BIOLOGICAL OVERVIEW

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

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

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

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

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

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

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

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

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

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

GENE STRUCTURE

cDNA clone length - 5838

Exons - 22

Bases in 3' UTR - 578

PROTEIN STRUCTURE

Amino Acids - 1752 (Unc13-A) and 1715 (Unc-13B)

Structural Domains

A Drosophila EST clone (LD11526) was used to design primers for RACE-PCR by which full-length unc-13 cDNA was isolated. This cDNA is 5824 nucleotides long and predicted to encode a 1752-amino-acid protein containing a single C1 domain and two C2 domains, similar to other UNC-13 family members. Alignment of Drosophila Unc-13 sequence with C. elegans Unc-13 and Munc 13-1 shows an overall 44% identity with C. elegans Unc-13 and 51% identity with Munc 13-1. Homologies among the C1 and C2 domains of Drosophila Inc-13, C. elegans Unc-13 and Munc13-1 are in the range of 70%-90%. This range is similar to that for other highly conserved synaptic proteins such as Synaptotagmin, Synaptobrevin and Syntaxin (Aravamudan, 1999).

Two cDNAs corresponding to Drosophila homologs of C. elegans Unc-13 (Dunc-13A and Dunc-13B) have been isolated in a screen for calmodulin-binding proteins (Xu, 1998). There is a non-consensus calmodulin-binding site in the N-terminus of Dunc-13A but not in Dunc-13B. Most of the protein sequence identified (for instance, amino acids 423-1752) is identical to that of Dunc-13A, including the calmodulin-binding domain (amino acids 487-511); however, an additional unique 422-amino-acid sequence has been identified in the N-terminus, demonstrating a third Dunc-13 isoform in Drosophila (Dunc-13C). Similarly, three C. elegans Unc-13 splice forms have been identified in C. elegans. However, Munc 13-1 and C. elegans Unc-13 contain a third C2 domain at the N-terminus of the protein that is not present in Drosophila Unc-13 (Aravamudan, 1999).

date revised: 30 August 2001

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

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