Because most neurons receive thousands of synaptic inputs, the neuronal membrane is a mosaic of specialized microdomains where neurotransmitter receptors cluster in register with the corresponding presynaptic neurotransmitter release sites. In many cases the coordinated differentiation of presynaptic and postsynaptic domains implicates trans-synaptic interactions between membrane-associated proteins such as neurexins and neuroligins1,2,3. The Caenorhabditis elegans neuromuscular junction (NMJ) provides a genetically tractable system in which to analyse the segregation of neurotransmitter receptors, because muscle cells receive excitatory innervation from cholinergic neurons and inhibitory innervation from GABAergic neurons4. Here we show that Ce-Punctin/madd-4 (ref. 5), the C. elegans orthologue of mammalian punctin-1 and punctin-2, encodes neurally secreted isoforms that specify the excitatory or inhibitory identity of postsynaptic NMJ domains. These proteins belong to the ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats)-like family, a class of extracellular matrix proteins related to the ADAM proteases but devoid of proteolytic activity6. Ce-Punctin deletion causes the redistribution of synaptic acetylcholine and GABAA (γ-aminobutyric acid type A) receptors into extrasynaptic clusters, whereas neuronal presynaptic boutons remain unaltered. Alternative promoters generate different Ce-Punctin isoforms with distinct functions. A short isoform is expressed by cholinergic and GABAergic motoneurons and localizes to excitatory and inhibitory NMJs, whereas long isoforms are expressed exclusively by cholinergic motoneurons and are confined to cholinergic NMJs. The differential expression of these isoforms controls the congruence between presynaptic and postsynaptic domains: specific disruption of the short isoform relocalizes GABAA receptors from GABAergic to cholinergic synapses, whereas expression of a long isoform in GABAergic neurons recruits acetylcholine receptors to GABAergic NMJs. These results identify Ce-Punctin as a previously unknown synaptic organizer and show that presynaptic and postsynaptic domain identities can be genetically uncoupled in vivo. Because human punctin-2 was identified as a candidate gene for schizophrenia7, ADAMTS-like proteins may also control synapse organization in the mammalian central nervous system.
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We thank C. Lena for expert assistance with analysis of postsynaptic receptor distributions; A. Boyanov for whole genome sequencing; L. Briseno-Roa for technical advice; T. Boulin and M. Jospin for critical reading of the manuscript; M. J. Caron for suggestions on the figure display; A. Valfort, H. Gendrot, P. Muller and B. Mathieu for technical help; J. Rand for the anti-UNC-17 antibodies; M. Hammarlund and E. Jorgensen for plasmids; and P. Roy, the Caenorhabditis Genetic Center (which is funded by National Institutes of Health Office of Research Infrastructure Programs, P40 OD010440) and the International C. elegans Gene Knockout Consortium for strains. B.P.-L. was supported by the Association Française contre les Myopathies; H.T. by the Fondation pour la Recherche Medicale and by the Neuropole de Recherche Francilien; M.P. by the Neuropole de Recherche Francilien; H.Z. by the Fondation Pierre-Gilles de Gennes; and C.S. by a Human Frontier Science Program Long-Term Fellowship. This work was funded by the Agence Nationale de la Recherche (ANR-2010-BLAN-1618-01 and ANR-11-BSV4-019), the Fondation pour la Recherche Medicale (‘Equipe FRM’ grant to J.-L.B.) and the Association Française contre les Myopathies (J.-L.B.).
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Structure of the madd-4 locus and of the fosmid reporters and domain composition of long and short MADD-4 isoforms.
