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, synaptic vesicle exocytosis

Symbol - unc-13

FlyBase ID: FBgn0025726

Genetic map position -

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

Cellular location - cytoplasmic

NCBI link: Entrez Gene

unc-13 orthologs: Biolitmine
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
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.
Fulterer, A., Andlauer, T. F. M., Ender, A., Maglione, M., Eyring, K., Woitkuhn, J., Lehmann, M., Matkovic-Rachid, T., Geiger, J. R. P., Walter, A. M., Nagel, K. I. and Sigrist, S. J. (2018). Active zone scaffold protein ratios tune functional diversity across brain synapses. Cell Rep 23(5): 1259-1274. PubMed ID: 29719243
High-throughput electron microscopy has started to reveal synaptic connectivity maps of single circuits and whole brain regions, for example, in the Drosophila olfactory system. However, efficacy, timing, and frequency tuning of synaptic vesicle release are also highly diversified across brain synapses. These features critically depend on the nanometer-scale coupling distance between voltage-gated Ca(2+) channels (VGCCs) and the synaptic vesicle release machinery. Combining light super resolution microscopy with in vivo electrophysiology, this study shows that two orthogonal scaffold proteins (ELKS family Bruchpilot, BRP, and Syd-1) cluster-specific (M)Unc13 release factor isoforms either close (BRP/Unc13A) or further away (Syd-1/Unc13B) from VGCCs across synapses of the Drosophila olfactory system, resulting in different synapse-characteristic forms of short-term plasticity. Moreover, BRP/Unc13A versus Syd-1/Unc13B ratios were different between synapse types. Thus, variation in tightly versus loosely coupled scaffold protein/(M)Unc13 modules can tune synapse-type-specific release features, and "nanoscopic molecular fingerprints" might identify synapses with specific temporal features.
Xu, S., Pany, S., Benny, K., Tarique, K., Al-Hatem, O., Gajewski, K., Leasure, J. L., Das, J. and Roman, G. (2018). Ethanol regulates presynaptic activity and sedation through presynaptic Unc13 proteins in Drosophila. eNeuro 5(3). PubMed ID: 29911175
One possible presynaptic effector for ethanol is the Munc13-1 protein. This study shows that ethanol binding to the rat Munc13-1 C1 domain, at concentrations consistent with binge exposure, reduces diacylglycerol (DAG) binding. The inhibition of DAG binding is predicted to reduce the activity of Munc13-1 and presynaptic release. In Drosophila, this study shows that sedating concentrations of ethanol significantly reduce synaptic vesicle release in olfactory sensory neurons (OSNs), while having no significant impact on membrane depolarization and Ca(2+) influx into the presynaptic compartment. These data indicate that ethanol targets the active zone in reducing synaptic vesicle exocytosis. Drosophila, haploinsufficent for the Munc13-1 ortholog Dunc13, are more resistant to the effect of ethanol on presynaptic inhibition. Genetically reducing the activity of Dunc13 through mutation or expression of RNAi transgenes also leads to a significant resistance to the sedative effects of ethanol. The neuronal expression of Munc13-1 in heterozygotes for a Dunc13 loss-of-function mutation can largely rescue the ethanol sedation resistance phenotype, indicating a conservation of function between Munc13-1 and Dunc13 in ethanol sedation. Hence, reducing Dunc13 activity leads to naive physiological and behavioral resistance to sedating concentrations of ethanol. It is propose that reducing Dunc13 activity, genetically or pharmacologically by ethanol binding to the C1 domain of Munc13-1/Dunc13, promotes a homeostatic response that leads to ethanol tolerance.
Bohme, M. A., McCarthy, A. W., Grasskamp, A. T., Beuschel, C. B., Goel, P., Jusyte, M., Laber, D., Huang, S., Rey, U., Petzoldt, A. G., Lehmann, M., Gottfert, F., Haghighi, P., Hell, S. W., Owald, D., Dickman, D., Sigrist, S. J. and Walter, A. M. (2019). Rapid active zone remodeling consolidates presynaptic potentiation. Nat Commun 10(1): 1085. PubMed ID: 30842428
Neuronal communication across synapses relies on neurotransmitter release from presynaptic active zones (AZs) followed by postsynaptic transmitter detection. Synaptic plasticity homeostatically maintains functionality during perturbations and enables memory formation. Postsynaptic plasticity targets neurotransmitter receptors, but presynaptic mechanisms regulating the neurotransmitter release apparatus remain largely enigmatic. By studying Drosophila neuromuscular junctions (NMJs) this study shows that AZs consist of nano-modular release sites and identify a molecular sequence that adds modules within minutes of inducing homeostatic plasticity. This requires cognate transport machinery and specific AZ-scaffolding proteins. Structural remodeling is not required for immediate potentiation of neurotransmitter release, but necessary to sustain potentiation over longer timescales. Finally, mutations in Unc13 disrupting homeostatic plasticity at the NMJ also impair short-term memory when central neurons are targeted, suggesting that both plasticity mechanisms utilize Unc13. Together, while immediate synaptic potentiation capitalizes on available material, it triggers the coincident incorporation of modular release sites to consolidate synaptic potentiation.
Woitkuhn, J., Ender, A., Beuschel, C. B., Maglione, M., Matkovic-Rachid, T., Huang, S., Lehmann, M., Geiger, J. R. P. and Sigrist, S. J. (2020). The Unc13A isoform is important for phasic release and olfactory memory formation at mushroom body synapses. J Neurogenet: 1-9. PubMed ID: 31980003
The cellular analysis of mushroom body (MB)-dependent memory forming processes is far advanced, whereas, the molecular and physiological understanding of their synaptic basis lags behind. Recent analysis of the Drosophila olfactory system showed that Unc13A, a member of the M(Unc13) release factor family, promotes a phasic, high release probability component, while isoform Unc13B supports a slower tonic release component, reflecting their different nanoscopic positioning within individual active zones. This study used STED super-resolution microscopy of MB lobe synapses to show that Unc13A clusters closer to the active zone centre than Unc13B. Unc13A specifically supported phasic transmission and short-term plasticity of Kenyon cell:output neuron synapses, measured by combining electrophysiological recordings of output neurons with optogenetic stimulation. Knockdown of unc13A within Kenyon cells provoked drastic deficits of olfactory aversive short-term and anaesthesia-sensitive middle-term memory. Knockdown of unc13B provoked milder memory deficits. Thus, a low frequency domain transmission component is probably crucial for the proper representation of memory-associated activity patterns, consistent with sparse Kenyon cell activation during memory acquisition and retrieval. Notably, Unc13A/B ratios appeared highly diversified across MB lobes, leaving room for an interplay of activity components in memory encoding and retrieval.
Pooryasin, A., Maglione, M., Schubert, M., Matkovic-Rachid, T., Hasheminasab, S. M., Pech, U., Fiala, A., Mielke, T. and Sigrist, S. J. (2021). Unc13A and Unc13B contribute to the decoding of distinct sensory information in Drosophila. Nat Commun 12(1): 1932. PubMed ID: 33771998
The physical distance between presynaptic Ca(2+) channels and the Ca(2+) sensors triggering the release of neurotransmitter-containing vesicles regulates short-term plasticity (STP). While STP is highly diversified across synapse types, the computational and behavioral relevance of this diversity remains unclear. In the Drosophila brain, at nanoscale level, distinct coupling distances can be distinguished between Ca(2+) channels and the (m)unc13 family priming factors, Unc13A and Unc13B. Importantly, coupling distance defines release components with distinct STP characteristics. This study shows that while Unc13A and Unc13B both contribute to synaptic signalling, they play distinct roles in neural decoding of olfactory information at excitatory projection neuron (ePN) output synapses. Unc13A clusters closer to Ca(2+) channels than Unc13B, specifically promoting fast phasic signal transfer. Reduction of Unc13A in ePNs attenuates responses to both aversive and appetitive stimuli, while reduction of Unc13B provokes a general shift towards appetitive values. Collectively, this study provides direct genetic evidence that release components of distinct nanoscopic coupling distances differentially control STP to play distinct roles in neural decoding of sensory information.
Jusyte, M., Blaum, N., Bohme, M. A., Berns, M. M. M., Bonard, A. E., Vámosi Á, B., Pushpalatha, K. V., Kobbersmed, J. R. L. and Walter, A. M. (2023). Unc13A dynamically stabilizes vesicle priming at synaptic release sites for short-term facilitation and homeostatic potentiation. Cell Rep 42(6): 112541. PubMed ID: 37243591
Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A's calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca(2+)/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity.

