luo, W., Guo, F., McMahon, A., Couvertier, S., Jin, H., Diaz, M., Fieldsend, A., Weerapana, E. and Rosbash, M. (2018). NonA and CPX link the circadian clockwork to locomotor activity in Drosophila. Neuron 99(4): 768-780.e763. PubMed ID: 30057203
Summary:
Drosophila NonA and its mammalian ortholog NONO are members of the Drosophila behavior and human splicing (DBHS) family. NONO also has a strong circadian connection: it associates with the circadian repressor protein Period (Per) and contributes to circadian timekeeping. This study investigated NonA, which is required for proper levels of evening locomotor activity as well as a normal free-running period in Drosophila. NonA is associated with the positive transcription factor Clock/Cycle (Clk/Cyc), interacts directly with complexin (cpx) pre-mRNA, and upregulates gene expression, including the gene cpx. Downregulation of cpx expression in circadian neurons phenocopies NonA downregulation, whereas cpx overexpression rescues the nonA RNAi phenotypes, indicating that cpx is an important NonA target gene. As the cpx protein contributes to proper neurotransmitter and neuropeptide release in response to calcium, these results and others indicate that this control is important for the normal circadian regulation of locomotor activity.

BIOLOGICAL OVERVIEW

SNARE-mediated synaptic exocytosis is orchestrated by facilitatory and inhibitory mechanisms. Genetic ablations of Complexins, 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 (Ishizuka, 1995; McMahon, 1995; Reim, 2005; Takahashi, 1995). 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 (Pabst, 2000). Complexins bind to the SNARE complex with high affinity (Bowen, 2005; Pabst, 2002). The central α helix of Complexins interacts with the SNARE motifs of Syntaxin-1 (see Drosophila Syntaxin) and Synaptobrevin-2 within the SNARE complex in an antiparallel fashion (Bracher, 2002; Chen, 2002). Complexins can also bind to the target-SNAREs' (Syntaxin-1 and SNAP-25) heterodimer with a lower affinity (Guan, 2008; Weninger, 2008; Yoon, 2008; Xue, 2009 and references therein).

Biophysical and physiological studies have indicated diverse functions for Complexins in vesicle fusion, some of which are incompatible (Brose, 2008). Complexins have been shown to inhibit SNARE-mediated cell fusion (Giraudo, 2006) and proteoliposome fusion in bulk ensemble assay (Schaub, 2006), and this inhibition is released by the Ca2+ sensor Synaptotagmin-1 (see Drosophila Synaptotagmin) 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 (Tang, 2006). 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 (Giraudo, 2006; Maximov, 2009; Schaub, 2006; Tang, 2006). However, Complexins have also been shown to stimulate proteoliposome fusion in both single-vesicle fusion assay (Yoon, 2008) and bulk ensemble assay (Malsam, 2009), 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 slices (Reim, 2001; Strenzke, 2009; Xue, 2008), and a decrease in Ca2+-triggered exocytosis in adrenal chromaffin cells (Cai, 2008), 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 (Huntwork, 2007), 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 (Maximov, 2009). 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 [Reim, 2001; Xue, 2008) versus mass cultures for knockdown study (Maximov, 2009), disavowing the fact that the knockout studies also employed mass cultures and acute brain slices (Xue, 2008), 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).

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 (Xue, 2007). A biophysical study reveals that CplxI inhibits SNARE complex formation, but strongly stimulates membrane fusion after the assembly of the SNARE complex in vitro (Yoon, 2008). 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 (Xue, 2007). 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 (Rizo, 2008). As a key regulator of the release machinery, Complexins play both facilitatory and inhibitory roles in vesicle fusion through distinct mechanisms (Xue, 2007; Yoon, 2008). 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 (Xue, 2007) 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 (Chen, 2002), but perhaps more importantly, can strategically position the accessory α helix and the N terminus for their actions (Xue, 2007; 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 (Xue, 2007). This model has recently been supported by the findings that Complexins can bind to Syntaxin-1 and SNAP-25 heterodimer in vitro (Guan, 2008; Weninger, 2008; Yoon, 2008) and may form an alternative four-helix bundle with target-SNAREs to inhibit fusion in a reconstituted fusion system (Giraudo, 2009; 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 (Maximov, 2009; Xue, 2007), 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 (Chen, 2002; Xue, 2007). 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 (Giraudo, 2009; Xue, 2007). 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 (Cai, 2008; Huntwork, 2007; Reim, 2001; Xue, 2008). 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 (Giraudo, 2006; Maximov, 2009; Schaub, 2006; Tang, 2006). 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 (Huntwork, 2007; Reim, 2005). 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).

Phosphorylation of Complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity

Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca(2+) entry. This study shows that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity (Cho, 2015).

