Systematic analysis of genes required for synapse structure and function

Article metrics


Chemical synapses are complex structures that mediate rapid intercellular signalling in the nervous system. Proteomic studies suggest that several hundred proteins will be found at synaptic specializations. Here we describe a systematic screen to identify genes required for the function or development of Caenorhabditis elegans neuromuscular junctions. A total of 185 genes were identified in an RNA interference screen for decreased acetylcholine secretion; 132 of these genes had not previously been implicated in synaptic transmission. Functional profiles for these genes were determined by comparing secretion defects observed after RNA interference under a variety of conditions. Hierarchical clustering identified groups of functionally related genes, including those involved in the synaptic vesicle cycle, neuropeptide signalling and responsiveness to phorbol esters. Twenty-four genes encoded proteins that were localized to presynaptic specializations. Loss-of-function mutations in 12 genes caused defects in presynaptic structure.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Summary of RNAi screens.
Figure 2: Functional profiling of 60 aldicarb-resistance genes.
Figure 3: Subcellular localization of proteins in motor neurons.
Figure 4: Synaptobrevin distribution in mutants.


  1. 1

    Timmons, L., Court, D. L. & Fire, A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103–112 (2001)

  2. 2

    Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003)

  3. 3

    Simmer, F. et al. Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. PLoS Biol. 1, E12 (2003)

  4. 4

    Kennedy, S., Wang, D. & Ruvkun, G. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427, 645–649 (2004)

  5. 5

    Simmer, F. et al. Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr. Biol. 12, 1317–1319 (2002)

  6. 6

    Miller, K. G. et al. A genetic selection for Caenorhabditis elegans synaptic transmission mutants. Proc. Natl Acad. Sci. USA 93, 12593–12598 (1996)

  7. 7

    Nguyen, M., Alfonso, A., Johnson, C. D. & Rand, J. B. Caenorhabditis elegans mutants resistant to inhibitors of acetylcholinesterase. Genetics 140, 527–535 (1995)

  8. 8

    Nurrish, S., Segalat, L. & Kaplan, J. M. Serotonin inhibition of synaptic transmission: Gα0 decreases the abundance of UNC-13 at release sites. Neuron 24, 231–242 (1999)

  9. 9

    Sonnichsen, B. et al. Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434, 462–469 (2005)

  10. 10

    Boulton, S. J. et al. Combined functional genomic maps of the C. elegans DNA damage response. Science 295, 127–131 (2002)

  11. 11

    Robatzek, M. & Thomas, J. H. Calcium/calmodulin-dependent protein kinase II regulates Caenorhabditis elegans locomotion in concert with a Go/Gq signaling network. Genetics 156, 1069–1082 (2000)

  12. 12

    Lackner, M. R., Nurrish, S. J. & Kaplan, J. M. Facilitation of synaptic transmission by EGL-30 Gqα and EGL-8 PLCβ: DAG binding to UNC-13 is required to stimulate acetylcholine release. Neuron 24, 335–346 (1999)

  13. 13

    Greener, T., Zhao, X., Nojima, H., Eisenberg, E. & Greene, L. E. Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells. J. Biol. Chem. 275, 1365–1370 (2000)

  14. 14

    Kass, J., Jacob, T. C., Kim, P. & Kaplan, J. M. The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans. J. Neurosci. 21, 9265–9272 (2001)

  15. 15

    Jacob, T. C. & Kaplan, J. M. The EGL-21 carboxypeptidase E facilitates acetylcholine release at Caenorhabditis elegans neuromuscular junctions. J. Neurosci. 23, 2122–2130 (2003)

  16. 16

    Jia, K., Chen, D. & Riddle, D. L. The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span. Development 131, 3897–3906 (2004)

  17. 17

    Tatar, M., Bartke, A. & Antebi, A. The endocrine regulation of aging by insulin-like signals. Science 299, 1346–1351 (2003)

  18. 18

    Ann, K., Kowalchyk, J. A., Loyet, K. M. & Martin, T. F. Novel Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated exocytosis. J. Biol. Chem. 272, 19637–19640 (1997)