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).

Rapid homeostatic modulation of transsynaptic nanocolumn rings

Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, it was found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. Distinct phases of structural transsynaptic homeostatic plasticity were revealed, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy (Muttathukunnel, 2022).

This study has identified a stereotypic arrangement of transsynaptically aligned molecular nanocolumns that is regulated in a modular and sequential fashion during homeostatic plasticity at the Drosophila NMJ. Moreover, a GluR subtype-specific nano-organization was revealed and it was discovered that the auxiliary GluR subunit Neto is required for rapid homeostatic modulation of transsynaptic nanocolumn number and neurotransmitter release (Muttathukunnel, 2022).

Previous work demonstrated that a cluster of voltage-gated Ca2+ channels localizes to the Brp ring center at the Drosophila NMJ. Furthermore, Unc13A, a molecule suggested as a molecular correlate of presynaptic release sites, forms ring-like arrays in close proximity to Brp C termini and GluRs. In light of these findings, the results are consistent with a model in which Ca2+ influx at the Brp/AZ center induces neurotransmitter release in the nanocolumn rings. Given that the neurotransmitter content released by a single synaptic vesicle does not activate all GluRs of a given PSD at the Drosophila NMJ and that Drosophila GluRs have a low glutamate affinity, neurotransmitter release may predominantly activate GluRs that are aligned to presynaptic release sites. Some evidence suggests that synaptic transmission predominantly occurs within transsynaptic nanocolumns. Hence, the transsynaptic nanocolumn rings discovered in this study may reflect subsynaptic transmission modules that are activated by a common Ca2+-channel cluster. Future work is needed to relate the molecular nanocolumn topography to synaptic physiology, for example, by assessing how many GluRs are activated by neurotransmitter release from a single synaptic vesicle. In this regard, the slight offset between Unc13A and GluR rings may indicate that a given release site may not only activate a single aligned GluR cluster but also neighboring GluR clusters, consistent with physiology data (Muttathukunnel, 2022).