These findings indicate that the SNARE-binding protein Cpx is a key PKA target that regulates spontaneous fusion rates and presynaptic plasticity at Drosophila NMJs. Cpx's function can be modified to regulate activity-dependent functional and structural plasticity. In vivo experiments using Cpx phosphomimetic rescues demonstrate that Cpx phosphorylation at residue S126 selectively alters its ability to act as a synaptic vesicle fusion clamp. In addition, S126 is critical for the expression of HFMR, an activity-dependent form of acute functional plasticity that modulates mini frequency at Drosophila synapses. These data indicate a Syt 4-dependent retrograde signaling pathway converges on Cpx to regulate its synaptic function. Additionally, it was found that elevated spontaneous fusion rates correlate with enhanced synaptic growth. This pathway requires Syt 4 retrograde signaling to enhance spontaneous release and to trigger synaptic growth. Moreover, the Cpx S126 PKA phosphorylation site is required for activity-dependent synaptic growth, suggesting acute regulation of minis via Cpx phosphorylation is likely to contribute to structural synaptic plasticity. Together, these data identify a novel mechanism of acute synaptic plasticity that impinges directly on the presynaptic release machinery to regulate spontaneous release rates and synaptic maturation (Cho, 2015).

How does acute phosphorylation of S126 alter Cpx's function? The Cpx C-terminus associates with lipid membranes through a prenylation domain (CAAX motif) and/or the presence of an amphipathic helix. The Drosophila Cpx isoform used in this study (Cpx 7B) lacks a CAAX-motif, but contains a C-terminal amphipathic helix flanked by the S126 phosphorylation site. S126 phosphorylation does not alter synaptic targeting of Cpx or its ability to associate with SNARE complexes in vitro. As such, phosphorylation may instead alter interactions of the amphipathic helix region with lipid membranes and/or alter Cpx interactions with other proteins that modulate synaptic release. Given the well-established role of the Cpx C-terminus in regulating membrane binding and synaptic vesicle tethering of Cpx, phosphorylation at this site would be predicted to alter the subcellular localization of the protein and its accessibility to the SNARE complex. However, no large differences between WT Cpx and CpxS126D were observed in liposome binding. This assay is unlikely to reveal subtle changes in lipid interactions by Cpx, as this study found that C-terminal deletions (CTD) maintained its ability to bind membranes. The ability of the CTD versions of Drosophila Cpx to associate with liposomes is unlike that observed with C. elegans Cpx, and indicate domains outside of Drosophila Cpx's C-terminus contribute to lipid membrane association as well, potentially masking effects from S126 phosphorylation that might occur in vivo. Alternatively, phosphorylation of the Cpx C-terminus could alter its association with other SNARE complex modulators such as Syt 1 (Cho, 2015).

The data indicate that enhanced minis regulate synaptic growth through several previously identified NMJ maturation pathways. The Wit signaling pathway is required for synaptic growth in the background of enhanced minis. The wit gene encodes a presynaptic type II BMP receptor that receives retrograde, transsynaptic BMP signals from postsynaptic muscles. Consistent with these data, other studies have demonstrated that downstream signaling components of the BMP pathway are necessary and sufficient for mini-dependent synaptic growth at the Drosophila NMJ. Additionally, it was found that the postsynaptic Ca++ sensor, Syt 4, is required for enhancing spontaneous release and increasing synaptic growth. The data does not currently distinguish the interdependence of the BMP and Syt 4 retrograde signaling pathways, and other retrograde signaling pathways might contribute to mini-dependent synaptic growth as well. Recently, several retrograde pathways have been identified that regulate functional homeostatic plasticity at the Drosophila NMJ. Future work will be required to fully define the retrograde signaling pathways necessary to mediate mini-dependent synaptic growth (Cho, 2015).

How might elevated spontaneous release through Cpx phosphorylation regulate synaptic growth? It is hypothesized that the switch in synaptic vesicle release mode to a constitutive fusion pathway that occurs over several minutes following stimulation serves as a synaptic tagging mechanism. By continuing to activate postsynaptic glutamate receptors in the absence of incoming action potentials, the elevation in mini frequency would serve to enhance postsynaptic calcium levels by prolonging glutamate receptor stimulation. This would prolong retrograde signaling that initiates downstream cascades to directly alter cytoskeletal architecture required for synaptic bouton budding. Given that elevated rates of spontaneous fusion still occur in cpx and syx3-69 in the absence of Syt 4 and BMP signaling, yet synaptic overgrowth is suppressed in these conditions, it is unlikely that spontaneous fusion itself directly drives synaptic growth. Rather, the transient enhancement in spontaneous release may serve to prolong postsynaptic calcium signals that engage distinct effectors for structural remodeling that fail to be activated in the absence of elevated spontaneous release. Results from mammalian studies indicate spontaneous release can uniquely regulate postsynaptic protein translation and activate distinct populations of NMDA receptors compared to evoked release, so it is possible that spontaneous fusion may engage unique postsynaptic effectors at Drosophila NMJs as well (Cho, 2015).

