InteractiveFly: GeneBrief

unc-13: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - unc-13

Synonyms -

Cytological map position - 102F6--7

Function - synaptic vesicle exocytosis, signaling

Keywords - neuromuscular junction, CNS

Symbol - unc-13

FlyBase ID: FBgn0025726

Genetic map position -

Classification - C1 lipid-binding motif and two C2 calcium-binding domains

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Reddy-Alla, S., et al. (2017). Stable positioning of Unc13 restricts synaptic vesicle fusion to defined release sites to promote synchronous neurotransmission. Neuron 95(6): 1350-1364.e1312. PubMed ID: 28867551
Summary:
Neural information processing depends on precisely timed, Ca2+-activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. This study found that the (M)Unc13-family member Unc13A generates release sites and showed the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release.
BIOLOGICAL OVERVIEW

Drosophila unc-13 is essential for a stage of neurotransmission following vesicle docking and before fusion. Exocytosis of synaptic vesicles is triggered by depolarization-dependent influx of calcium and modulated by downstream second messenger cascades involving calcium-binding proteins, diacylglycerol and PKC activators such as phorbol esters, among other effector molecules. Proteins that bind such second-messenger signals and localize at presynaptic vesicle fusion sites probably regulate synaptic efficacy. The UNC-13 family of presynaptic proteins interact closely with multiple components of the fusion machinery, and they have domains that can bind both Ca2+ and diacylglycerol. Thus, Unc-13 proteins may mediate Ca2+ and/or diacylglycerol signals to control synaptic vesicle exocytosis (Aravamudan, 1999).

Unc-13 was first identified in C. elegans based on an uncoordinated mutant phenotype (Brenner, 1976). The protein contains a zinc finger-like C1 domain that binds diacylglycerol and phorbol esters and two C2 domains similar to the Ca2+-binding regulatory regions (Maruyama, 1991) of PKC and Synaptotagmin. The four aspartates in the first C2 domain of Synaptotagmin that are essential for Ca2+ binding are conserved in the middle C2 domain of Unc-13, indicating that Unc-13 should also bind calcium (Aravamudan, 1999 and references therein).

There are three mouse homologs of Unc-13 (Munc 13-1, 2 and 3). Munc 13-1, the best studied mammalian homolog, has the same domain structure as Unc-13 and is expressed specifically in the nervous system, similar to Unc-13. Munc 13-1 is a high-affinity receptor for phorbol esters and mediates neurotransmitter release induced by phorbol esters when overexpressed at frog neuromuscular junctions. In addition, Munc 13-1 interacts with the core-complex protein Syntaxin, Munc-18, the synaptic vesicle-associated Doc 2alpha, and the brain-specific spectrin, ß-spIIIsigma. Thus Unc-13 and Munc-13 bind plasma membrane, vesicular proteins, and cytosolic proteins central to the process of neurotransmission. To determine the synaptic role of Unc-13, the Drosophila homolog has been identified and its null-mutant phenotype characterized (Aravamudan, 1999).

Like its C. elegans and mammalian homologs, Drosophila unc-13 contains a C1 lipid-binding motif and two C2 calcium-binding domains, and its expression is restricted to neurons. Elimination of unc-13 expression abolishes synaptic transmission, an effect comparable only to removal of the core complex proteins Syntaxin and Synaptobrevin. Ultrastructurally, mutant terminals accumulate docked vesicles at presynaptic release sites. It is concluded that Drosophila unc-13 is essential for a stage of neurotransmission following vesicle docking and before fusion (Aravamudan, 1999).

Drosophila Unc-13 expression is neural-specific, similar to both Munc 13-1 and C. elegans Unc-13, and the expression patterns of Unc-13 and other proteins important for neurotransmission are temporally similar. Drosophila Unc-13 is essential for synaptic transmission, a conclusion supported by similar observations in mouse and C. elegans. Without Unc-13, neurotransmission is effectively abolished, a phenotype mimicked only by complete removal of the core-complex proteins Synaptobrevin and Syntaxin. The frequency of miniature excitatory junctional current (mEJCs) in unc-13 mutants is also greatly decreased, indicating severe impairment of spontaneous fusion of synaptic vesicles with the plasma membrane. Because most mEJCs at the Drosophila neuromuscular junction (NMJ) are calcium dependent, this suggests loss of essentially all Ca2+-dependent synaptic vesicle fusion events. It is not known if the persistent mEJCs represent a small population of Ca2+-dependent events or are actual Ca2+-independent synaptic vesicle fusions. Elevation of the calcium signal with high-frequency stimulation or elevated external Ca2+ can not restore transmission in unc-13 mutants. Likewise, a Ca2+-independent fusion trigger, hyperosmotic saline, fails to effectively bypass the blockage. Thus, it is concluded that Unc-13 is centrally important for synaptic vesicle fusion competence (Aravamudan, 1999).