GluR subunit composition and GluR location with regard to release sites are important factors determining synaptic efficacy. At the Drosophila NMJ, the ratio of slowly and rapidly desensitizing GluRIIA- and GluRIIB-containing receptors is a key regulator of quantal size. This study revealed that transsynaptic nanocolumns harbor a mix of GluRIIA- and GluRIIB-containing receptors, and that ambient receptors, which represent almost half of the GluRs within a PSD, mainly incorporate the GluRIIB subunit. The persistence of transsynaptic nanocolumn rings in GluRIIA and GluRIIB mutants implies that neither of these subunits alone is sufficient for ring formation or transsynaptic alignment. Previous work revealed no defects in spontaneous or AP-evoked synaptic transmission upon GluRIIA overexpression or after GluRIIB loss . Thus, two genetic manipulations that mainly decrease ambient receptor abundance, but not receptors inside the nanocolumn ring, do not induce a corresponding decrease in synaptic transmission. This indicates that synaptic transmission is largely confined to transsynaptic nanocolumn rings and/or that synaptic transmission outside the rings is dominated by rapidly desensitizing GluRIIB-containing receptors. Moreover, the observation of increased mEPSP amplitudes in GluRIIBSP5 mutants suggests that GluRIIB-containing receptors surrounding the nanocolumns have the potential to negatively regulate synaptic transmission by replacing GluRIIA-containing receptors within the nanocolumns (Muttathukunnel, 2022).

A variety of auxiliary subunits control GluR assembly, trafficking, and function. The auxiliary GluR subunit Neto has been implicated in GluR clustering at the Drosophila NMJ (Kim, 2021). This uncovered modular ring arrays of Neto-β that transsynaptically align with Brp C termini, suggesting that this auxiliary GluR subunit is a postsynaptic element of transsynaptic nanocolumn rings. The persistence of transsynaptic nanocolumn rings in hypomorphic neto109 mutants suggests that neto is not crucial for ring formation or transsynaptic alignment, or that the remaining Neto was sufficient for transsynaptic nanocolumn ring formation. In contrast to neto109 mutants, in which both Neto-α and Neto-β levels are reduced, loss of Neto-α does not decrease GluR levels or mEPSP amplitude, suggesting that this Neto isoform either does not stabilize GluRs at the Drosophila NMJ or that there is a compensation by Neto-β. While reduced levels of ambient receptors do not impair synaptic transmission in case of GluRIIA overexpression or in GluRIIBSP5 mutants, the decreased GluR abundance within the rings of neto109 mutants correlates with a decrease in spontaneous and AP-evoked synaptic transmission, again implying that synaptic transmission predominantly occurs within the rings (Muttathukunnel, 2022).

GluR impairment at the Drosophila NMJ induces a homeostatic increase in release, and there is evidence for the modulation of presynaptic nano-architecture during this form of homeostatic plasticity. A previous study reported increased GluR levels upon sustained pharmacological GluR inhibition for several days. This study demonstrates GluR modulation within 5 min after pharmacological GluR impairment that precedes the modulation of Brp, as well as Neto-β. Although it cannot be excluded that other molecules are modulated prior to GluRs, or that small changes in Brp or Neto-&beta could not be resolved; after PhTX treatment for 5 min, the data imply that GluR modulation precedes Neto-β and presynaptic regulation during homeostatic plasticity. Furthermore, GluR and Brp fluorescence intensity changes detected with confocal microscopy preceded the increase in GluR and Brp cluster numbers at STED resolution. This could either indicate that small nanostructural changes could not be detected with STED microscopy or that the modulation of transsynaptic nano-architecture lags behind the regulation of GluR and Brp levels or distribution. Similar to the data obtained with confocal microscopy, the increase in GluR cluster number preceded Brp cluster regulation upon GluR perturbation, again indicative of a temporal sequence of transsynaptic changes during PHP. Interestingly, while GluR, but not Brp cluster number increased 15 min after PhTX treatment, a larger fraction of transsynaptically aligned Brp clusters was noted. This suggests that transsynaptic nanocolumn formation likely precedes Brp cluster formation. The temporal sequence of GluR and Brp regulation may also explain the existence of GluR clusters within the ring that are not opposed by Brp. Together, these findings are consistent with a model of coordinated, transsynaptic, and modular structural plasticity during PHP that results in the addition of transsynaptic nanocolumns to the ring (Muttathukunnel, 2022).

Apparent changes in GluR fluorescence intensity, GluR cluster number, or homeostatic potentiation of release upon pharmacological GluR perturbation in hypomorphic neto109 mutants were not observed. This shows that wild-type Neto levels are required for homeostatic control of GluRs and presynaptic release. GluR inhibition also led to a slight but significant increase in Brp fluorescence intensity in neto109 mutants, which was less pronounced than in wild type. The defect in PHP seen in neto109 mutants could thus arise from impaired GluR and/or Brp regulation. Although the genetic data establish a causal relationship between the homeostatic regulation of transsynaptic nanocolumns and presynaptic release, future work is required to scrutinize the relationship between transsynaptic nano-architecture and synaptic transmission, and to dissect the molecular mechanisms controlling transsynaptic nano-architecture and its homeostatic regulation. In this regard, it will be exciting to explore which molecules are involved in transsynaptic alignment and ring formation. Synaptic cell-adhesion molecules, such as neurexins and neuroligins, represent obvious candidates (Muttathukunnel, 2022).

Transient active zone remodeling in the Drosophila mushroom body supports memory

Elucidating how the distinct components of synaptic plasticity dynamically orchestrate the distinct stages of memory acquisition and maintenance within neuronal networks remains a major challenge. Specifically, plasticity processes tuning the functional and also structural state of presynaptic active zone (AZ) release sites are widely observed in vertebrates and invertebrates, but their behavioral relevance remains mostly unclear. This study provides evidence that a transient upregulation of presynaptic AZ release site proteins supports aversive olfactory mid-term memory in the Drosophila mushroom body (MB). Upon paired aversive olfactory conditioning, AZ protein levels (ELKS-family BRP/(m)unc13-family release factor Unc13A) increased for a few hours with MB-lobe-specific dynamics. Kenyon cell (KC, intrinsic MB neurons)-specific knockdown (KD) of BRP did not affect aversive olfactory short-term memory (STM) but strongly suppressed aversive mid-term memory (MTM). Different proteins crucial for the transport of AZ biosynthetic precursors (transport adaptor Aplip1/Jip-1; kinesin motor IMAC/Unc104; small GTPase Arl8) were also specifically required for the formation of aversive olfactory MTM. Consistent with the merely transitory increase of AZ proteins, BRP KD did not interfere with the formation of aversive olfactory long-term memory (LTM; i.e., 1 day). These data suggest that the remodeling of presynaptic AZ refines the MB circuitry after paired aversive conditioning, over a time window of a few hours, to display aversive olfactory memories (Turrel, 2022).

Synapses are key sites of information processing and storage in the brain. Notably, synaptic transmission is not hardwired but adapts through synaptic plasticity to provide appropriate input-output relationships as well as to process and store information on a circuit level. Still, there are fundamental gaps in understanding of exactly how the dynamic changes of synapse performance intersect with circuit operation and consequently define behavioral states. This is partly due to the inherent complexity of synaptic plasticity mechanisms, which operate across a large range of timescales (sub-second to days) and use a rich spectrum of both pre- and post-synaptic molecular and cellular mechanisms. Lately, refinement processes following the immediate engram formation have been described, which might promote specific neuronal activity patterns to select neurons for longer-term information display and storage (Turrel, 2022).

Synaptic transmission across chemical synapses is evoked by action potentials that activate presynaptic Ca2+ influx through voltage-gated Ca2+ channels to trigger the fusion of synaptic vesicles (SVs) containing neurotransmitter at sites called active zones (AZs). AZs assemble from conserved scaffold proteins, including ELKS (Drosophila ortholog: BRP), RIM, and the RIM-binding protein (RBP) family. Recent work in Drosophila showed that discrete SV release sites form at AZ. In the AZ, the ELKS-family BRP master scaffold protein localizes the critical Munc13 family release factor Unc13A in defined nanoscopic clusters around Ca2+ channels (BRP/Unc13A nanomodules). This AZ architecture of the nanoscale organization between BRP/Unc13 release machinery and the AZ-centric Ca2+ channels is present across all Drosophila synapses, including Kenyon cell (KC) derived AZs, and munc13-clusters also define release sites at central mammalian synapses. Importantly, AZ structure and function is dynamic and can remodel within 10 min, as shown at Drosophila neuromuscular junction (NMJ) synapses (Turrel, 2022).

The Drosophila mushroom body (MB) forms and subsequently stores olfactory memories. Importantly, a depression of SV release from the AZ of intrinsic KCs within specific compartments of the MB lobes was found to promote the formation of olfactory memories within a few minutes of paired conditioning. Indeed, Ca2+ in vivo imaging experiments indicate that dopamine bidirectionally tunes the strength of KC synapses to output neurons, with forward conditioning driving depression of those synapses and backward conditioning generally driving potentiation. How this tuning is executed at AZ level is not yet known (Turrel, 2022).

This study present evidence for AZ remodeling (BRP, Syd1, and Unc13A) to take place within MB lobes after paired conditioning for a few hours and provide genetic evidence that this AZ remodeling within the MB-intrinsic KCs is crucial for mid-term aversive olfactory memories. To identify candidate mechanisms of presynaptic remodeling to then be tested in MB-dependent olfactory memory, the role of AZ remodeling was studied during extended larval NMJ plasticity and relevant transport factors were identified. These data suggest that broad but transient changes of presynaptic AZs depending on the transport of new biosynthetic material support refinement processes within KC and MB circuitry and are specifically needed for stable formation of mid-term olfactory memories (Turrel, 2022).