In the last few decades, intense research efforts have elucidated several molecular mechanisms of classic Hebbian forms of synaptic plasticity that include long-term potentiation (LTP) and long-term depression (LTD), alterations in synaptic function that lasts last minutes to hours. The most widely studied expression mechanism for these forms of synaptic plasticity involve modulation of postsynaptic AMPA-type glutamate receptor (AMPAR) function and membrane trafficking. In contrast, molecular mechanisms of short-term synaptic plasticity remain poorly understood. Several forms of short-term plasticity have been described, such as post-tetanic potentiation (PTP), which involves stimulation-dependent increases in synaptic strength, including changes in mini frequency. Short-term plasticity expression is likely to impinge on the alterations to the presynaptic release machinery downstream of activated effector molecules. For example, Munc 18, a presynaptic protein involved in the priming step of vesicle exocytosis via its ability to associate with members of the SNARE machinery, is dynamically regulated by Ca++-dependent protein kinase C (PKC), and its regulation is required to express PTP at the Calyx of Held. This study demonstrates that the presynaptic vesicle fusion machinery can also be directly modified to alter spontaneous neurotransmission via activity-dependent modification of Cpx function by PKA. Protein kinase CK2 and PKC phosphorylation sites within the C-terminus of mammalian and C. elegans Cpx have been identified. Therefore, activity-dependent regulation of Cpx function via C-terminal phosphorylation may be an evolutionarily conserved mechanism to regulate synaptic plasticity. Moreover, Cpx may lie downstream of multiple effector pathways to modulate various forms of short-term plasticity, including PTP, in a synapse-specific manner. Interestingly, Cpx is expressed both pre- and postsynaptically in mammalian hippocampal neurons and is required to express LTP via regulation of AMPAR delivery to the synapse, suggesting Cpx-mediated synaptic plasticity expression mechanisms may also occur postsynaptically (ACho, 2015 and references therein).

In summary, these results indicate minis serve as an independent and regulated neuronal signaling pathway that contributes to activity-dependent synaptic growth. Previous studies found Cpx’s function as a facilitator and clamp for synaptic vesicle fusion is genetically separable, demonstrating distinct molecular mechanisms regulate evoked and spontaneous release. Evoked and spontaneous release are also separable beyond Cpx regulation, as other studies have demonstrated that minis can utilize distinct components of the SNARE machinery, distinct vesicle pools, and distinct individual synaptic release sites . These findings suggest diverse regulatory mechanisms for spontaneous release that might be selectively modulated at specific synapses (Cho, 2015).

Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability

Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. This study interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. NO was found to compromise synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, these data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release (Robinson, 2018).

Throughout the central nervous system (CNS), the volume transmitter nitric oxide (NO) has been implicated in controlling synaptic function by multiple mechanisms, including modulation of transmitter release, plasticity, or neuronal excitability. NO-mediated posttranslational modifications (PTMs) in particular have become increasingly recognized as regulators of specific target proteins. S-nitrosylation is a nonenzymatic and reversible PTM resulting in the addition of a NO group to a cysteine (Cys) thiol/sulfhydryl group, leading to the generation of S-nitrosothiols (SNOs). In spite of the large number of SNO-proteins thus far identified, the functional outcomes and mechanisms of the underlying specificity of S-nitrosylation in terms of target proteins and Cys residues within these proteins are not clear (Robinson, 2018).

Synaptic transmitter release is controlled by multiple signaling proteins and involves a cascade of signaling steps. This process requires the assembly of the soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) complex and associated proteins, the majority of which can be regulated to modulate synapse function. Regulatory mechanisms include phosphorylation of SNARE proteins as well as SNARE-binding proteins such as Complexin (Cpx), which have been reported at different synapses such as the Drosophila neuromuscular junction (NMJ) or in the rat CNS (Robinson, 2018).

Several contrasting effects on transmitter release are induced by NO-mediated PTMs. Other forms of protein modification to modulate cellular signaling include prenylation, an attachment of a farnesyl or geranyl-geranyl moiety to a Cys residue in proteins harboring a C-terminal CAAX prenylation motif. This process renders proteins attached to endomembrane/endoplasmic reticulum (ER) and Golgi structures until further processing, as shown for Rab GTPases. Farnesylation also regulates mouse cpx 3/4 and Drosophila Cpx function. The Cys within CAAX motifs can also undergo S-nitrosylation, which interferes with the farnesylation signaling; however, direct evidence in a physiological environment is lacking. Cpx function has been studied in many different systems and there is controversy regarding its fusion-clamp activity. Cpx supports Ca2+-triggered exocytosis but also exhibits a clamping function. Analysis of mouse cpx double-knockout neurons lacking cpx 1 and 2 found only a facilitating function for cpx on release, and different D. melanogaster and Caenorhabditis elegans cpx mutant lines exhibit altered phenotypes in clamping or priming/fusion function, illustrating the controversial actions of Cpx (Robinson, 2018).

This study investigated the effects of NO on synaptic transmission and found that NO reduces Ca2+-triggered release as well as the size of the functional vesicle pool, which was reversed by glutathione (GSH) signaling. At the same time, spontaneous release rates were negatively affected by NO. It was confirmed that cpx is S-nitrosylated and that NO changes the synaptic localization of cpx, as also seen following genetic and pharmacological inhibition of farnesylation. Thus, it is proposed that the function of cpx is regulated by S-nitrosylation of Cys within the CAAX motif to prevent farnesylation. This increases cpx-SNARE-protein interactions, thereby rendering cpx with a dominant clamping function, which suppresses both spontaneous and evoked release (Robinson, 2018).