a, madd-4 genomic organization (black boxes represent exons), and position of different mutants used in this study (open arrowheads represent insertion and point mutations; brackets represent deletions). One allele disrupts the long isoform specifically: madd-4(ttTi103747) has a Mos1 insertion in the fifth exon of madd-4A and madd-4C (Mos1 insertion at position 601 of madd-4A cDNA, based on Wormbase release WS240). One allele disrupts the short isoform madd-4(tr185) specifically: M1I (ref. 5). The other alleles affect all isoforms: madd-4(kr249), W374stop (A instead of G at position 1121 of madd-4A cDNA, this allele was isolated based on a L-AChR distribution defect following mutagenesis); madd-4(ok2862), 631-bp deletion at position 1438 of madd-4A cDNA leading to an insertion of 16 amino acids and a premature stop5; and madd-4(kr270), 11,042-bp full deletion of the locus (from the ATG of madd-4A to the stop codon) engineered by homologous recombination. Because madd-4(kr249) shows the same L-AChR and GABAAR clustering defects as madd-4(kr270), it is probably a null allele. Scale bar, 1 kb. b, The three mRNAs transcribed from the madd-4 locus. In this paper, MADD-4L refers to both MADD-4A and MADD-4C, in instances where they cannot be distinguished. c, Structure of the madd-4 locus in the three fosmids obtained by recombineering of the WRM0626CA02 fosmid (see Methods). Arrows indicate the beginning of the open reading frame of madd-4L (black) and madd-4B (grey). Green exons correspond to the sequence of the GFP. Red crosses indicate removal of exons. d, The different domains of MADD-4A, MADD-4C and MADD-4B proteins. The two long isoforms (MADD-4A and MADD-4C, globally referred to as MADD-4L) have the same domain composition. e, Sequence alignment of six TSP-1 repeats of MADD-4L shows six conserved cysteine residues, characteristic of these domains. The TSP-1 no. 9 was not described previously5. The sequence alignment was generated with Clustal W (EBI).
Extended Data Figure 2 Both L- and N-AChRs are redistributed at extrasynaptic areas at the surface of muscle cells, and L-AChRs remain associated with scaffolding proteins in madd-4 mutant.
a–f, SAB head motoneurons differentiate wild-type cholinergic boutons (immunofluorescence, anti-VAChT/UNC-17 antibody) in a madd-4(kr270) null mutant (a, d) but synaptic L-AChRs (immunofluorescence, anti-UNC-38) are not detectable (b, e). g, h, Transgenic animals expressing a Myc-tagged UNC-38 L-AChR subunit were injected with fluorescent anti-Myc antibodies into the body cavity to reveal L-AChRs at the muscle cell surface, in the wild type (g) and in madd-4(ok2862) mutant background (h). i, j, N-AChRs (ACR-16–GFP) are redistributed to muscle arms in a madd-4(kr270) null mutant (j), in contrast with the wild type (i). k–p, At the dorsal cord of a madd-4(kr249) mutant, synaptic and non-synaptic L-AChR clusters (unc-29–RFP in red) (k, n) co-localize with LEV-10 detected by immunofluorescence (cyan) (l) and with LEV-9 (o) stained with anti-T7 antibody in unc-29–RFP madd-4(kr249); lev-9-T7 knock-in animals. q–s, L-AChR clusters (unc-29–RFP) remaining in madd-4(kr249) are lost in lev-9 and lev-10 mutant backgrounds. Arrowheads point to extrasynaptic AChR clusters. c, f, m, p, Channel overlays along with a colour mixing guide. Scale bars, 10 μm.
Extended Data Figure 3 Synapse morphology is normal at the ultrastructural level in the madd-4 mutant.
Electron microscopy tomogram sections of cholinergic NMJs in the dorsal nerve cords of wild-type and madd-4(kr249) null mutant. ma, muscle arm; dp, dense projection; n, neuron. Scale bars, 100 nm.
Extended Data Figure 4 Analysis of L-AChR and GABAAR localization in transgenic lines expressing MADD-4L or MADD-4B.
Immunofluorescence analysis of transgenic madd-4(kr249) null mutants containing the recombineered fosmids for revealing MADD-4–GFP (a–c, j–l), MADD-4L–GFP (d–f, m–o) or MADD-4B–GFP (g–i, p–r) (see Extended data Fig. 1c). L-AChRs (anti-UNC-38 antibody (b, e, h)) are localized opposite cholinergic boutons (anti-VAChT/UNC-17 antibody (a, d, g)) at a wild-type level when MADD-4–GFP or MADD-4L–GFP is expressed, and at a lower level when MADD-4B–GFP is expressed. Immunofluorescence staining of UNC-49 shows a rescue of GABAARs for MADD-4–GFP (k) and for MADD-4B–GFP (q) but not for MADD-4L–GFP (n). GABA boutons are revealed with a Punc-47::snb-1-myc marker (j, m, p). c, f, i, l, o, r, Channel overlays along with a colour mixing guide. Scale bars, 10 μm.
