Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Drosophila Unc-13 is essential for synaptic transmission

Nature Neuroscience volume 2, pages 965–971 (1999)Cite this article

Abstract

The UNC-13 protein family has been suggested to be critical for synaptic vesicle dynamics based on its interactions with Syntaxin, Munc-18 and Doc 2α. We cloned the Drosophila homolog (Dunc-13) and characterized its function using a combination of electrophysiology and ultrastructural analyses. Dunc-13 contained a C1 lipid-binding motif and two C2 calcium-binding domains, and its expression was restricted to neurons. Elimination of dunc-13 expression abolished synaptic transmission, an effect comparable only to removal of the core complex proteins Syntaxin and Synaptobrevin. Transmitter release remained impaired under elevated calcium influx or application of hyperosmotic saline. Ultrastructurally, mutant terminals accumulated docked vesicles at presynaptic release sites. We conclude that Dunc-13 is essential for a stage of neurotransmission following vesicle docking and before fusion.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Dunc-13 is the Drosophila homolog of UNC-13 and Munc-13.
The alternative text for this image may have been generated using AI.
Figure 2: Expression of dunc-13 is restricted to the nervous system.
The alternative text for this image may have been generated using AI.
Figure 3: Identification of an RNA null mutant in dunc-13.
The alternative text for this image may have been generated using AI.
Figure 4: Null dunc-13 mutants have normal synaptic morphology but highly impaired synaptic function.
The alternative text for this image may have been generated using AI.
Figure 5: Electrophysiological analysis of dunc-13 mutant synaptic function.
The alternative text for this image may have been generated using AI.
Figure 6: Synaptic vesicles accumulated in dunc-13 mutants.
The alternative text for this image may have been generated using AI.

Similar content being viewed by others

References

  1. Bennett, M. K. Ca2+ and the regulation of neurotransmitter secretion. Curr. Opin. Neurobiol. 7, 316–322 (1997).

    Article  CAS  Google Scholar 

  2. Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y. C. & Scheller, R. H. SNARE complex formation is triggered by Ca2+ and drives membrane fusion. Cell 97, 165–174 (1999).

    Article  CAS  Google Scholar 

  3. Valtorta, F. & Meldolesi, J. The presynaptic compartment: signals and targets. Semin. Cell Biol. 5, 211– 219 (1994).

    Article  CAS  Google Scholar 

  4. Egea, G., Guitart, X. & Marsal, J. Ultrastructural changes induced by 12-O-tetradecanoylphorbol 13-acetate in pure cholinergic synaptosomes of Torpedo electric organ. J. Neurochem. 57, 1593– 1598 (1991).

    Article  CAS  Google Scholar 

  5. Chavez, R. A., Miller, S. G. & Moore, H. P. A biosynthetic regulated secretory pathway in constitutive secretory cells. J. Cell. Biol. 133, 1177 –1191 (1996).

    Article  CAS  Google Scholar 

  6. Betz, A., Okamoto, M., Benseler, F. & Brose, N. Direct interaction of the rat unc-13 homologue Munc13-1 with the N terminus of syntaxin. J. Biol. Chem. 272, 2520–2526 (1997).

    Article  CAS  Google Scholar 

  7. Sassa, T. et al. Regulation of the UNC-18-Caenorhabditis elegans syntaxin complex by UNC-13. J. Neurosci. 19, 4772 –4777 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Sutton, R. B., Davletov, B. A., Berghuis, A. M., Sudhof, T. C. & Sprang, S. R. Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold. Cell 80, 929–938 (1995).

    Article  CAS  Google Scholar 

  11. Sutton, R. B. & Sprang, S. R. Structure of the protein kinase Cbeta phospholipid-binding C2 domain complexed with Ca2+. Structure 6, 1395–1405 ( 1998).

    Article  CAS  Google Scholar 

  12. Anderson, W. B. & Gopalakrishna, R. Functional and regulatory importance of calcium-mediated hydrophobic regions of calmodulin, protein kinase C, and other calcium-binding proteins. Curr. Top. Cell Regul. 27, 455–469 (1985).

    Article  CAS  Google Scholar 

  13. Brose, N., Hofmann, K., Hata, Y. & Sudhof, T. C. Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. J. Biol. Chem. 270, 25273– 25280 (1995).

    Article  CAS  Google Scholar 

  14. Betz, A. et al. Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release. Neuron 21, 123 –136 (1998).

    Article  CAS  Google Scholar 

  15. Mochida, S., Orita, S., Sakaguchi, G., Sasaki, T. & Takai, Y. Role of the Doc2 alpha-Munc13-1 interaction in the neurotransmitter release process. Proc. Natl. Acad. Sci. USA 95, 11418–11422 (1998).

    Article  CAS  Google Scholar 

  16. Orita, S. et al. Physical and functional interactions of Doc2 and Munc13 in Ca2+- dependent exocytotic machinery. J. Biol. Chem. 272, 16081–16084 (1997).

    Article  CAS  Google Scholar 

  17. Sakaguchi, G. et al. A novel brain-specific isoform of beta spectrin: isolation and its interaction with Munc13. Biochem. Biophys. Res. Commun. 248, 846–851 ( 1998).

    Article  CAS  Google Scholar 

  18. Keshishian, H., Broadie, K., Chiba, A. & Bate, M. The Drosophila neuromuscular junction: a model system for studying synaptic development and function. Annu. Rev. Neurosci. 19, 545 –575 (1996).