NO regulates a multitude of physiological and pathological pathways in neuronal function via generation of cGMP, thiol-nitrosylation, and 3-nitrotyrosination in health and disease. This study shows by employing biochemical and genetic tools in Drosophila, mouse, and HEK cells that NO can S-nitrosylate Cpx and modulate (in a cGMP-independent manner) neurotransmitter release at the NMJ by interfering with its prenylation status, thereby affecting the localization and function of this fusion-clamp protein. These nitrergic effects are reversed by GSH application or overexpression of GSH-liberating and de-nitrosylating enzymes (GCLm/c, GSNOR). GSH is the major endogenous scavenger for the NO moiety by the formation of S-nitrosoglutathione (GSNO) and consequently reduces protein-SNO levels via trans- and de-nitrosylation. The suppression of NOS activity facilitates synaptic function and the data support the notion that endogenous or exogenous NO enhances S-nitrosylation, reduces cpx farnesylation, and diminishes release (Robinson, 2018).

Of the numerous synaptic molecules involved in release, Cpx in particular has been implicated in the regulation of both evoked and spontaneous release due to its fusion-clamp activity. Despite the seemingly simple structure of Cpx, its physiological function is highly controversial, as this small SNARE-complex binding protein can both facilitate but also diminish fast Ca2+-dependent and spontaneous release, depending on the system studied. In addition, there are different mammalian isoforms of Cpx (1-4), which differ in their C-terminal region, with only cpx 3/4 containing the CAAX prenylation motif. Farnesylation in general determines protein membrane association and protein-protein interactions, and some cpx isoforms, such as muscpx 3/4 and Dmcpx 7A, are regulated in this manner. However, muscpx 1/2 does not possess a CAAX motif, suggesting differential regulatory pathways to modulate cpx function. In Drosophila, there are alternative splice variants resulting from a single cpx gene, but the predominant isoform contains the CAAX motif (Dmcpx 7A), implicating the importance of this signaling molecule. The other splice isoform (Dmcpx 7B) lacks the CAAX motif and is expressed at about 1,000-fold lower levels at the larval stage [15], thus making Dmcpx 7A the dominant isoform to be regulated by farnesylation. However, the lack of Dmcpx 7B phosphorylation by PKA induces similar phenotypes as seen in the current experiments when assessed following an induction of activity-dependent potentiation of mEJC frequency, which also may involve cpx-synaptotagmin 1 interactions (Robinson, 2018).

Interestingly, both depletion and excessive levels of cpx suppress Ca2+-dependent and -independent exocytosis. Cpx may promote SNARE complex assembly and simultaneously block completion of fusion by retaining it in a highly fusogenic state. Ca2+-dependent fusion is promoted below a concentration of 100 nM of cpx, whereas above 200 nM, it exhibits a clamping function resulting in a bell-shaped response curve. Previous work suggests that synaptotagmin 1, once bound to Ca2+, relieves the cpx block and allows fusion. Another study reported that selective competition between cpx and synaptotagmin 1 for SNARE binding allows regulation of release. The data are in agreement with the latter findings, as reduced synaptotagmin 1-Cpx interactions was observed following the block of farnesylation, indicating fewer synaptotagmin molecules binding to the SNARE complex to displace Cpx. This limited replacement of Cpx by synaptotagmin has been implicated in biochemical studies showing that local excess of Cpx inhibits release, presumably by outcompeting synaptotagmin binding. Thus, synaptotagmin-SNARE binding is strongly dependent upon the local concentration of Cpx. Alternatively, and this possibility cannot be excluded, the modulation of Cpx may simply alter its binding to the SNARE complex without directly displacing synaptotagmin, but interpretation of the data from the current assays (PLA, co-localization) would not allow distinguishing between these possibilities (Robinson, 2018).

The data are compatible with the idea that Cpx binds to the SNARE complex, facilitates assembly, and then exerts its clamping function by preventing full fusion due to SNARE complex stabilization and subsequent increased energy barrier to allow fusion. The current model could provide an explanation of how Cpx can be regulated to signal downstream to modulate transmitter release. So far, there are no data available, apart from mutation studies, as to how Cpx function can be altered. This study provides data indicating a physiologically relevant mechanism to adjust Cpx function, possibly to the requirements of the neuron to adjust synaptic transmission. This likely occurs due to Cys S-nitrosylation and suppression of farnesylation, allowing greater amounts of hydrophilic Cpx, not bound to endomembranes, to be available for binding with the SNARE complex in an altered configuration. This cross signaling between nitrosylation/farnesylation has been proposed to act as a molecular switch to modulate Ras activity. The current data show that enhanced nitrergic activity and blocking farnesylation, either genetically (CpxΔX) or pharmacologically, alters the localization of Cpx at the Drosophila NMJ and that of GFP-CAAX in HEK cells. Furthermore, by using a nitroso-mimetic cpx mutant, this study found enhanced co-localization of Cpx with the AZ protein Brp, implying a localization-function relationship. This consequently increases the net-clamping function because of elevated local concentrations of Cpx. Dmcpx specifically exhibits a strong clamping function, as shown following overexpression in hippocampal neurons, which causes suppression of evoked and spontaneous release accompanied by a reduction of the release probability or reduced vesicle fusion efficiency in in vitro assays (Robinson, 2018).