Ultrastructural observations of unc-13 mutants show an increased accumulation of vesicles throughout the synapse, consistent with a block in synaptic vesicle exocytosis. The increase in synaptic vesicle density in all synaptic 'compartments' (docked, clustered or removed from the active zone) is consistent with a maintained dynamic equilibrium of the vesicle population. These data are essentially identical to the effects of removing Syntaxin or Synaptobrevin. In all three cases, vesicles accumulate to a level 30%-50% above that of the normal population, suggesting that this feature is diagnostic of blockage in synaptic vesicle fusion in this system, and that feedback mechanisms must prevent further accumulation of vesicles following a fusion block. Together, these data suggest that vesicles are morphologically docked but prevented from fusing without Unc-13. Therefore, it is concluded that Unc-13 is essential in, or immediately before, calcium-triggered synaptic vesicle fusion (Aravamudan, 1999).

The data are consistent with the molecular description and phenotypes of unc-13 mutants of C. elegans. Knockouts of munc 13-1 also produce similar transmission defects in mice, indicating that Munc 13-1 is important in vesicle maturation. The munc 13-1 knockout does not, however, change synaptic vesicle density or distribution, suggesting more efficient feedback mechanisms controlling synaptic vesicle dynamics in mouse and/or partial redundancy with other members of the Munc-13 family. However, overall, unc-13 mutant phenotype is concordant with both unc-13 and munc 13-1 phenotypes, supporting the conclusion that the Unc-13 proteins have a highly conserved function in evolutionarily distant organisms at both cholinergic and GABAergic (C.elegans) and glutamatergic (Drosophila, mouse) synapses (Aravamudan, 1999).

Where does Unc-13 act in the exocytotic process? The severity of synaptic vesicle fusion defects resulting from the removal of Unc-13 have been seen only in animals lacking essential core complex proteins. Eliminating Syntaxin abolishes all fusion in both neuronal and non-neuronal cells. Removal of n-Synaptobrevin generates slightly less severe presynaptic transmission defects, essentially identical to those in unc-13 mutants. Transmission in n-Synaptobrevin mutants can be slightly increased by conditions that increase presynaptic Ca2+, such as introduction of a Ca2+ ionophore, application of black-widow-spider venom, elevation of extracellular Ca2+ or increased frequency of stimulation. Both high frequency stimulation and increased external [Ca2+] cause a slight increase in transmission in unc-13 mutants as well. These observations indicate that Unc-13 is as essential for synaptic vesicle fusion as is the core complex protein Synaptobrevin, and that these two proteins may act in the same process (Aravamudan, 1999).

Similarly, severely reduced responses to hyperosmotic saline in unc-13 mutants are comparable to those observed in syntaxin or n-synaptobrevin mutants. The hyperosmotic response requires core complex formation, suggesting that Unc-13 might regulate the formation and/or fusion competence of the core complex. The observations are consistent with impaired formation and/or competence of the core complex in unc-13 mutants, possibly leading to a defect in fusion and abnormal accumulation of docked, pre-fusion vesicles in the presynaptic terminal (Aravamudan, 1999).

Neither Syntaxin nor Synaptobrevin binds Ca2+. Therefore, the Ca2+-binding component or 'Ca2+ sensor', required to mediate the signal that triggers evoked synaptic transmission must be located elsewhere. Unc-13 contains potential Ca2+-binding C2 domains, and the unc-13 mutant phenotype indicates a specialized role for Unc-13 in neural-specific synaptic vesicle exocytosis rather than ubiquitous fusion machinery. Moreover, C. elegans Unc-13 clearly associates with components of the core fusion complex, and the dunc-13/unc-13/munc 13-1 null mutations result in a neural-specific block in synaptic vesicle fusion equivalent to the disruption of this complex. Thus, Unc-13 may mediate the calcium dependence of synaptic vesicle fusion, a role also proposed for another non-core complex protein, Synaptotagmin. However, it remains to be determined if the putative Ca2+-sensing ability of Unc-13 proteins are required for the Ca2+ dependence of triggered synaptic vesicle fusion and/or other events downstream of core-complex assembly. Future work is needed to discern the molecular interactions of Unc-13 governing the synaptic vesicle exocytotic process in vivo (Aravamudan, 1999).

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca2+ channel, but molecular mechanisms controlling this are unknown. This study found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-alpha, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca2+ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A null mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. Isoform-specific Unc13-AZ scaffold interactions were identified, regulating SV-Ca2+-channel topology whose developmental tightening optimizes synaptic transmission (Bohme, 2016).