  19. 19

    Lindberg, I., Tu, B., Muller, L. & Dickerson, I. M. Cloning and functional analysis of C. elegans 7B2. DNA Cell Biol. 17, 727–734 (1998)

  20. 20

    Nathoo, A. N., Moeller, R. A., Westlund, B. A. & Hart, A. C. Identification of neuropeptide-like protein gene families in Caenorhabditis elegans and other species. Proc. Natl Acad. Sci. USA 98, 14000–14005 (2001)

  21. 21

    Rhee, J. S. et al. Beta phorbol ester- and diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs. Cell 108, 121–133 (2002)

  22. 22

    Gillis, K. D., Mossner, R. & Neher, E. Protein kinase C enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool of secretory granules. Neuron 16, 1209–1220 (1996)

  23. 23

    Colbert, H. A., Smith, T. L. & Bargmann, C. I. OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J. Neurosci. 17, 8259–8269 (1997)

  24. 24

    Premkumar, L. S. & Ahern, G. P. Induction of vanilloid receptor channel activity by protein kinase C. Nature 408, 985–990 (2000)

  25. 25

    Reboul, J. et al. C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nature Genet. 34, 35–41 (2003)

  26. 26

    White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Tran. R. Soc. Lond. B 314, 1–340 (1986)

  27. 27

    Paganoni, S. & Ferreira, A. Expression and subcellular localization of Ror tyrosine kinase receptors are developmentally regulated in cultured hippocampal neurons. J. Neurosci. Res. 73, 429–440 (2003)

  28. 28

    Hall, D. H. & Hedgecock, E. M. Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans. Cell 65, 837–847 (1991)

  29. 29

    Zahn, T. R. et al. Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104. Traffic 5, 544–559 (2004)

  30. 30

    Schuske, K. R. et al. Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin. Neuron 40, 749–762 (2003)

  31. 31

    Fuchs, F. & Westermann, B. Role of Unc104/KIF1-related motor proteins in mitochondrial transport in Neurospora crassa. Mol. Biol. Cell 16, 153–161 (2004)

  32. 32

    Kohn, R. et al. Expression of multiple UNC-13 proteins in the C. elegans nervous system. Mol. Biol. Cell 11, 3441–3452 (2000)

  33. 33

    Zhen, M. & Jin, Y. The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans. Nature 401, 371–375 (1999)

  34. 34

    Zhen, M., Huang, X., Bamber, B. & Jin, Y. Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exchanger with a RING-H2 finger domain. Neuron 26, 331–343 (2000)

  35. 35

    Koushika, S. P. et al. A post-docking role for active zone protein Rim. Nature Neurosci. 4, 997–1005 (2001)

  36. 36

    Sankaranarayanan, S., Atluri, P. P. & Ryan, T. A. Actin has a molecular scaffolding, not propulsive, role in presynaptic function. Nature Neurosci. 6, 127–135 (2003)

  37. 37

    Dunaevsky, A. & Connor, E. A. F-actin is concentrated in nonrelease domains at frog neuromuscular junctions. J. Neurosci. 20, 6007–6012 (2000)

  38. 38

    Sone, M. et al. Synaptic development is controlled in the periactive zones of Drosophila synapses. Development 127, 4157–4168 (2000)

  39. 39

    Wan, H. I. et al. Highwire regulates synaptic growth in Drosophila. Neuron 26, 313–329 (2000)

  40. 40

    Oliver, C. J. et al. Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology. Mol. Cell. Biol. 22, 4690–4701 (2002)

  41. 41

    Zito, K., Knott, G., Shepherd, G. M., Shenolikar, S. & Svoboda, K. Induction of spine growth and synapse formation by regulation of the spine actin cytoskeleton. Neuron 44, 321–334 (2004)

  42. 42

    Bloom, O. et al. Colocalization of synapsin and actin during synaptic vesicle recycling. J. Cell Biol. 161, 737–747 (2003)

  43. 43

    Crump, J. G., Zhen, M., Jin, Y. & Bargmann, C. I. The SAD-1 kinase regulates presynaptic vesicle clustering and axon termination. Neuron 29, 115–129 (2001)

  44. 44

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

  45. 45

    Weimer, R. M. et al. Defects in synaptic vesicle docking in unc-18 mutants. Nature Neurosci. 6, 1023–1030 (2003)