Two independent studies eliminating the CAAX motif in Dmcpx (cpx572 and cpx1257) investigated localization-function interactions and showed disagreeing effects on both release and cpx localization. In particular, it has also been shown that the truncated Cpx (cpx572, lacking the last 25 amino acids) does not co-localize with Brp. Interestingly, this mutant causes a strong decrease in C-terminal hydrophobicity and a modest physiological response (increased mini frequency, decreased evoked amplitudes equivalent to a loss of clamping and loss of fusion function) relative to the total knock-out (KO). In contrast, the cpx mutant with single amino acid deletion (cpx1275) causes no effect on evoked but identical effects on the frequency of spontaneous release, suggesting a lack of clamping but no lack of fusion function. In addition, this mutant now co-localizes with the AZ at the NMJ. These two studies indicate that the different mutations cause contrasting electrophysiological and morphological phenotypes, indicating that it is due to the nature of the mutation (lack of the last 25 amino acids versus 1 amino acid), which highlights the importance of a functional C-terminus. More recent studies have shown that deletions of the final amino acids (6 or 12 residues) completely abolished the membrane binding of Cpx-1, impairing its inhibitory function and confirming the requirement of an intact C-terminus for inhibition of release. This study used an endogenous Cpx with intact hydrophobic C-terminus, allowing physiological membrane binding. This is essential for inhibitory function, as the C-terminus is required for selective binding to highly curved membranes, such as those of vesicles. Thus, as different approaches are used to alter farnesylation, and a single a single amino acid mutant cpx (Dmcpx 7AC140W) was created, leaving the C-terminus intact, these studies were performed under conditions of endogenous regulation of Cpx function and thus provide new functional data on Cpx signaling. Importantly, the data show that this regulation alters cpx function, and this is the first study to provide an explanation for the differential effects observed using cpx mutants or even Cpx protein fragments in mammals, worm, and fly in various cross-species rescue experiments (Robinson, 2018).

The data are in agreement with a model that non-farnesylated hydrophilic and soluble cytosolic Cpx binds to the vesicular membrane via its C-terminal interactions, thereby exerting its inhibitory effect. When proteins are farnesylated, they are likely tethered to endomembranes, other than vesicle membranes. It has to be distinguished between Cpx interaction with the vesicle membrane as a result of the hydrophobic C-terminus, allowing Cpx to become in close proximity to the AZ, and Cpx endomembrane binding following farnesylation, which prevents Cpx interactions with the AZ. However, in the current, SNO modification may enhance the binding to other proteins (e.g., SNAREs), thereby augmenting the effects. These additional interactions with unknown binding partners may affect proper Cpx function and explain some of the discrepancies seen in studies using other genetically altered Cpxs (Robinson, 2018).

In summary, this study provides new data to illustrate a potential mechanism to regulate cpx function in a physiological environment, and it was shown that NO acts as an endogenous signaling molecule that regulates synaptic membrane targeting of Cpx, a pathway that may reconcile some of the controversial findings regarding Cpx function. It is suggested that increased S-nitrosylation and consequent lack of farnesylation leads to enhanced cytosolic levels of a soluble hydrophilic Cpx and less endomembrane-bound fractions, because farnesylation-incompetent proteins remain in the cytosol. These novel observations advance understanding of similar nitrergic regulation of farnesylation that may be relevant for mammalian cpx-dependent synaptic transmission at the retina ribbon synapse and other brain regions. Finally, this work has broader implications for physiological or pathological regulation of the prenylation pathway not only during neurodegeneration and aging, when enhanced S-nitrosylation might contribute to abnormal farnesylation signaling, but also in other biological systems in which nitrergic activity and prenylation have important regulatory functions such as in cardio-vasculature or cancer signaling (Robinson, 2018).

Differential regulation of evoked and spontaneous neurotransmitter release by C-terminal modifications of complexin

Complexins are small alpha-helical proteins that modulate neurotransmitter release by binding to SNARE complexes during synaptic vesicle exocytosis. They have been found to function as fusion clamps to inhibit spontaneous synaptic vesicle fusion in the absence of Ca2+, while also promoting evoked neurotransmitter release following an action potential. Complexins consist of an N-terminal domain and an accessory alpha-helix that regulates the activating and inhibitory properties of the protein, respectively, and a central alpha-helix that binds the SNARE complex and is essential for both functions. In addition, complexins contain a largely unstructured C-terminal domain whose role in synaptic vesicle cycling is poorly defined. This study demonstrates that the C-terminus of Drosophila complexin (DmCpx) regulates localization to synapses and that alternative splicing of the C-terminus can differentially regulate spontaneous and evoked neurotransmitter release. Characterization of the single DmCpx gene by mRNA analysis revealed expression of two alternatively expressed isoforms, DmCpx7A and DmCpx7B, which encode proteins with different C-termini that contain or lack a membrane tethering prenylation domain. The predominant isoform, DmCpx7A, is further modified by RNA editing within this C-terminal region. Functional analysis of the splice isoforms showed that both are similarly localized to synaptic boutons at larval neuromuscular junctions, but have differential effects on the regulation of evoked and spontaneous fusion. These data indicate that the C-terminus of Drosophila complexin regulates both spontaneous and evoked release through separate mechanisms and that alternative splicing generates isoforms with distinct effects on the two major modes of synaptic vesicle fusion at synapses (Behl, 2013).

Complexin controls spontaneous and evoked neurotransmitter release by regulating the timing and properties of synaptotagmin activity

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).

A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth

Neuronal signaling occurs through both action potential-triggered synaptic vesicle fusion and spontaneous release, although the fusion clamp machinery that prevents premature exocytosis of synaptic vesicles in the absence of calcium is unknown. This study demonstrates that spontaneous release at synapses is regulated by complexin, a SNARE complex-binding protein. Analysis of Drosophila complexin null mutants showed a marked increase in spontaneous fusion and a profound overgrowth of synapses, suggesting that complexin functions as the fusion clamp in vivo and may modulate structural remodeling of neuronal connections by controlling the rate of spontaneous release (Huntwork, 2007).

To determine the function of complexin in vivo, a complete knockout of complexin was generated. Drosophila has a single gene encoding the complexin protein (Tokumaru, 2001). Antisera was generated to recombinant Drosophila complexin protein that recognized a 16-kDa protein in brain extracts that is enriched in both CNS and peripheral synapses. Costaining for complexin and Discs large (Dlg) or the active-zone protein Bruchpilot demonstrated that complexin is expressed diffusely in presynaptic terminals. A 17-kb intragenic deletion within complexin (cpxSH1) that removed most of the coding region was generated by imprecise excision of a P element. A precise excision lacking any deletion was also generated, and served as a control for genetic background in all experiments. Western analysis and immunocytochemistry confirmed the loss of expression of complexin in cpxSH1. Null mutants lacking complexin are semilethal, with most animals dying before adult eclosion. Escaper adults are infertile, show severe motor defects and lack the on- and off-transients that represent normal synaptic transmission in the visual system. Pan-neuronal expression of a UAS-complexin transgene with the elav-GAL4 neuronal driver rescues the reduced viability and abnormal synaptic transmission of cpxSH1 mutants (Huntwork, 2007).

To analyze the role of complexin in neurotransmitter release, electrophysiological recordings were performed at Drosophila third-instar larval abdominal muscle 6 synapses. cpxSH1 mutants had profound defects in spontaneous vesicle fusion. The frequency of miniature excitatory junctional potentials (minis) was greatly enhanced at cpxSH1 mutant synapses, demonstrating continuous exocytosis of synaptic vesicles in the absence of any stimulation. Neuronal expression of a UAS-complexin transgene rescued the enhanced frequency of spontaneous release. In high extracellular [Ca2+], evoked responses in cpxSH1 mutants after nerve stimulation were significantly reduced compared with controls, whereas in low extracellular [Ca2+] no difference was observed. As described below, complexin mutant synapses showed a 64% increase in active zone number, indicating that neuromuscular junction (NMJ) synapses lacking complexin had a reduction in evoked fusion events per active zone that was exacerbated at high calcium levels, where large numbers of synchronous synaptic vesicle fusion events are required (Huntwork, 2007).

To confirm that the increase in miniature postsynaptic potentials resulted from increased spontaneous vesicle fusion, as opposed to nonvesicular dumping of glutamate at the synapse, a temperature-sensitive allele of dynamin (shibireTS1) was used that blocks endocytosis at 32°C. High-frequency stimulation at 32°C depletes the synaptic vesicle pool in shibire mutants and would be predicted to eliminate the enhanced spontaneous vesicle fusion in double mutants. After a 5-min 10-Hz stimulation train at 32°C in shibireTS1; cpxSH1 double mutants, which abolished evoked release, the elevated mini frequency observed at permissive temperatures was eliminated. The frequency of spontaneous release in cpxSH1 mutants remained strongly elevated in 0 mM extracellular calcium, ruling out an aberrant influx of calcium into the presynaptic nerve terminal as the cause for the enhanced spontaneous release. These observations suggest that complexin functions as the synaptic vesicle fusion clamp in vivo, and with its loss, synaptic vesicles continuously fuse in the absence of stimulation (Huntwork, 2007).

The large increase in spontaneous fusion observed at complexin mutant NMJs provided an opportunity to determine the consequences of altered complexin function and increased mini frequency on synaptic growth. To analyze synaptic structure, synaptic varicosities were counted in age-matched control and cpxSH1 third-instar NMJs. cpxSH1 mutants showed a two-fold overproliferation of boutons at each muscle examined, as well as 64% more active zones. Neuronal expression of a complexin transgene reverted the synaptic overgrowth phenotype. These data eliminate any structural considerations that could account for the enhanced minis, as the 64% increase in the number of active zones is insufficient by far to explain the >20-fold increase in spontaneous release (Huntwork, 2007).

This analysis demonstrates that the interactions of complexin with SNAREs provide a molecular fusion clamp to prevent spontaneous release at synapses in the absence of stimulation. These in vivo observations match well with the predicted fusion clamp model based on the in vitro reduction of SNARE-mediated fusion by complexin. Although analysis of cultured hippocampal autaptic neurons lacking two of the four mouse complexin genes showed reduced neurotransmitter release, which matches the observations in Drosophila, mini frequency in the mouse system was reported to be unchanged. Although it is unclear what underlies the discrepancy in mini frequency between models, one potential explanation is that in mouse either complexin 3 or 4 or other unknown components of the fusion clamp machinery compensate for the loss of complexin (Huntwork, 2007).

It has long been known that minis represent single vesicle fusion events, but their underlying function has remained unclear. The current results unexpectedly uncovered a marked effect on synaptic growth at the Drosophila NMJ in complexin mutants. Although previous studies in hyperexcitable Drosophila mutants have demonstrated that increased neuronal activity promotes synaptic growth, the contributions of spontaneous versus evoked signaling are unknown. Activity-dependent retrograde signaling by the postsynaptic calcium sensor Synaptotagmin 4 has also been shown to transiently increase mini frequency 100-fold and trigger enhanced synaptic growth at Drosophila embryonic NMJs. It is tempting to speculate that regulation of complexin during retrograde signaling underlies activity-dependent enhancement of spontaneous fusion and synaptic growth. There is at present no evidence that the increased minis cause the morphological overgrowth, and further studies will be required to dissect the mechanism by which complexin mutants enhance synaptic growth. Recent work in mammals suggests that spontaneous release can regulate dendritic spine morphogenesis and dendritic protein synthesis. Complexin dysfunction has also been implicated in human diseases, including schizophrenia, indicating that abnormal spontaneous fusion may contribute to certain neurological diseases. Given that complexin and the synaptic vesicle calcium sensor Synaptotagmin 1 may compete for binding to SNARE complexes, an attractive model for synaptic vesicle exocytosis is that complexin stabilizes a hemifused intermediate that can complete full fusion upon calcium activation of Synaptotagmin 1. Together with Synaptotagmin 1, complexin provides a key neuronal modulator of SNARE function that adapts the ubiquitous membrane trafficking machinery for synaptic vesicle fusion (Huntwork, 2007).

Complexin activates exocytosis of distinct secretory vesicles controlled by different synaptotagmins

Complexins are SNARE-complex binding proteins essential for the Ca(2+)-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. This study shows that, in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a cofactor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, the data indicate that complexins are essential for activation of multiple types of Ca(2+)-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved (Cao, 2013).

Synaptic vesicles position complexin to block spontaneous fusion

Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin can both promote and inhibit fusion through interactions between its alpha-helical domain and the SNARE complex. In addition, complexin's C-terminal half is required for the inhibition of spontaneous fusion in worm, fly, and mouse, although the molecular mechanism remains unexplained. This study shows that complexin's C-terminal domain binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. It is proposed that the SV pool serves as a platform to sequester and position complexin where it can intercept the rapidly assembling SNAREs and control the rate of spontaneous fusion (Wragg, 2013).


REFERENCES

Search PubMed for articles about Drosophila Complexin

Bowen, M. E., et al. (2005). Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex. Biophys. J. 89: 690-702. PubMed ID: 15821166

Bracher, A., et al. (2002). X-ray structure of a neuronal complexin-SNARE complex from squid. J. Biol. Chem. 277: 26517-26523. PubMed ID: 12004067

Brose, N. (2008). For better or for worse: complexins regulate SNARE function and vesicle fusion. Traffic 9: 1403-1413. PubMed ID: 18445121

Buhl, L. K., Jorquera, R. A., Akbergenova, Y., Huntwork-Rodriguez, S., Volfson, D. and Littleton, J. T. (2013). Differential regulation of evoked and spontaneous neurotransmitter release by C-terminal modifications of complexin. Mol Cell Neurosci 52: 161-172. PubMed ID: 23159779

Cai, H., et al. (2008). Complexin II plays a positive role in Ca2+-triggered exocytosis by facilitating vesicle priming. Proc. Natl. Acad. Sci. 105: 19538-19543. PubMed ID: 19033464

Cao, P., Yang, X. and Sudhof, T. C. (2013). Complexin activates exocytosis of distinct secretory vesicles controlled by different synaptotagmins. J Neurosci 33: 1714-1727. PubMed ID: 23345244

Cho, R. W., Buhl, L. K., Volfson, D., Tran, A., Li, F., Akbergenova, Y. and Littleton, J. T. (2015). Phosphorylation of Complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity. Neuron 88: 749-761. PubMed ID: 26590346