All presynaptic AZs accumulate scaffold proteins from a canonical set of few protein families, which are characterized by extended coiled-coil stretches, intrinsically unstructured regions and a few classical interaction domains, particularly PDZ and SH3 domains. These multidomain proteins collectively form a compact 'cytomatrix' often observable by electron-dense structures covering the AZ membrane, which have been found to physically contact SVs, and thus have been suggested to promote SV docking and priming as well as to recruit Ca2+ channels. Still, how the structural scaffold components (ELKS, RBP, RIM and Liprin-α) tune the functionality of the SV-release machinery has remained largely enigmatic. Liprin-α is crucial for the AZ assembly process and at Drosophila NMJ AZs, Liprin-α-Syd-1 cluster formation initializes the assembly of an 'early' scaffold complex, which subsequently guides the accumulation of a 'late' RBP-BRP scaffold complex. This study provides evidence that these scaffold complexes together operated as 'molecular rulers' that confer a remarkable degree of order, patterning AZ composition and function in space and time: the 'early' Liprin-α-Syd-1 clusters recruit Unc13B, and this scaffold serves as a template to accumulate the 'late' BRP-RBP scaffold, which recruits Unc13A. Unc13 isoforms are precisely organized in the tens of nanometers range, which the data suggest to be instrumental to control SV release probability and SV-Ca2+ channel coupling. As a molecular basis of this patterning and recruitment, this study identified a multitude of molecular contacts between the Unc13 N termini and the respective scaffold components using systematic Y2H analysis. As one out of several interactions, a cognate PxxP motif was identified in the N terminus of Unc13A to interact with the second and third SH3 domains of RBP. Point mutants within the PxxP motif interfered with the binding of the RBP-SH3 domains II and III on the Y2H level but did not have a major impact on Unc13A localization and function when introduced into an Unc13 genomic transgene. Nonetheless, elimination of the scaffold components BRP and RBP on the one hand or Liprin-α on the other hand drastically impaired the accumulation of Unc13A or Unc13B. It is suggested that these results are explained by a multitude of parallel interactions that provide the avidity needed to enrich the respective Unc13 isoforms in their specific 'niches' and may cause a functional redundancy among interaction motifs, as was likely observed in the case of the Unc13A PxxP motif. Future analysis will be needed to investigate these interaction surfaces in greater detail, and address how exactly 'early' and 'late' scaffolds coordinate AZ assembly (Bohme, 2016).

Unc13 proteins have well-established functions in SV docking and priming. Accordingly, it was observed that loss of Unc13A resulted in overall reduced SV docking without affecting T-bar-tethered SVs, which is qualitatively opposite to a function of BRP in SV localization, whose C-terminal amino acids function in T-bar-tethering, but not docking. Variants lacking these residues suffer from increased synaptic depression, suggesting a role in SV replenishment. Therefore, in addition to its role in localizing Unc13A to the AZ reported in this study, BRP may also cooperate functionally with Unc13A by facilitating SV delivery to docking sites (Bohme, 2016).

Synapses are highly adapted to their specific features, varying widely concerning their release efficacy and short-term plasticity. These features impact information transfer and may provide neurons with the ability to detect input coherence, maintain stability and promote synchronization. Differences in the biochemical milieu of SVs can tune priming efficacy and release probability, which largely affects short-term plasticity. In the current experiments, it was found that loss of Unc13A resulted in dramatically (~90%) reduced synaptic transmission, which exceeded the (~50%) reduction in SV docking, pointing to an additional function in enhancing release efficacy. These changes were paralleled by drastically increased short-term facilitation as well as EGTA hypersensitivity and could be due to decreased Ca2+ sensitivity of the molecular release machinery, for example, mediated by different Synaptotagmin-type Ca2+ sensors, or different numbers of SNARE complexes. However, although a rightward shift of the dependence of normalized release amplitudes on extracellular Ca2+ concentration was observed at Unc13A-deficient synapses, its slope and thus Ca2+ cooperativity was unaltered, arguing against fundamentally different Ca2+-sensing mechanisms. Instead a scenario is favored in which SV Ca2+ sensing is conserved, but local Ca2+ signals at SV positions are attenuated because of their larger distances to Ca2+ channels upon loss of Unc13A. Both Unc13 isoforms were clearly segregated physically with different distances to the Ca2+ channel cluster, and loss of Unc13A selectively reduced the number of docked SVs in the AZ center. These findings are best explained by Unc13A promoting the docking and priming of SVs closer to Ca2+ channels than Unc13B. In fact, mathematical modeling reproduced the data by merely assuming release from two independent pathways with identical Ca2+ sensing and fusion mechanisms that only differed in their physical distance to the Ca2+ source in the AZ center. The distances estimated by the model were in very good agreement with the positions of the two Unc13 isoforms defined by STED microscopy. Thus, the data suggest that differences in the distance of SVs in the tens of nanometer range to the Ca2+ channels mediated by the two Unc13 isoforms likely contributed profoundly to the observed phenotypes. It is proposed that the role of the N terminus is to differentially target the isoforms into specific zones of the AZ, while the conserved C terminus confers identical docking and priming functions at both locations. Notably, recent work in Caenorhabditis elegans also characterized two Unc13 isoforms, with fast release being mediated by UNC-13L, whereas slow release required both UNC-13L and UNC-13S (Hu, 2013). The proximity of the UNC-13L isoform to Ca2+ entry sites was mediated by the protein's N-terminal C2A-domain (not present in Drosophila) and was critical for accelerating neurotransmitter release, and for increasing/maintaining the probability of evoked release assayed by the fraction of AP- to sucrose-induced release. In contrast, the slow SV release form dominantly localized outside AZ regions. Thus it would be interesting to investigate the sub-AZ distribution of C. elegans Unc-13 isoforms and test whether the same scaffold complexes as in Drosophila mediate the localization of the different Unc-13 isoforms (Bohme, 2016).

Notable differences in short-term plasticity have been reported for mammalian Unc13 isoforms. The mammalian genome harbors five Munc13 genes. Of those, Munc13-1, -2 and -3 are expressed in the brain, and function in SV release; differential expression of Munc13 isoforms at individual synapses may represent a mechanism to control short-term plasticity. Thus, it might be warranted to analyze whether differences in the sub-active zone distribution of Munc13 isoforms contribute to these aspects of synapse diversity in the rodent brain (Bohme, 2016).