Historically, postsynaptic plasticity mechanisms have been analyzed extensively, and molecular and cellular processes targeting postsynaptic neurotransmitter receptors have been convincingly connected to learning and memory. At the same time, the necessity of using postsynaptic neurons as reporters of presynaptic activity (and, thus, setup paired recordings) has imposed an additional obstacle specific to the functional study of presynaptic forms of mid- and long-term plasticity. Furthermore, the cellular and molecular processes remodeling presynaptic AZs are not characterized as extensively as those at the postsynapse. Consequently, although widely expressed by excitatory and inhibitory synapses of mammalian brains, the behavioral relevance of longer-term presynaptic plasticity remains largely obscure (Turrel, 2022).

This study combined the possibility of genetically analyzing memory formation and stabilization within discrete neuron populations of the Drosophila MB with the identification of molecular machinery remodeling presynaptic AZs in vivo. Evidence is provided for an extended but temporally restricted (a few hours post training) upregulation of presynaptic AZ proteins across the MB lobes, a process seemingly needed in MB intrinsic neurons to display olfactory MTM (Turrel, 2022).

Notably, the acute formation of aversive STM was previously shown to trigger synaptic depression at the KC::MBON synapse in the respective MB compartments. It is emphasized that the exact relation of the AZ remodeling described in this study to this STM-controlling short-term depression is presently unknown. Particularly, it is not possible to tell whether the conditioning-associated presynaptic remodeling described in this study is indeed potentiating KCs and MB AZs or whether overlapping sets of synapses are involved in STM and MTM formation and display. What can be concluded, however, is that molecular machinery that executes structural remodeling at NMJ AZs is critically needed for MTM within the MB intrinsic neurons. Establishing the degree to which synaptic weight changes are associated with the mechanism of MB presynaptic remodeling will have to await the development of protocols to directly follow synapses in vivo for hours after conditioning. Different from presynaptic remodeling being part of the memory trace or engram itself, the idea is favored that synaptic upregulation might instead execute a refinement function extending over larger parts of the MB AZ populations. Refinement is an emerging concept stating that stable propagation and maintenance of memory traces might depend on homeostatic regulations of neuronal circuitry. Sleep-dependent synaptic plasticity is suggested to similarly play an important role in neuronal circuit refinement after learning (Turrel, 2022).

Notably, it has been recently shown that a similar upregulation of AZ proteins (BRP/Unc13A) is indeed a functional part of Drosophila sleep homeostasis, where it suffices to trigger rebound sleep patterns. It thus appears conceivable that the AZ changes associated with conditioning reported in this study might promote specific MB activity patterns instrumental for MTM. An alternative, not mutually exclusive, option is that the initial synaptic depression associated with aversive conditioning must, on a longer term, be compensated by the MB AZ changes (and potential potentiation) described in this study (Turrel, 2022).

Notably, compartment-specific synaptic changes occur in the MB in response to sheer odor presentation or DAN activity although AZ remodeling in this study behaved strictly conditioning dependent, meaning it was not observed after unpaired conditioning, and appeared broadly distributed. It cannot be excluded, however, that smaller size, compartment-specific AZ changes, have been missed, given the limited resolution of the staining assays (Turrel, 2022).

Cell biological processes remodeling presynaptic AZs at larval NMJ synapses can also be of relevance for memory formation in the adult fly KCs. Concretely, this study found that the MB KC-specific KD of transport factors, which at the NMJ level provoked plasticity profiles similar to BRP, also specifically affected MTM but spared STM. Given that several molecular factors, including transport proteins not directly physically associated with the AZ, fulfilled this relation, it indeed appears likely that retrieving axon-transported biosynthetic AZ precursor material is what is critical here (Turrel, 2022).

Speaking of the specificity of rthe MTM phenotypes in relation to AZ remodeling, this study found STM formation undisturbed, but at the same time, MTM to be severely affected after BRP and transport factor KD. This is strong evidence against the possibility of baseline synaptic defects being responsible for the observed MTM deficits. It is also emphasized that this study achieved behavioral phenotypes by comparatively mild and strictly post-developmental KD and that odor Ca2+ responses in MBON neurons postsynaptic to KC appeared normal in BRP KD flies (Turrel, 2022).

When analyzing in a MB-lobe-specific manner, α/&betal and α'/β' neurons showed stronger and more sustained upregulation of BRP/Unc13A than the γ lobes. This might indicate that the extent and role of refinement across the MB lobes is adapted to their specific roles in memory acquisition and retrieval. This is also in accordance with previous observations showing heterogeneity in the exact AZ protein composition across synapses of the Drosophila brain (Turrel, 2022).

Interestingly, Syd-1 levels are significantly increased 1 h after conditioning in the α/β and α'/β' lobes, whereas it has been shown that Syd-1 levels are not increased 10 min after PhTx treatment at the NMJ. This finding indicates that some of the AZ proteins may be affected differently in those two plasticity processes (Turrel, 2022).