a–f, In transgenic strains expressing MADD-4A–GFP (a, b) and MADD-4B–GFP (c, d) from recombineered fosmids, fluorescence is detected in intracellular vesicles of coelomocytes (highlighted with white dotted circles). This indicates that MADD-4–GFP proteins were secreted in the pseudocoelomic cavity and endocytosed by these scavenger cells, which filter the pseudocoelomic fluid and are not fluorescent in the wild type (e, f). g–l, Transgenic animals expressing GFP-tagged MADD-4L (g) or MADD-4B (j) in the madd-4(kr270) mutant background were injected into the body cavity with red-fluorescent anti-GFP antibodies (conjugated with Alexa 555) (h, k) to reveal MADD-4 isoforms secreted at synapses. m–o, Immunofluorescence staining shows that L-AChRs (anti-UNC-38 antibody) (n) are redistributed at extrasynaptic areas at the dorsal side in the unc-104(e1265) mutant, as in a madd-4 null mutant. Cholinergic boutons are revealed with an anti-VAChT/UNC-17 antibody (m). p, Immunofluorescence staining of UNC-49 in unc-104(e1265) mutants shows a redistribution of GABAARs in muscle arms at the dorsal side, which mimics the loss of madd-4. Arrowheads show extrasynaptic clustering. i, l, o, Channel overlays along with a colour mixing guide. Scale bars, 10 μm.
Extended Data Figure 6 MADD-4 isoforms are retained at synapses independently from cholinergic and GABAergic receptors.
a–d, MADD-4A–GFP (krSi4[Pmadd-4L::madd-4A–GFP]) (a,c) and MADD-4B–GFP (krEx1069[madd-4B–GFP fosmid]) (b, d) are unaffected by the absence of L-AChRs and N-AChRs in an unc-29(x29); acr-16(ok789) double mutant (c, d), when compared with the wild type (a, b). Arrows indicate the position of synapses made with SAB motoneurons. AF, non-specific autofluorescence of muscle cells. e–h, MADD-4B–GFP (f) is juxtaposed to dorsal cord GABA boutons (Punc-47::snb-1-myc marker) (e) in the absence of GABAARs in the unc-49(e407) mutant (e–h), as in the wild type (Fig. 2m–p). g, Channel overlay along with a colour mixing guide. Scale bars, 10 μm (a–d) and 5 μm (e–g).
Immunofluorescence staining shows that L-AChRs (anti-UNC-38 antibody) (b, e, h, k) are clustered opposite acetylcholine release sites (anti-VAChT/UNC-17 antibody) (a, d, g, j) at a wild-type level in madd-4B(0) mutants (madd-4(tr185)) (a–f) and at a lower level in madd-4L(0) mutants (madd-4(ttTi103747)) (g–l), both at synapses made with SAB motoneurons (a–c, g–i) and at the dorsal nerve cord (d–f, j–l). c, f, i, l, Channel overlays along with a colour mixing guide. Scale bars, 10 μm.
Extended Data Figure 8 Analysis of L-AChR and GABAAR distribution in transgenic lines expressing different combinations of MADD-4 isoforms.