Fast and slow phases of release have recently been attributed to parallel release pathways operating in the calyx of Held of young rodents (56 nm and 135 nm) qualitatively matching the coexistence of two differentially positioned release pathways described in this study. The finding of discretely localized release pathways with distances larger than 60 nm is further in line with the recent suggestion that, at some synapses, SVs need to be positioned outside an 'exclusion zone' from the Ca2+ source (~50 nm distance to the center of the SV for the calyx of Held). At mammalian synapses, developmental changes in the coupling of SVs and Ca2+ channels have been described, which qualitatively matches the sequential arrival of loosely and tightly coupled Unc13B and Unc13A isoforms during synaptogenesis described here. Thus, this this work suggests that differential positioning of Unc13 isoforms couples functional and structural maturation of AZs. To what degree modulation of this process contributes to the functional diversification of synapses is an interesting subject of future analysis (Bohme, 2016).


DEVELOPMENTAL BIOLOGY

Embryonic

From RNA in-situ hybridization in Drosophila embryos, unc-13 seems to be expressed throughout the nervous system but not elsewhere. The sole exception is faint, transient expression in regions of the gut that disappears following development stage 12. Neural expression is first detected at stages 11-12, coincident with the onset of expression of other synaptic proteins such as postsynaptic glutamate receptors and presynaptic Synaptotagmin and n-Synaptobrevin. This expression precedes initial synapse formation by three to four hours. In late-stage embryos (Stage 16), the unc-13 message is restricted to the central and peripheral nervous system. Thus, its temporal and spatial expression suggest that unc-13 encodes a neural-specific synaptic protein, as reported for its homologs in other species (Aravamudan, 1999).

Adult

Ca2+ influxes regulate multiple events in photoreceptor cells including phototransduction and synaptic transmission. An important Ca2+ sensor in Drosophila vision appears to be calmodulin since a reduction in levels of retinal calmodulin causes defects in adaptation and termination of the photoresponse. These functions of calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the TRP and TRPL ion channels, NinaC and INAD. To identify additional calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, a screen was conducted for retinal calmodulin-binding proteins. Eight additional calmodulin-binding proteins were found that are expressed in the Drosophila retina. These include six targets that are related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein, UNC-13, and a protein, CRAG, related to Rab3 GTPase exchange proteins. Other calmodulin-binding proteins include Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission (Xu, 1998).


EFFECTS OF MUTATION

Homozygous null unc-13 mutants die in late embryonic stages (20-22hours AF), just before the normal time of hatching. The unc-13 embryos have normal gross morphology including properly developed epidermis, trachea, alimentary tract, musculature and nervous systems. This suggests that Unc-13 is not essential in the morphogenesis of any of these tissues. In particular, there is no evidence for a role in non-neuronal secretion. However, the unc-13 mutant embryos are completely paralyzed and show no muscular peristalsis or neurally coordinated movement, required for hatching and locomotion (Aravamudan, 1999).

Effects of the unc-13 null mutation on neuromuscular cytoarchitecture were assayed. Confocal analysis of nervous systems of homozygous mutant animals visualized with anti-HRP antibodies (recognizing a neuronal-membrane marker) and of synaptic structure with antibodies against the synaptic-vesicle-associated protein CSP reveals no defects in the arrangement of neuronal cell bodies, processes or synapses. At the neuromuscular junction, no significant alteration was detected in synaptic branching, differentiation of presynaptic boutons or distribution of synaptic vesicle markers. Thus the paralysis leading to embryonic death results from a functional rather than a morphological defect (Aravamudan, 1999).

Electrophysiology at the neuromuscular synapse at the end of embryogenesis (22-24 hours AF) was used to understand the role of Unc-13 in neurotransmission. Low-frequency (1 Hz) electrical stimulation of the motor nerve at physiological calcium levels (1.8 mM Ca2+) in wild-type animals demonstrates robust (over 1.5 nA), high-fidelity synaptic transmission that is essentially eliminated in unc-13 mutants. Most stimuli (over 97%) failed to elicit any detectable postsynaptic response at the resolution of single quantal events. Rare responses were limited to a few quanta and lacked tight temporal coupling to the presynaptic stimulus, thus severely reducing average excitatory junctional current (EJC) amplitude in unc-13 to less than 1% of normal. Similarly, the rate of spontaneous transmitter release, or miniature EJCs (mEJCs), is significantly decreased. However, the average mEJC amplitude of the rare, persisting mEJCs was not significantly altered in the mutants, demonstrating that the defect is unlikely to result from postsynaptic alteration or changes in amount of neurotransmitter contained in synaptic vesicles (Aravamudan, 1999).

Removal of Unc-13 severely impairs coupling of Ca2+ influx with synaptic vesicle fusion. Attempts to alleviate the unc-13 transmission defect were made by increasing the presynaptic calcium signal. First, stimulation was carried out at elevated frequencies (5-20 Hz): this did not significantly increase transmission. Second, junctions were stimulated in elevated extracellular Ca2+. Average EJC amplitude in 5 mM calcium is slightly, but significantly, greater than in 1.8 mM Ca2+ in unc-13 mutants but is not improved over control levels in similar conditions, and the quantal content of transmission remains similarly impaired. Therefore, it seems that unc-13 synaptic terminals lack significant stimulus-induced synaptic vesicle fusion (Aravamudan, 1999).

Attempts were made to stimulate fusion in unc-13 with hyperosmotic saline application. A 3-second focal application of 1175 mOsm saline to a wild-type junction evoked a prolonged synaptic response composed of many repetitive secretion events, whereas responses of unc-13 synapses were extremely depressed relative to controls and similar to those of mutants lacking the essential secretory proteins, Syntaxin and Synaptobrevin. Calculation of the total charge elicited in response to hypersomotic saline revealed significant and similar lack of response in unc-13, synaptobrevin and syntaxin. However, in response to hyperosmotic saline, unc-13 has significantly more vesicle fusion events than syntaxin or synaptobrevin mutants. Thus, unc-13 mutants show severely reduced neurotransmission in response to normal and elevated Ca2+ influx and severely reduced responses to hyperosmotic saline (Aravamudan, 1999).

At what step does presynaptic transmission require Unc-13? To further characterize synaptic defects associated with unc-13 mutation, an ultrastructural analysis of the neuromuscular junction was conducted. The appearance of typical presynaptic boutons containing active zones of transmitter release is similar at synapses in controls and unc-13 mutants at the end of embryogenesis. No alteration was detected in conformation of the T-bars or the overall active zone, the size or appearance of individual synaptic vesicles or any other component of the pre- or post-synaptic terminal, further suggesting normal development of neuromuscular synapses in unc-13 mutants (Aravamudan, 1999).

In contrast, a 50% increase in the number of synaptic vesicles was observed throughout the boutons of unc-13. Likewise, the number of vesicles clustered within 250 nm of active zones in mutants was increased by 50%. The number of docked vesicles within the active zone radius also was 50% higher in unc-13 mutants than in controls. Finally, the percentage of the total number of clustered vesicles that were docked was also significantly higher in unc-13 mutants. These results, similar to findings in syntaxin and synaptobrevin mutants, indicate that synaptic vesicle exocytosis is specifically blocked in unc-13 mutants (Aravamudan, 1999).


EVOLUTIONARY HOMOLOGS

C. elegans unc-13

Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode C. elegans. Molecular cloning of the gene and sequencing of the cDNA reveal that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C. The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium: diacylglycerol inhibited the binding in a competitive manner (Maruyama, 1991).

The C. elegans unc-13 mutant is a member of a class of mutants that exhibit un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein is zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detects a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. It is concluded that UNC-13 represents a novel class of phorbol ester receptor (Ahmed, 1992).

The C. elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. The cysteine-rich region of Unc-13 was expressed in Escherichia coli and its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity were quantitatively analyzed. [3H]Phorbol 12,13-dibutyrate [3H]PDBu binds with high affinity to the cysteine-rich region of Unc-13. This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 binds diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis reveals significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 requires slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, is also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although these results reveal some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some 'specific' antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13 (Kazanietz, 1995).

The C. elegans unc-13, unc-18, and unc-64 genes are required for normal synaptic transmission. The UNC-18 protein binds to the unc-64 gene product C. elegans syntaxin (Ce syntaxin: see Drosophila Syntaxin). However, it is not clear how this protein complex is regulated. UNC-13 has been shown to transiently interact with the UNC-18-Ce syntaxin complex, resulting in rapid displacement of UNC-18 from the complex. Genetic and biochemical evidence is presented that UNC-13 contributes to the modulation of the interaction between UNC-18 and Ce syntaxin (Sassa, 1999).

Serotonin inhibits synaptic transmission at C. elegans neuromuscular junctions, and a signaling pathway is described that mediates this effect. Exogenous serotonin inhibits acetylcholine release, whereas serotonin antagonists stimulates release. The effects of serotonin on synaptic transmission are mediated by GOA-1 (a Galpha0 subunit) and DGK-1 (a diacylglycerol [DAG] kinase), both of which act in the ventral cord motor neurons. Mutants lacking goa-1 accumulate abnormally high levels of the DAG-binding protein UNC-13 at motor neuron nerve terminals, suggesting that serotonin inhibits synaptic transmission by decreasing the abundance of UNC-13 at release sites (Nurrish, 1999).

Neurotransmitter release at C. elegans neuromuscular junctions is facilitated by a presynaptic pathway composed of a Gqalpha (EGL-30), EGL-8 phospholipase Cbeta (PLCbeta), and the diacylglycerol- (DAG-) binding protein UNC-13. Activation of this pathway increases release of acetylcholine at neuromuscular junctions, whereas inactivation decreases release. Phorbol esters stimulate acetylcholine release, and this effect is blocked by a mutation that eliminates phorbol ester binding to UNC-13. Expression of a constitutively membrane-bound form of UNC-13 restores acetylcholine release to mutants lacking the egl-8 PLCbeta. Activation of this pathway with muscarinic agonists causes UNC-13 to accumulate in punctate structures in the ventral nerve cord. These results suggest that presynaptic DAG facilitates synaptic transmission and that part of this effect is mediated by UNC-13 (Lackner, 1999).

The synaptic physiology of unc-13 mutants was analyzed in the nematode C. elegans. Mutants of unc-13 have normal nervous system architecture, and the densities of synapses and postsynaptic receptors were normal at the neuromuscular junction. However, the number of synaptic vesicles at neuromuscular junctions is two- to three-fold greater in unc-13 mutants than in wild-type animals. Most importantly, evoked release at both GABAergic and cholinergic synapses is almost absent in unc-13 null alleles, as determined by whole-cell, voltage-clamp techniques. Although mutant synapses have morphologically docked vesicles, these vesicles are not competent for release as assayed by spontaneous release in calcium-free solution or by the application of hyperosmotic saline. These experiments support models in which UNC-13 mediates either fusion of vesicles during exocytosis or priming of vesicles for fusion (Richmond, 1999).

The priming step of synaptic vesicle exocytosis is thought to require the formation of the SNARE complex, which comprises the proteins synaptobrevin, SNAP-25 and syntaxin. In solution syntaxin adopts a default, closed configuration that is incompatible with formation of the SNARE complex. Specifically, the amino terminus of syntaxin binds the SNARE motif and occludes interactions with the other SNARE proteins. The N terminus of syntaxin also binds the presynaptic protein UNC-13. Studies in mouse, Drosophila (Aravamudan, 1999) and Caenorhabditis elegans suggest that UNC-13 functions at a post-docking step of exocytosis, most likely during synaptic vesicle priming. Therefore, UNC-13 binding to the N terminus of syntaxin may promote the open configuration of syntaxin. To test this model, mutations were engineered into C. elegans syntaxin that cause the protein to adopt the open configuration constitutively. The open form of syntaxin can bypass the requirement for UNC-13 in synaptic vesicle priming. Thus, it is likely that UNC-13 primes synaptic vesicles for fusion by promoting the open configuration of syntaxin (Richmond, 2001).

Syntaxin adopts a closed configuration in solution. However, mutations in two highly conserved amino acids (L165A, E166A) cause syntaxin to adopt a constitutively open configuration in vitro. The corresponding mutations were made in C. elegans syntaxin (L166A, E167A; open syntaxin). Similar to vertebrate open syntaxin, the mutated C. elegans protein can bind synaptobrevin but not UNC-18 in pull-down assays (Richmond, 2001).

Expression of open syntaxin can fully rescue null mutations of syntaxin. unc-64(js115) is a null allele of the gene encoding C. elegans syntaxin. Homozygotes of unc-64(js115) are completely paralysed and arrest development after hatching. This developmental defect is fully rescued by expression of wild-type syntaxin or the open form of syntaxin in null mutants. Expression of either form of syntaxin rescues the behavioural phenotypes associated with unc-64(js115). Furthermore, expression of open syntaxin does not affect neuronal development (Richmond, 2001).

UNC-13 contains several C2 domains that are calcium-binding motifs. The presence of these domains suggest that UNC-13 might be a calcium sensor for synaptic vesicle exocytosis. If UNC-13 were the sole calcium sensor, then in the absence of UNC-13 there should be no calcium-dependent release. Consistent with this hypothesis, unc-13(s69) mutants completely lack calcium-dependent evoked responses, and overexpression of wild-type syntaxin fails to rescue evoked release in the unc-13(s69) mutants. However, overexpression of open syntaxin completely restores evoked responses to wild-type levels. These normal responses to calcium in the absence of UNC-13 are consistent with the observation that overexpression of rat UNC-13 in chromaffin cells does not affect the calcium sensitivity of release. Together, these data demonstrate that UNC-13 is not the calcium sensor that triggers fusion of synaptic vesicles (Richmond, 2001).

Vesicles become fusion competent at the priming step of exocytosis. At the molecular level, priming is thought to be mediated by the formation of the SNARE complex. Overexpression of Munc13-1 in bovine chromaffin cells accelerates the forward rate constant for the priming of morphologically docked, large dense-core vesicles without affecting the rate of fusion or the calcium sensitivity of release. This stage of dense-core vesicle exocytosis coincides with the association of the SNARE proteins. Although Munc13-1 levels are normally very low in chromaffin cells, these observations suggest that UNC-13 can function to promote dense-core vesicle priming, possibly by promoting formation of the SNARE complex. The data confirm and extend these studies by demonstrating that UNC-13 promotes the priming of synaptic vesicles by acting through syntaxin. Specifically, the role of UNC-13 may be to bind the autoinhibitory domain of syntaxin to promote or maintain the open state and thus facilitate formation of the SNARE complex (Richmond, 2001).

The rescue of the Unc-13 phenotype from no evoked responses to wild-type levels of evoked responses by open syntaxin is a dramatic result; however, these animals are not completely wild type. First, body thrashing and locomotory activity of the mutant is greatly reduced compared with the wild type. Second, measures of endogenous release of synaptic vesicles in the presence of calcium is also greatly reduced compared with the wild type. There are several possible explanations for these results. One potential explanation is that the L166A and E167A mutations do not completely mimic the conformation of syntaxin when it is bound to UNC-13. Alternatively, UNC-13 may have an additional role in vesicle exocytosis, possibly to tether synaptic vesicles near calcium channels. Nevertheless, these data suggest that UNC-13 stimulates priming by opening syntaxin either through the direct interaction previously demonstrated or by acting on another protein, such as UNC-18 (Richmond, 2001).

The C. elegans UNC-13 protein and its mammalian homologs are important for normal neurotransmitter release. A set of transcripts identified from the unc-13 locus in C. elegans results from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. The sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 have been analyzed alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. A deletion allele has been isolated that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products (Kohn, 2000).