Given the generally observed sparse representation of odors within the MB KCs, one might expect initial synaptic changes to be specific to only a few odor-response KCs. Still, this analysis apparently reveals more extended changes of synaptic AZs across the lobes. Potentially, upon successful conditioning, the initial, more restricted, synaptic changes might be followed by an extended communication between the neurons involved in the memory circuit, potentially including KC::KC communication. Indeed, there is ample evidence for a transfer of requirement between different subsets of KCs in the temporal evolution of olfactory memory. This communication seemingly involves gap junctions between KCs but might in parallel also use chemical synapses and their AZs. Concerning the broad distribution of the AZ changes across compartments, it is interesting to mention that KC-global, conditioning-dependent metabolic changes have been observed, being critical for LTM but also MTM (Turrel, 2022).

It is tempting to speculate that the initial, compartment-specific changes, confined to a few odor-responding KCs, might overcome a threshold to also trigger more global synaptic changes. Also interesting in this context, dorsal paired medial (DPM) neurons' odor response increase following spaced conditioning, also indicating that opposite synaptic strength changes might counterbalance the initial synaptic changes occurring in the memory-relevant compartment or depending on post-synaptic partner neurons provoke either potentiation or depression (Turrel, 2022).

As mentioned above, this study found that KD of BRP in the adult MB lobes did not affect LTM, whereas MTM was decreased both at 1 and 3 h. Such a phenotype, a deficit of MTM but subsequent memory phases being intact, was only rarely observed before (Nep2-RNAi in adult DPM neurons, synapsin mutants with memory deficits up to 1 h but normal memory later on). On one hand, this reinforces the idea that MTM and LTM might form using separate circuits, and on the other hand, that cell types other than KCs might contribute to aversive olfactory LTM formation. Different sets of proteins in the same lobes might operate in parallel circuits similar to what has been observed in the honeybee. However, it might also well be that the presynaptic AZ remodeling observed in this study is indeed specific for the display of MTM and that the synaptic memory traces orchestrating the later recall of LTM are mediated by independent parallel molecular/synaptic mechanisms or distinct circuit (Turrel, 2022).



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).


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).


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).


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).

A comparative analysis of the mobility of 45 proteins in the synaptic bouton

Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, the motion of 45 GFP-tagged synaptic proteins expressed in cultured hippocampal neurons, was analyzed using fluorescence recovery after photobleaching, particle tracking, and modeling. Synaptic vesicle proteins, endo- and exocytosis cofactors, cytoskeleton components, and trafficking proteins were compared. Movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. Protein movement was analyzed thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology (Reshetniak, 2020).

This study provides a first comparative study of protein mobility in the synaptic bouton, encompassing 45 different proteins, from different types and classes. The results confirm several expectations, including the lower mobility of membrane proteins when compared to soluble proteins, or the lower mobility of virtually all proteins in the synapse, when compared to the axon. Other expected observations were that the movement rates of the same proteins in the axon and in the synapse correlated little, presumably due to the different conditions encountered there, or that the mobility of soluble proteins was only little controlled by their molecular size. Several other observations could be made, including relations between protein mobility and structural parameters, mRNA composition, or protein lifetimes (Reshetniak, 2020).

The measurements, as indicated above, were performed with the caveat that the proteins that were measured were more abundant than under normal (wild-type) conditions. The overexpression levels that were observed were mild, but they may nevertheless contribute to artifacts, namely to an over-estimation of the mobility of individual proteins. If one protein is expressed too highly, its copy numbers saturate all binding to interacting partners, and the un-bound molecules end up moving randomly in the synapse, presumably at the highest possible speeds. The various validations that performed suggest that this is not a major problem. At the same time, the reported values should be taken as maximal mobility estimates, due to this issue. Native (non-tagged) proteins have not been investigated often, with only a handful of studies available. Several such studies were reproduced well by the current data. At the same time, a FRAP measurement of knock-in Munc13 provided a substantially lower mobility, with the shortest time constant measured in cultured mouse cortical neurons being around ~3 min, as opposed to a few seconds in the current measurements. This difference probably has both technical and biological grounds. The previous work imaged the synapses at intervals of a few minutes, to avoid photobleaching. Analyzing the current Munc13 data at 20-30 s of intervals (as opposed to two times per second, as in original data) raised the time constant from ~4 to ~30 s. Analyzing such data every few minutes would presumably result in an even longer time constant. Also, the previous work bleached multiple boutons in the same area, which probably resulted in bleaching a considerable proportion of the fast-moving molecules in the respective axons, which will reduce the fluorescence recovery. Nevertheless, it is still possible that Munc13 mobility is particularly sensitive to overexpression (Reshetniak, 2020).