  46. 46

    Renden, R. et al. Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion. Neuron 31, 421–437 (2001)

  47. 47

    Helliwell, S. B., Losko, S. & Kaiser, C. A. Components of a ubiquitin ligase complex specify polyubiquitination and intracellular trafficking of the general amino acid permease. J. Cell Biol. 153, 649–662 (2001)

  48. 48

    Rotin, D., Staub, O. & Haguenauer-Tsapis, R. Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J. Membr. Biol. 176, 1–17 (2000)

  49. 49

    Shenoy, S. K., McDonald, P. H., Kohout, T. A. & Lefkowitz, R. J. Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin. Science 294, 1307–1313 (2001)

  50. 50

    Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001)

  51. 51

    Nakata, K. et al. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell 120, 407–420 (2005)

  52. 52

    Allen, P. B., Ouimet, C. C. & Greengard, P. Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines. Proc. Natl Acad. Sci. USA 94, 9956–9961 (1997)

  53. 53

    Muly, E. C., Smith, Y., Allen, P. & Greengard, P. Subcellular distribution of spinophilin immunolabeling in primate prefrontal cortex: localization to and within dendritic spines. J. Comp. Neurol. 469, 185–197 (2004)

  54. 54

    Grant, S. G. Systems biology in neuroscience: bridging genes to cognition. Curr. Opin. Neurobiol. 13, 577–582 (2003)

  55. 55

    Wang, D. et al. Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants. Nature doi:10.1038/nature04010 (this issue)

Download references


We thank the C. elegans Genetic Stock Center, the knockout consortia and S. Mitani for strains; J. Hodgkin for help with naming genes; A. Rogers, R. Lee and K. Van Auken for assistance with WormBase; A. Frand, J. Kim and members of the Kaplan laboratory for advice and for critically reading the manuscript; J. Dittman for developing fluorescence analysis software; D. Simon for the unc-10 and syd-2::gfp constructs; the Ahringer laboratory for RNAi clones and databases; A. Zolotova and J. Xu for technical assistance; and W. Wong and J. Suen for RNAi plates. This work was supported by postdoctoral fellowships from Damon Runyon Cancer Research Foundation (D.S.) and Jane Coffin Childs Memorial Fund (Q.C.), by a predoctoral fellowship from Howard Hughes Medical Institute (M.D.) and by grants from the National Institutes of Health.

Author information

Correspondence to Joshua M. Kaplan.

Ethics declarations

Competing interests

Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Methods (DOC 37 kb)

Supplementary Notes

References, including those for Methods and for Supplementary Table S2. (DOC 47 kb)

Supplementary Figure S1

Classification of the 2072 RNAi genes selected to screen for aldicarb resistance, and classification of the 185 positive genes from the RNAi screens (PDF 604 kb)

Supplementary Figure 2

Aldicarb and phorbol ester resistance of mutants corresponding to positive genes. (PDF 697 kb)

Supplementary Figure S3

Images of the subcellular localization of dorsal punctate proteins, their co localization with SNB-1 and their localization in unc-104 KIF1A mutants. (PDF 2332 kb)

Supplementary Figure S4

Images of motoneuron expression patterns of synaptic proteins. (PDF 1216 kb)

Supplementary Table S1

List of 2072 genes screened. (PDF 119 kb)

Supplementary Table S2

Complete list of positives from the aldicarb resistant (Ric) and dgk-1 suppressor (Dgk) screens. (PDF 48 kb)

Supplementary Table S3

Homologs of positive genes from the RNAi screens. (PDF 285 kb)

Supplementary Table S4

Validation of RNAi screens with known C. elegans neurotransmission mutants. (PDF 28 kb)

Supplementary Table S5

Aldicarb resistance of mutants corresponding to positive genes. (PDF 22 kb)

Supplementary Table S6

Summary of protein localization data. (PDF 22 kb)

Supplementary Table S7

Expression patterns of transcriptional fusions. (PDF 18 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sieburth, D., Ch'ng, Q., Dybbs, M. et al. Systematic analysis of genes required for synapse structure and function. Nature 436, 510–517 (2005) doi:10.1038/nature03809

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.