a, Synaptic clustering of L-AChRs (unc-29–RFP knock-in) was evaluated in the control madd-4(+) and in transgenic madd-4(kr249) mutants animals expressing MADD-4A, MADD-4C or MADD-4A–GFP in cholinergic motoneurons. Data are presented as means and s.e.m.; N = number of worms; Student’s t-test compared with the wild type (black) or compared with madd-4(kr249) (grey): ***P ≤ 0.001; *P ≤ 0.01; NS, not significant. b–k, Labelling of cholinergic terminals (anti-UNC-17 immunofluorescence) (b, g), L-AChRs (UNC-29–RFP) (c, h) and GABAARs (anti-UNC-49 immunofluorescence) (d, i) in madd-4(kr249) null mutants expressing the long isoform MADD-4A [madd-4(0); PACh::madd-4A] or both MADD-4A and MADD-4B [madd-4(0); PACh::madd-4A PACh::madd-4B] in cholinergic motoneurons. The relative fluorescence intensity of L-AChRs and GABAARs along the cord is shown (f, k). Co-localization of L-AChRs and GABAARs was quantified in 25 and 33 worms, respectively. r2 values are the means of squares of Pearson’s coefficient, r2 (mean ± s.e.m.). A value close to 0 indicates a lack of co-localization, whereas a value close to 1 indicates complete co-localization because all calculated r values were positive. The r2 of the four genotypes (also presented in Fig. 5q, v) is statistically different by ANOVA. Scale bars, 10 μm. l–n, L-AChRs (UNC-29–RFP) and GABAARs (anti-UNC-49 immunofluorescence) were detected together in madd-4(+) and in madd-4(kr249) mutants expressing the long isoform MADD-4A in GABAergic motoneurons [madd-4(0); PGABA::madd-4A], in cholinergic motoneurons [madd-4(0); PACh::madd-4A] or expressing both MADD-4A and MADD-4B in cholinergic motoneurons [madd-4(0); PACh::madd-4A PACh::madd-4B]. l, Example of the fluorescence profile of GABAARs (green) and L-AChRs (red) of madd-4(+); receptor clusters are detected as local peaks in the fluorescence profile 1 s.d. above the mean fluorescence (see Methods). The threshold for the green and red profiles is indicated by the dashed lines; the circles indicate the position of the peaks used for the co-localization study. m, Average-density histogram of L-AChR clusters around GABAAR clusters for the four genotypes. In madd-4(+), the flat curve reveals a random distribution of cholinergic NMJs relative to GABAergic NMJs. The co-localization of GABAARs and L-AChRs is revealed by a central peak in the histogram. Number of worms used: N = 23, N = 19, N = 25 and N = 33, respectively. Bin = 0.125 μm (2 pixels); mean (plain line) ± s.e.m. (grey area). n, Quantification of the amplitude of the central peaks in the histogram density of b (expressed as a deviation from the average density). The genotypes are statistically different (ANOVA on the factor genotype: F(3,96) = 10.46, P < 10−4). Post-hoc Student’s test: *P < 0.05; **P < 0.01. e, f (lower), j, k (lower), Channel overlays along with a colour mixing guide.
Extended Data Figure 9 madd-4B(0) mutants show muscle arm extension defects but madd-4L(0) mutants do not.
The average number of muscle arms for the ventral left muscle 11 and the dorsal right muscle 15 is indicated for the wild type and the madd-4(ttTi103747) (madd-4L(0)) and madd-4(tr185) (madd-4B(0)) mutants. Distal muscle cells were revealed with a Phim-4::yfp muscle arm reporter (trIs25). Results are shown as means ± s.e.m.; N = number of worms; Mann–Whitney test: ***P ≤ 0.0001; *P ≤ 0.05; NS, not significant.
a, Immunoprecipitation of MADD-4A–GFP (lane 2) or MADD-4B–GFP (lane 4) with anti-GFP antibodies was followed by western blot analysis with anti-Myc antibodies to detect MADD-4A–Myc or MADD-4B–Myc. A specific MADD-4A or MADD-4B signal was detected in the immunoprecipitation products of MADD-4A–GFP or MADD-4B–GFP, respectively, but not when GFP alone was co-expressed as a control (lanes 3 and 5). The GFP-tagged and Myc-tagged proteins were co-expressed in HEK cells. Lane 1, mock control. b, By using a similar procedure, a specific MADD-4A or MADD-4B signal was detected in the immunoprecipitation products of MADD-4B–GFP or MADD-4A–GFP, respectively. These experiments were repeated independently three times.
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Pinan-Lucarré, B., Tu, H., Pierron, M. et al. C. elegans Punctin specifies cholinergic versus GABAergic identity of postsynaptic domains. Nature 511, 466–470 (2014). https://doi.org/10.1038/nature13313
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