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).
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
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
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
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.
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
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
Xu, X. Z. et al. (1998). Retinal targets for calmodulin include proteins implicated in synaptic transmission. J. Biol. Chem. 273: 31297-31307. 9813038
date revised: 30 August 2001
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