A secreted complement-control-related protein ensures acetylcholine receptor clustering

Abstract

Efficient neurotransmission at chemical synapses relies on spatial congruence between the presynaptic active zone, where synaptic vesicles fuse, and the postsynaptic differentiation, where neurotransmitter receptors concentrate. Diverse molecular systems have evolved to localize receptors at synapses, but in most cases, they rely on scaffolding proteins localized below the plasma membrane1,2,3. A few systems have been suggested to control the synaptic localization of neurotransmitter receptors through extracellular interactions, such as the pentraxins that bind AMPA receptors and trigger their aggregation4. However, it is not yet clear whether these systems have a central role in the organization of postsynaptic domains in vivo or rather provide modulatory functions5. Here we describe an extracellular scaffold that is necessary to cluster acetylcholine receptors at neuromuscular junctions in the nematode Caenorhabditis elegans. It involves the ectodomain of the previously identified transmembrane protein LEV-10 (ref. 6) and a novel extracellular protein, LEV-9. LEV-9 is secreted by the muscle cells and localizes at cholinergic neuromuscular junctions. Acetylcholine receptors, LEV-9 and LEV-10 are interdependent for proper synaptic localization and physically interact based on biochemical evidence. Notably, the function of LEV-9 relies on eight complement control protein (CCP) domains. These domains, also called ‘sushi domains’, are usually found in proteins regulating complement activity in the vertebrate immune system7. Because the complement system does not exist in protostomes, our results suggest that some of the numerous uncharacterized CCP proteins expressed in the mammalian brain might be directly involved in the organization of the synapse, independently from immune functions.

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Figure 1: lev-9 encodes a muscle-expressed protein containing a WAP domain and eight CCP domains.
Figure 2: LEV-9 localizes at cholinergic neuromuscular junctions.
Figure 3: LEV-9 is specifically required to localize L-AChRs at the neuromuscular junction.
Figure 4: LEV-9 and LEV-10 are interdependent for proper localization of L-AChRs.
Figure 5: LEV-9 physically interacts with LEV-10 and LEV-10 physically interacts with L-AChR.

Accession codes

Primary accessions

EMBL/GenBank/DDBJ

Data deposits

The EMBL database accession number for lev-9 cDNA is FN433774.

References

  1. 1

    Elias, G. M. & Nicoll, R. A. Synaptic trafficking of glutamate receptors by MAGUK scaffolding proteins. Trends Cell Biol. 17, 343–352 (2007)

    CAS  Article  Google Scholar 

  2. 2

    Kneussel, M. & Loebrich, S. Trafficking and synaptic anchoring of ionotropic inhibitory neurotransmitter receptors. Biol. Cell 99, 297–309 (2007)

    CAS  Article  Google Scholar 

  3. 3

    Sanes, J. R. & Lichtman, J. W. Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nature Rev. Neurosci. 2, 791–805 (2001)

    CAS  Article  Google Scholar 

  4. 4

    O’Brien, R. J. et al. Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp. Neuron 23, 309–323 (1999)

    Article  Google Scholar 

  5. 5

    Bjartmar, L. et al. Neuronal pentraxins mediate synaptic refinement in the developing visual system. J. Neurosci. 26, 6269–6281 (2006)

    CAS  Article  Google Scholar 

  6. 6

    Gally, C., Eimer, S., Richmond, J. E. & Bessereau, J. L. A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans . Nature 431, 578–582 (2004)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Kirkitadze, M. D. & Barlow, P. N. Structure and flexibility of the multiple domain proteins that regulate complement activation. Immunol. Rev. 180, 146–161 (2001)

    CAS  Article  Google Scholar 

  8. 8

    Lewis, J. A., Wu, C. H., Berg, H. & Levine, J. H. The genetics of levamisole resistance in the nematode Caenorhabditis elegans . Genetics 95, 905–928 (1980)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Fleming, J. T. et al. Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits. J. Neurosci. 17, 5843–5857 (1997)