Mammalian unc-13 homologs

The unc-13 gene in Caenorhabditis elegans is essential for normal presynaptic function and encodes a large protein with C1- and C2-domains. In protein kinase C and synaptotagmin, C1- and/or C2-domains are regulatory domains for Ca2+, phospholipids, and diacylglycerol, suggesting a role for unc-13 in regulating neurotransmitter release. To determine if a similar protein is a component of the presynaptic machinery for neurotransmitter release in vertebrates, unc-13 homologs were studied in rat. Molecular cloning revealed that three homologs of unc-13 called Munc13-1, -13-2, and -13-3 are expressed in rat brain. Munc13s are large, brain-specific proteins with divergent N termini but conserved C termini containing C1- and C2-domains. Specific antibodies demonstrated that Munc13-1 is a peripheral membrane protein that is enriched in synaptosomes and localized to plasma membranes but absent from synaptic vesicles. These data suggest that the function of unc-13 in C. elegans is conserved in mammals and that Munc13s act as plasma membrane proteins in nerve terminals. The presence of C1- and C2-domains in these proteins and the phenotype of the C. elegans mutants raises the possibility that Munc13s may have an essential signaling role during neurotransmitter release (Brose, 1995).

Munc13 proteins constitute a family of three highly homologous molecules (Munc13-1, Munc13-2 and Munc13-3). With the exception of a ubiquitously expressed Munc13-2 splice variant, Munc13 proteins are brain-specific. Munc13-1 has a central priming function in synaptic vesicle exocytosis from glutamatergic synapses. In order to identify Munc13-like proteins that may regulate secretory processes in non-glutamatergic neurons or non-neuronal cells, protein profiles were developed for two Munc13-homology-domains (MHDs). MHDs are present in a wide variety of proteins, some of which have previously been implicated in membrane trafficking reactions. Taking advantage of partial sequences in the human expressed sequence tag (EST) database, a novel, ubiquitously expressed, rat protein (Munc13-4) was characterized that belongs to a subfamily of Munc13-like molecules, in which the typical Munc13-like domain structure is conserved. Munc13-4 is predominantly expressed in lung where it is localizes to goblet cells of the bronchial epithelium and to alveolar type II cells, both of which are cell types with secretory function. In the present study a group of novel proteins has been identified; some of these proteins may function in a Munc13-like manner to regulate membrane trafficking. The MHD profiles described in the present study are useful tools for the identification of Munc13-like proteins, which would otherwise have remained undetected (Koch, 2000).

Munc13-1 is one of three closely related rat homologs of C. elegans unc-13. Based on the high degree of similarity between unc-13 and Munc13 proteins, it is thought that their essential function has been conserved from C. elegans to mammals. Munc13-1 is a brain-specific peripheral membrane protein with multiple regulatory domains that may mediate diacylglycerol, phospholipid, and calcium binding. The C-terminus of Munc13-1 interacts directly with a putative coiled coil domain in the N-terminal part of syntaxin. Syntaxin is a component of the exocytotic synaptic core complex, a heterotrimeric protein complex with an essential role in transmitter release. Through this interaction, Munc13-1 binds to a subpopulation of the exocytotic core complex containing synaptobrevin, SNAP25 (synaptosomal-associated protein of 25 kDa), and syntaxin, but to no other tested syntaxin-interacting or core complex-interacting protein. The site of interaction in syntaxin is similar to the binding site for the unc-18 homolog Munc18, but different from that of all other known syntaxin interactors. These data indicate that unc-13-related proteins may indeed be involved in the mediation or regulation of synaptic vesicle exocytosis by modulating or regulating core complex formation. The similarity between the unc-13 and unc-18 phenotypes is paralleled by the coincidence of the binding sites for Munc13-1 and Munc18 in syntaxin. It is possible that the phenotype of unc-13 and unc-18 mutations is caused by the inability of the respective mutated gene products to bind to syntaxin (Betz, 1997).

Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering -- priming to fusion competence, and Ca2+-triggered fusion -- are taking place in a highly coordinated manner. The active zone components Munc13-1 (an essential vesicle priming protein) and RIM1 (a Rab3 effector with a putative role in vesicle tethering) interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles (Betz, 2001).

Munc13-1 and DOC2 have been implicated in the regulation of exocytosis. In vivo these two proteins undergo a transient phorbol ester-mediated and protein kinase C-independent interaction, resulting in the translocation of DOC2 from a vesicular localization to the plasma membrane. The translocation of DOC2 is dependent upon the DOC2 Munc interacting domain that binds specifically to Munc13-1, whereas the association of DOC2 with intracellular membranes is dependent on its C2 domains. This is the first direct in vivo demonstration of a protein-protein interaction between two presynaptic proteins and may represent a molecular basis for phorbol ester-dependent enhancement of exocytosis (Duncan, 2000).