In spite of these caveats, several conclusions could nevertheless be drawn. First, synaptic protein mobility seems to be influenced by the interaction of the proteins with the vesicles. For soluble proteins, it has been hypothesized that strong interactions to the vesicle cluster cause their enrichment in synapses. This was observed especially for synapsin, whose slow movement in synapses was paralleled by strong binding to vesicles. This effect was even more strongly visible for membrane proteins and is mostly explained by the fact that molecules that are more highly enriched in synaptic vesicles are present at lower levels on the plasma membrane. This implies that large fractions of these proteins will recover slowly during FRAP, through the infrequent active transport of synaptic vesicles. This will result in large time constants for the respective proteins. However, this is not the only explanation for this observation. The time constants of bona fide synaptic vesicle proteins are also higher in the axon, when compared to non-vesicular proteins. As all of these molecules are found in axons mainly as molecules fused to the plasma membrane, an explanation based on the transport of synaptic vesicles seems unlikely. A potential solution to this question is that synaptic vesicle proteins may diffuse in the axon in the form of assemblies composed of multiple molecules. This issue has been discussed for several decades, and it is still open for further interpretation. However, a series of recent observations, made mainly through super-resolution imaging of fused synaptic vesicles, suggested that such assemblies are indeed present in the axon, and may even be the dominant form in which vesicle proteins are found in the axonal compartment (Reshetniak, 2020).

Second, soluble unstructured proteins also appeared to move more slowly in synapses. This observation is especially interesting in the context of a recently proposed mechanism of synaptic vesicle cluster segregation. It has been suggested that synaptic vesicles, together with synaptic vesicle binding proteins, form a distinct liquid phase via liquid-liquid phase separation within the synapses. By definition, material exchange between liquid phases is slower than free diffusion; therefore, it is expected that soluble proteins of synaptic vesicle cluster would have slower recovery rates. Since the presence of multiple disordered coils is one of the main structural characteristics of proteins known to take part in liquid phase separation, the current observations fit very well with this model (Reshetniak, 2020).

Third, several correlations could be found to the presence of different amino acids in the protein sequence, or to the presence of particular nucleotides in the mRNA sequence. While the correlations to specific amino acids were relatively easy to interpret, the links to mRNA composition are less obvious. Different parameters of protein homeostasis have been linked to the mRNA composition in mammalian cells, and especially to the mRNA secondary structure or to the GC contents. At the same time, the mRNA composition has been suggested to control the folding conformation of specific proteins. It is still unclear whether the relations between mRNA composition and cell biology parameters are causative in nature, but they are sufficiently strong to enable reasonable predictions of protein abundance, lifetime, and translation rate. Overall, it is therefore not entirely surprising that protein mobility also correlates with mRNA composition, albeit it is difficult to explain why a high percentage of adenine correlates with higher mobility. One hypothesis could be based on the observation that proteins related to specialized function, including synapse formation, are encoded by GC-rich genes. In contrast, proteins involved in cell proliferation and in general cellular metabolism are encoded by AU-rich genes. This implies that “less synaptic” proteins would have mRNAs containing higher adenine percentages than bona fide synaptic proteins. The former would interact less with synaptic vesicles or other synaptic components, and would therefore be more mobile than true synaptic proteins. This hypothesis is supported by the fact that the top adenine-containing targets, which influence mostly these correlations, are SNAP23, Rab5, VAMP4, and SNAP29, trafficking molecules that are not specific to synapses in any fashion. At the opposite end, the top adenine-lacking proteins are the synaptic vesicle markers synaptophysin, synaptogyrin, and vGlut (glutamate transporter), along with the endocytosis cofactor epsin, further confirming this hypothesis (Reshetniak, 2020).

Finally, the FRAP data was analyzed thoroughly, to provide diffusion coefficients for the different proteins. These coefficients were validated by several types of measurements, as described above, and should provide a good starting point for models of synaptic physiology. Laboratories specialized in neuronal and synaptic modeling could exploit the entire dataset by introducing the different protein amounts and mobilities in multi-reaction synaptic models. Importantly, the data could be compared and combined with any dataset on hippocampal cultured synapses, which are a commonly used experimental model, for which large numbers of functional datasets are available (Reshetniak, 2020).

It is concluded that the current work provides a novel resource for the analysis of synaptic function, which should enable synaptic modeling with substantially higher precision than in the past (Reshetniak, 2020).


Search PubMed for articles about Drosophila unc-13

Ahmed, S., et al. (1992). The C. elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor. Biochem. J. 287 (Pt 3): 995-9. 1445255

Aravamudan, B., Fergestad, T., Davis, W. S., Rodesch1, C. J. and Broadie, K. (1999). Drosophila Unc-13 is essential for synaptic transmission. Nature Neurosci. 2: 965-971. 10526334

Ashery, U., et al. (2000). Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells. EMBO J. 19(14): 3586-96. 10899113

Augustin, I., et al. (1999a). Differential expression of two novel Munc13 proteins in rat brain. Biochem. J. 337(Pt 3): 363-71. 9895278

Augustin, I., et al. (1999b). Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400(6743): 457-61. 10440375

Augustin, I., et al. (2001). The cerebellum-specific Munc13 isoform Munc13-3 regulates cerebellar synaptic transmission and motor learning in mice. J. Neurosci. 21(1): 10-17. 11150314

Betz, A., et al. (1997). Direct interaction of the rat unc-13 homologue Munc13-1 with the N terminus of syntaxin. J. Biol. Chem. 272: 2520-2526.