    CAS  Article  Google Scholar 

  10. 10

    Boulin, T. et al. Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor. Proc. Natl Acad. Sci. USA 105, 18590–18595 (2008)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Touroutine, D. et al. acr-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the Caenorhabditis elegans neuromuscular junction. J. Biol. Chem. 280, 27013–27021 (2005)

    CAS  Article  Google Scholar 

  12. 12

    Francis, M. M. et al. The Ror receptor tyrosine kinase CAM-1 is required for ACR-16-mediated synaptic transmission at the C. elegans neuromuscular junction. Neuron 46, 581–594 (2005)

    CAS  Article  Google Scholar 

  13. 13

    Bessereau, J. L. et al. Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413, 70–74 (2001)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Williams, D. C., Boulin, T., Ruaud, A. F., Jorgensen, E. M. & Bessereau, J. L. Characterization of Mos1-mediated mutagenesis in Caenorhabditis elegans: a method for the rapid identification of mutated genes. Genetics 169, 1779–1785 (2005)

    CAS  Article  Google Scholar 

  15. 15

    Bingle, C. D. & Vyakarnam, A. Novel innate immune functions of the whey acidic protein family. Trends Immunol. 29, 444–453 (2008)

    CAS  Article  Google Scholar 

  16. 16

    Soares, D. C. et al. Large-scale modelling as a route to multiple surface comparisons of the CCP module family. Protein Eng. Des. Sel. 18, 379–388 (2005)

    CAS  Article  Google Scholar 

  17. 17

    Fares, H. & Greenwald, I. Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants. Genetics 159, 133–145 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Robert, V. & Bessereau, J. L. Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks. EMBO J. 26, 170–183 (2007)

    CAS  Article  Google Scholar 

  19. 19

    Qian, H., Robertson, A. P., Powell-Coffman, J. A. & Martin, R. J. Levamisole resistance resolved at the single-channel level in Caenorhabditis elegans . FASEB J. 22, 3247–3254 (2008)

    CAS  Article  Google Scholar 

  20. 20

    Zheng, Y., Mellem, J. E., Brockie, P. J., Madsen, D. M. & Maricq, A. V. SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans . Nature 427, 451–457 (2004)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Ng, D. et al. Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning. PLoS Biol. 7, e41 (2009)

    Google Scholar 

  22. 22

    Zhang, W. et al. A transmembrane accessory subunit that modulates kainate-type glutamate receptors. Neuron 61, 385–396 (2009)

    CAS  Article  Google Scholar 

  23. 23

    Arlaud, G. J., Barlow, P. N., Gaboriaud, C., Gros, P. & Narayana, S. V. Deciphering complement mechanisms: the contributions of structural biology. Mol. Immunol. 44, 3809–3822 (2007)

    CAS  Article  Google Scholar 

  24. 24

    Hoshino, M., Suzuki, E., Nabeshima, Y. & Hama, C. Hikaru genki protein is secreted into synaptic clefts from an early stage of synapse formation in Drosophila . Development 122, 589–597 (1996)

    CAS  PubMed  Google Scholar 

  25. 25

    Hoshino, M. et al. Neural expression of hikaru genki protein during embryonic and larval development of Drosophila melanogaster . Dev. Genes Evol. 209, 1–9 (1999)

    CAS  Article  Google Scholar 

  26. 26

    Roll, P. et al. SRPX2 mutations in disorders of language cortex and cognition. Hum. Mol. Genet. 15, 1195–1207 (2006)

    CAS  Article  Google Scholar 

  27. 27

    Nonaka, M. & Yoshizaki, F. Primitive complement system of invertebrates. Immunol. Rev. 198, 203–215 (2004)

    CAS  Article  Google Scholar 

  28. 28

    Robert, V. J., Katic, I. & Bessereau, J. L. Mos1 transposition as a tool to engineer the Caenorhabditis elegans genome by homologous recombination. Methods 10.1016/j.ymeth.2009.02.013 (in the press)

