Systematic analysis of genes required for synapse structure and function

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Abstract

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.

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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.

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Acknowledgements

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.

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Correspondence to Joshua M. Kaplan.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. 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)

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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

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