Betz, A., et al. (2001). Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming. Neuron 30(1): 183-96. 11343654

Bohme, M. A., Beis, C., Reddy-Alla, S., Reynolds, E., Mampell, M. M., Grasskamp, A. T., Lutzkendorf, J., Bergeron, D. D., Driller, J. H., Babikir, H., Gottfert, F., Robinson, I. M., O'Kane, C. J., Hell, S. W., Wahl, M. C., Stelzl, U., Loll, B., Walter, A. M. and Sigrist, S. J. (2016). Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling. Nat Neurosci [Epub ahead of print]. PubMed ID: 27526206

Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77: 71-94

Brose, N., et al. (1995). Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. J. Biol. Chem. 270(42): 25273-80. 7559667

Duncan, R. R., et al. (2000). Transient, phorbol ester-induced DOC2-Munc13 interactions in vivo. 7: J. Biol. Chem. 274(39): 27347-50. 10488064

Hu, Z., Tong, X. J. and Kaplan, J. M. (2013). UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans. Elife 2: e00967. PubMed ID: 23951547

Kazanietz, M. G., et al. (1995). Characterization of the cysteine-rich region of the Caenorhabditis elegans protein Unc-13 as a high affinity phorbol ester receptor. Analysis of ligand-binding interactions, lipid cofactor requirements, and inhibitor sensitivity. J. Biol. Chem. 270(18): 10777-83. 7537738

Kim, Y. J., Bao, H., Bonanno, L., Zhang, B. and Serpe, M. (2012). Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction. Genes Dev 26(9): 974-987. PubMed ID: 22499592

Koch, H., Hofmann, K. and Brose, N. (2000). Definition of Munc13-homology-domains and characterization of a novel ubiquitously expressed Munc13 isoform. Biochem. J. 349(Pt 1): 247-53. 10861235

Kohn, R. E., et al. (2000). Expression of multiple UNC-13 proteins in the Caenorhabditis elegans nervous system. Mol. Biol. Cell 11(10): 3441-52. 11029047

Lackner, M. R., Nurrish, S. J. and Kaplan, J. M. (1999). Facilitation of synaptic transmission by EGL-30 Gqalpha and EGL-8 PLCbeta: DAG binding to UNC-13 is required to stimulate acetylcholine release. Neuron 24(2): 335-46. 10571228

Maruyama, I. N. and Brenner, S. (1991). A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans. Proc. Natl. Acad. Sci. 88: 5729-5733.

Muttathukunnel, P., Frei, P., Perry, S., Dickman, D. and Muller, M. (2022). Rapid homeostatic modulation of transsynaptic nanocolumn rings. Proc Natl Acad Sci U S A 119(45): e2119044119. PubMed ID: 36322725

Neeb, A., et al. (1999). Direct interaction between the ARF-specific guanine nucleotide exchange factor msec7-1 and presynaptic Munc13-1. Eur. J. Cell Biol. 78(8): 533-8. 10494859

Nurrish, S., Segalat, L. and Kaplan, J. M. (1999). Serotonin inhibition of synaptic transmission: Galpha(0) decreases the abundance of UNC-13 at release sites. Neuron 24(1): 231-42. 10677040

Reshetniak, S., Ussling, J. E., Perego, E., Rammner, B., Schikorski, T., Fornasiero, E. F., Truckenbrodt, S., Koster, S. and Rizzoli, S. O. (2020). A comparative analysis of the mobility of 45 proteins in the synaptic bouton. EMBO J 39(16): e104596. PubMed ID: 32627850

Richmond, J. E., Davis, W. S. and Jorgensen, E. M. (1999). UNC-13 is required for synaptic vesicle fusion in C. elegans. Nat. Neurosci. 2(11): 959-64. 10526333

Richmond, J. E., Weimer, R. M. and Jorgensen, E. M. (2001). An open form of syntaxin bypasses the requirement for C. elegans Unc-13 in vesicle priming Nature 412: 338-341. 11460165

Sassa, T., et al. (1999). Regulation of the UNC-18-Caenorhabditis elegans syntaxin complex by UNC-13. J. Neurosci. 19(12): 4772-7. 10366611

Schmitz, F., Augustin, I. and Brose, N. (2001). The synaptic vesicle priming protein Munc13-1 is absent from tonically active ribbon synapses of the rat retina. Brain Res. 895(1-2): 258-63. 11259787

Song, Y., Ailenberg, M. and Silverman M. (1999). Human munc13 is a diacylglycerol receptor that induces apoptosis and may contribute to renal cell injury in hyperglycemia. Mol. Biol. Cell 10(5): 1609-19. 10233166

Turrel, O., Ramesh, N., Escher, M. J. F., Pooryasin, A. and Sigrist, S. J. (2022). Transient active zone remodeling in the Drosophila mushroom body supports memory. Curr Biol 32(22): 4900-4913. PubMed ID: 36327980

Xu, X. Z. et al. (1998). Retinal targets for calmodulin include proteins implicated in synaptic transmission. J. Biol. Chem. 273: 31297-31307. 9813038

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date revised: 15 December 2023

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