  29. 29

    Boulin, T., Etchberger, J. F. & Hobert, O. Reporter gene fusions. WormBook doi/10.1895/wormbook.1.106.1. 1–23 (2006)

  30. 30

    Liegeois, S., Benedetto, A., Garnier, J. M., Schwab, Y. & Labouesse, M. The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans . J. Cell Biol. 173, 949–961 (2006)

    CAS  Article  Google Scholar 

  31. 31

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

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Ruaud, A. F. & Bessereau, J. L. Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C. elegans . Development 133, 2211–2222 (2006)

    CAS  Article  Google Scholar 

  33. 33

    Coudreuse, D. Y., Roel, G., Betist, M. C., Destree, O. & Korswagen, H. C. Wnt gradient formation requires retromer function in Wnt-producing cells. Science 312, 921–924 (2006)

    ADS  CAS  Article  Google Scholar 

  34. 34

    Flanagan, J. G. et al. Alkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues, and embryos. Methods Enzymol. 327, 19–35 (2000)

    CAS  Article  Google Scholar 

  35. 35

    Mello, C. C., Kramer, J. M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991)

    CAS  Article  Google Scholar 

  36. 36

    Miller, K. G., Emerson, M. D., McManus, J. R. & Rand, J. B. RIC-8 (Synembryn): a novel conserved protein that is required for Gqα signaling in the C. elegans nervous system. Neuron 27, 289–299 (2000)

    CAS  Article  Google Scholar 

  37. 37

    Gally, C. & Bessereau, J. L. GABA is dispensable for the formation of junctional GABA receptor clusters in Caenorhabditis elegans . J. Neurosci. 23, 2591–2599 (2003)

    CAS  Article  Google Scholar 

  38. 38

    Duerr, J. S. et al. The cat-1 gene of Caenorhabditis elegans encodes a vesicular monoamine transporter required for specific monoamine-dependent behaviors. J. Neurosci. 19, 72–84 (1999)

    CAS  Article  Google Scholar 

  39. 39

    Duerr, J. S., Gaskin, J. & Rand, J. B. Identified neurons in C. elegans coexpress vesicular transporters for acetylcholine and monoamines. Am. J. Physiol. Cell Physiol. 280, C1616–C1622 (2001)

    CAS  Article  Google Scholar 

  40. 40

    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)

    CAS  Article  Google Scholar 

  41. 41

    Richmond, J. E. Electrophysiological recordings from the neuromuscular junction of C. elegans . WormBook 10.1895/wormbook.1.112.1, 1–8 (2006)

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Acknowledgements

We thank E. M. Jorgensen and D. Williams for the lev-9(ox171::Mos1) strain, M. Labouesse for the anti-VHA-5 antibodies, J. Rand for the anti-UNC-17 antibodies, A. Fire for the GFP vectors, the Caenorhabditis Genetic Center and W. R. Schafer for strains, I. Katic, M. Zhen and S. Marty for critical reading of the manuscript, and H. Gendrot and B. Mathieu for technical help. M.G. was supported by a fellowship from the Ministère de la Recherche and by the Association Française contre les Myopathies. G.R. is a Ministère de la Recherche fellow. This work was funded by an INSERM Avenir grant, the Agence Nationale de la Recherche (ANR-07-NEURO-032-01) and the Association Française contre les Myopathies. J.E.R. was supported by NIH RO1 MH073156.

Author Contributions M.G. performed most of the experiments. J.E.R. performed all the electrophysiology experiments (Fig. 3j, k and Supplementary Fig. 9d). G.R. generated and characterized the unc-63::YFP knock-in strain. M.G. and J.-L.B. wrote the manuscript. J.-L.B. supervised the project.

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Correspondence to Jean-Louis Bessereau.

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Gendrel, M., Rapti, G., Richmond, J. et al. A secreted complement-control-related protein ensures acetylcholine receptor clustering. Nature 461, 992–996 (2009). https://doi.org/10.1038/nature08430

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