Chen, X., et al. (2002). Three-dimensional structure of the complexin/SNARE complex. Neuron 33: 397-409. PubMed ID: 11832227

Giraudo, C. G., et al. (2006). A clamping mechanism involved in SNARE-dependent exocytosis, Science 313: 676-680. PubMed ID: 16794037

Giraudo, C. G., et al. (2009). Alternative zippering as an on-off switch for SNARE-mediated fusion. Science 323: 512-516. PubMed ID: 19164750

Guan, R., Dai, H. and Rizo, J. (2008). Binding of the Munc13-1 MUN domain to membrane-anchored SNARE complexes. Biochemistry 47: 1474-1481. PubMed ID: 18201107

Huntwork, S. and Littleton, J. T. (2007). A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth. Nat. Neurosci. 10: 1235-1237. PubMed ID: 17873870

Ishizuka, T., et al. (1995). Synaphin: a protein associated with the docking/fusion complex in presynaptic terminals. Biochem. Biophys. Res. Commun. 213: 1107-1114. PubMed ID: 7654227

Jorquera, R. A., Huntwork-Rodriguez, S., Akbergenova, Y., Cho, R. W. and Littleton, J. T. (2012). Complexin controls spontaneous and evoked neurotransmitter release by regulating the timing and properties of synaptotagmin activity. J Neurosci 32: 18234-18245. PubMed ID: 23238737

Malsam, J., et al. (2009). The carboxy-terminal domain of complexin I stimulates liposome fusion. Proc. Natl. Acad. Sci. 106: 2001-2006. PubMed ID: 19179400

Maximov, A., et al. (2009). Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323: 516-521. PubMed ID: 19164751

McMahon, H. T., et al. (1995). Complexins: cytosolic proteins that regulate SNAP receptor function, Cell 83: 111-119. PubMed ID: 7553862

Pabst, S., et al. (2000). Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions. J. Biol. Chem. 275: 19808-19818. PubMed ID: 10777504

Pabst, S., et al. (2002). Rapid and selective binding to the synaptic SNARE complex suggests a modulatory role of complexins in neuroexocytosis. J. Biol. Chem. 277: 7838-7848. PubMed ID: 11751907

Reim, K., et al. (2005). Structurally and functionally unique complexins at retinal ribbon synapses, J. Cell Biol. 169: 669-680. PubMed ID: 15911881

Rizo, J. and Rosenmund, C. (2008). Synaptic vesicle fusion. Nat. Struct. Mol. Biol. 15: 665-674. PubMed ID: 18618940

Robinson, S. W., Bourgognon, J. M., Spiers, J. G., Breda, C., Campesan, S., Butcher, A., Mallucci, G. R., Dinsdale, D., Morone, N., Mistry, R., Smith, T. M., Guerra-Martin, M., Challiss, R. A. J., Giorgini, F. and Steinert, J. R. (2018). Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability. PLoS Biol 16(4): e2003611. PubMed ID: 29630591

Schaub, J. R., et al. (2006). Hemifusion arrest by complexin is relieved by Ca(2+)-synaptotagmin I. Nat. Struct. Mol. Biol. 13: 748-750. PubMed ID: 16845390

Strenzke, N., et al. (2009). Complexin-I is required for high-fidelity transmission at the endbulb of held auditory synapse. J. Neurosci. 29: 7991-8004. PubMed ID: 19553439

Takahashi, S., et al. (1995). Identification of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses. FEBS Lett. 368: 455-460. PubMed ID: 7635198

Tang, J., et al. (2006). A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126: 1175-1187. PubMed ID: 16990140

Tokumaru, H. et al. (2001). SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis. Cell 104: 421-432. PubMed ID: 11239399

Weninger, K., et al. (2008). Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1:1 Syntaxin/SNAP-25 complex. Structure 16: 308-320. PubMed ID: 18275821

Wragg, R. T., Snead, D., Dong, Y., Ramlall, T. F., Menon, I., Bai, J., Eliezer, D. and Dittman, J. S. (2013). Synaptic vesicles position complexin to block spontaneous fusion. Neuron 77: 323-334. PubMed ID: 23352168

Xue, M., et al. (2007). Distinct domains of complexin I differentially regulate neurotransmitter release. Nat. Struct. Mol. Biol. 14: 949-958. PubMed ID: 17828276

Xue, M., et al. (2008). Complexins facilitate neurotransmitter release at excitatory and inhibitory synapses in mammalian central nervous system. Proc. Natl. Acad. Sci. 105: 7875-7880. PubMed ID: 18505837

Xue, M., et al. (2009). Tilting the balance between facilitatory and inhibitory functions of mammalian and Drosophila Complexins orchestrates synaptic vesicle exocytosis. Neuron 64: 367-380. PubMed ID: 19914185

Yoon, T. Y., et al. (2008). Complexin and Ca2+ stimulate SNARE-mediated membrane fusion. Nat. Struct. Mol. Biol. 15: 707-713. PubMed ID: 18552825


Biological Overview

date revised: 22 July 2013

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.