Msec7-1, a mammalian homolog of yeast sec7p, is a specific GDP/GTP exchange factor for small G-proteins of the ARF family. Overexpression of msec7-1 in Xenopus neuromuscular junctions leads to an increase in synaptic transmitter release that is most likely caused by an increase in the pool of readily releasable vesicles. However, the molecular mechanisms by which msec7-1 is targeted to presynaptic compartments and enhances neurotransmitter release are not known. Msec7-1 is shown to interact directly with Munc13-1, a phorbol ester-dependent enhancer of neurotransmitter release that is specifically localized to presynaptic transmitter release zones. Given that Munc13-1 and msec7-1 participate in very similar presynaptic processes and because Munc13-1 is specifically targeted to presynaptic active zones, it is suggested that the msec7-1/Munc13-1 interaction serves to colocalize the two proteins at the active zone, a subcellular compartment with extremely high membrane turnover (Neeb, 1999).

Human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, the subcellular localization of hmunc13 was investigated in a transiently transfected renal cell line, opossum kidney cells. It was found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant, in which the diacylglycerol (C1) binding domain is absent, exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. It is concluded that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, these data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia (Song, 1999).

In chromaffin cells the number of large dense-core vesicles (LDCVs) that can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, Munc13-1 was overexpressed in chromaffin cells and secretion was stimulated by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, are increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, is increased by the same factor. The response to a second flash, however, is greatly reduced, indicating a depletion of release-competent vesicles. Since there is no apparent change in the number of docked vesicles, it is concluded that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles (Ashery, 2000).

Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release. In the present study the regional, cellular and subcellular expression patterns in rat of two novel Munc13 proteins, Munc13-2 and Munc13-3, were examined. Munc13-1 mRNA is expressed throughout the brain, whereas Munc13-2 mRNA is preferentially present in rostral brain regions, and Munc13-3 mRNA in caudal areas. The novel Munc13 proteins are enriched in synapses. Munc13-3, like Munc13-1, is concentrated in presynaptic terminals. Thus Munc13 proteins are members of a family of neuron-specific, synaptic molecules that bind to syntaxin, an essential mediator of neurotransmitter release. Munc13-2 and Munc13-3 are expressed in a complementary fashion and might act in concert with Munc13-1 to modulate neurotransmitter release (Augustin, 1999a).

Neurotransmitter release at synapses between nerve cells is mediated by calcium-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at the presynaptic release site where they mature to a fusion-competent state. Munc13-1, a brain-specific presynaptic phorbol ester receptor, has been identified as an essential protein for synaptic vesicle maturation. Glutamatergic hippocampal neurons from mice lacking Munc13-1 form ultrastructurally normal synapses whose synaptic-vesicle cycle is arrested at the maturation step. Transmitter release from mutant synapses cannot be triggered by action potentials, calcium-ionophores or hypertonic sucrose solution. In contrast, release evoked by alpha-latrotoxin is indistinguishable from wild-type controls, indicating that the toxin can bypass Munc13-1-mediated vesicle maturation. A small subpopulation of synapses of any given glutamatergic neuron as well as all synapses of GABA-containing neurons are unaffected by Munc13-1 loss, demonstrating the existence of multiple and transmitter-specific synaptic vesicle maturation processes in synapses (Augustin, 1999b).

Munc13 proteins form a family of three, primarily brain-specific phorbol ester receptors (Munc13-1/2/3) in mammals. Munc13-1 is a component of presynaptic active zones in which it acts as an essential synaptic vesicle priming protein. In contrast to Munc13-1, which is present in most neurons throughout the rat and mouse CNS, Munc13-3 is almost exclusively expressed in the cerebellum. Munc13-3 mRNA is present in granule and Purkinje cells but absent from glia cells. Munc13-3 protein is localized to the synaptic neuropil of the cerebellar molecular layer but is not found in Purkinje cell dendrites, suggesting that Munc13-3, like Munc13-1, is a presynaptic protein at parallel fiber-Purkinje cell synapses. To examine the role of Munc13-3 in cerebellar physiology, Munc13-3-deficient mutant mice were generated. Munc13-3 deletion mutants exhibit increased paired-pulse facilitation at parallel fiber-Purkinje cell synapses. In addition, mutant mice display normal spontaneous motor activity but have an impaired ability to learn complex motor tasks. These data demonstrate that Munc13-3 regulates synaptic transmission at parallel fiber-Purkinje cell synapses. It is proposed that Munc13-3 acts at a similar step of the synaptic vesicle cycle as does Munc13-1, albeit with less efficiency. In view of the present data and the well established vesicle priming function of Munc13-1, it is likely that Munc13-3-loss leads to a reduction in release probability at parallel fiber-Purkinje cell synapses by interfering with vesicle priming. This, in turn, would lead to increases in paired-pulse facilitation and could contribute to the observed deficit in motor learning (Augustin, 2001).

Ribbon synapses, for example of the retina, are specialized synapses that differ from conventional, phasically active synapses in several aspects. Ribbon synapses can tonically and yet very rapidly release neurotransmitter via synaptic vesicle exocytosis. This requires an optimization of the synaptic machinery and is at least partly due to the presence of synaptic ribbons that bind large numbers of synaptic vesicles and which are believed to participate in priming synaptic vesicles for exocytosis. This paper analyzes whether ribbon synapses of the retina employ similar priming factors, i.e. Munc13-1, as do conventional, non-ribbon containing phasically active synapses. Though present in conventional synapses of the retina Munc13-1 is completely absent from ribbon-containing synapses of the retina, both in the outer as well as in the inner plexiform layer. This indicates that ribbon synapses of the retina employ other, possibly more potent priming factors than phasically active conventional synapses (Schmitz, 2001).


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