    Article  CAS  Google Scholar 

  19. Broadie, K. S. Genetic dissection of the molecular mechanisms of transmitter vesicle release during synaptic transmission. J. Physiol. (Paris) 89 , 59–70 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Currie, D. A., Truman, J. W. & Burden, S. J. Drosophila glutamate receptor RNA expression in embryonic and larval muscle fibers. Dev. Dyn. 203 , 311–316 (1995).

    Article  CAS  Google Scholar 

  22. Littleton, J. T., Bellen, H. J. & Perin, M. S. Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse. Development 118, 1077–1088 (1993).

    CAS  PubMed  Google Scholar 

  23. Chin, A. C., Burgess, R. W., Wong, B. R., Schwarz, T. L. & Scheller, R. H. Differential expression of transcripts from syb, a Drosophila melanogaster gene encoding VAMP (synaptobrevin) that is abundant in non-neuronal cells. Gene 131, 175–181 (1993).

    Article  CAS  Google Scholar 

  24. Broadie, K. S. & Bate, M. Development of the embryonic neuromuscular synapse of Drosophila melanogaster. J. Neurosci. 13, 144–166 (1993).

    Article  CAS  Google Scholar 

  25. Rosenmund, C. & Stevens, C. F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16 , 1197–1207 (1996).

    Article  CAS  Google Scholar 

  26. Fergestad, T., Davis, W. S. & Broadie, K. The stoned proteins regulate synaptic vesicle recycling in the presynaptic terminal. J. Neurosci. 19, 5847–5860 (1999).

    Article  CAS  Google Scholar 

  27. Broadie, K. et al. Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. Neuron 15, 663–673 (1995).

    Article  CAS  Google Scholar 

  28. Richmond, J., Davis, W. S., & Jorgensen, E. M. UNC-13 is required for synaptic vesicle fusion. Nat. Neurosci. 2, 959–964 (1999).

    Article  CAS  Google Scholar 

  29. Augustin, I., Rosenmund, C., Sudhof, T. C. & Brose. Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400, 457–461 (1999).

    Article  CAS  Google Scholar 

  30. Deitcher, D. L. et al. Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin. J. Neurosci. 18, 2028– 2039 (1998).

    Article  CAS  Google Scholar 

  31. Schulze, K. L., Broadie, K., Perin, M. S. & Bellen, H. J. Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission. Cell 80, 311–320 ( 1995).

    Article  CAS  Google Scholar 

  32. Sweeney, S. T., Broadie, K., Keane, J., Niemann, H. & O'Kane, C. J. Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14, 341– 351 (1995).

    Article  CAS  Google Scholar 

  33. Li, J. & Schwarz, T. L. Genetic evidence for an equilibrium between docked and undocked vesicles. Phil. Trans. R. Soc. Lond. B Biol. Sci. 354, 299–306 (1999).

    Article  CAS  Google Scholar 

  34. Yoshihara, M. et al. Selective effects of neuronal-synaptobrevin mutations on transmitter release evoked by sustained versus transient Ca2+ increases and by cAMP. J. Neurosci. 19, 2432– 2441 (1999).

    Article  CAS  Google Scholar 

  35. Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717 –727 (1994).

    Article  CAS  Google Scholar 

  36. Lehmann, R. & Tautz, D. In situ hybridization to RNA. Methods Cell. Biol. 44, 575–598 (1994).

    Article  CAS  Google Scholar 

  37. Broadie, K. & Bate, M. Muscle development is independent of innervation during Drosophila embryogenesis. Development 119, 533–543 ( 1993).

    CAS  PubMed  Google Scholar 

  38. Jan, L. Y. & Jan, Y. N. Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos. Proc. Natl. Acad. Sci. USA 79, 2700– 2704 (1982).

    Article  CAS  Google Scholar 

  39. Broadie, K. S. & Bate, M. Development of larval muscle properties in the embryonic myotubes of Drosophila melanogaster. J. Neurosci. 13, 167–180 (1993).

    CAS  PubMed  Google Scholar 

  40. Prokop, A., Landgraf, M., Rushton, E., Broadie, K. & Bate, M. Presynaptic development at the Drosophila neuromuscular junction: assembly and localization of presynaptic active zones. Neuron 17, 617– 626 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by NIH grant GM54544, a Searle Scholarship to K.B. and an NIH Developmental Biology Training Grant (5T32 HD07491) to T.F and C.R. We thank K. Zinsmaier for providing antibodies to CSP, D. Featherstone and J. Rohrbough for comments on the manuscript and K. Clark for technical advice.

Author information

Author notes

  1. Bharathi Aravamudan and Tim Fergestad: The first two authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, 84112-0840, Utah, USA

    Bharathi Aravamudan, Tim Fergestad, Warren S. Davis, Chris K. Rodesch & Kendal Broadie

Authors
  1. Bharathi Aravamudan
    View author publications

    Search author on:PubMed Google Scholar

  2. Tim Fergestad
    View author publications

    Search author on:PubMed Google Scholar

  3. Warren S. Davis
    View author publications

    Search author on:PubMed Google Scholar

  4. Chris K. Rodesch
    View author publications

    Search author on:PubMed Google Scholar

  5. Kendal Broadie
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Kendal Broadie.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aravamudan, B., Fergestad, T., Davis, W. et al. Drosophila Unc-13 is essential for synaptic transmission. Nat Neurosci 2, 965–971 (1999). https://doi.org/10.1038/14764

Download citation

  • Received:

  • Accepted:

  • Issue date:

  • DOI: https://doi.org/10.1038/14764

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing