A single immunoglobulin-domain protein required for clustering acetylcholine receptors in C. elegans

Abstract

At Caenorhabditis elegans neuromuscular junctions (NMJs), synaptic clustering of the levamisole-sensitive acetylcholine receptors (L-AChRs) relies on an extracellular scaffold assembled in the synaptic cleft. It involves the secreted protein LEV-9 and the ectodomain of the transmembrane protein LEV-10, which are both expressed by muscle cells. L-AChRs, LEV-9 and LEV-10 are part of a physical complex, which localizes at NMJs, yet none of its components localizes independently at synapses. In a screen for mutants partially resistant to the cholinergic agonist levamisole, we identified oig-4, which encodes a small protein containing a single immunoglobulin domain. The OIG-4 protein is secreted by muscle cells and physically interacts with the L-AChR/LEV-9/LEV-10 complex. Removal of OIG-4 destabilizes the complex and causes a loss of L-AChR clusters at the synapse. Interestingly, OIG-4 partially localizes at NMJs independently of LEV-9 and LEV-10, thus providing a potential link between the L-AChR-associated scaffold and local synaptic cues. These results add a novel paradigm for the immunoglobulin super-family as OIG-4 is a secreted protein required for clustering ionotropic receptors independently of synapse formation.

Figure 1

oig-4 mutant alleles confer partial resistance to levamisole. (A) oig-4, lev-10 and lev-9 mutants paralyse after 2 h exposure to levamisole but at higher concentrations than the wild-type (WT) animals. (B) After overnight exposure to the drug they regain motility in contrast to the WT (right graph) (mean±s.e.m., n=3 independent experiments, total number of animals tested per point: 300).

Figure 2

oig-4 encodes a single immunoglobulin (Ig)-domain protein expressed predominantly in body-wall muscle. (A) Structure of the oig-4 genomic locus. Grey line: 5′ untranslated region, SA: splice acceptor site, black box: coding regions, vertical black lines: point mutations, grey box: deletion. kr39 is a missense mutation, kr193 is a change of the fifth base pair of the first intron of oig-4, the tm3753 is a deletion of 239 base pairs. (B) OIG-4 is predicted to be a secreted protein. It contains a signal peptide (sp) and one immunoglobulin domain (Ig). The mutation in the kr39 mutant allele (vertical line) causes a glycine to arginine amino-acid change (G84R). (C) An oig-4 genomic fragment or an oig-4 cDNA expressed by muscle can rescue the oig-4 mutant phenotype. Graphs represent the percentage of dead animals after overnight exposure to levamisole 0.6 mM (mean±s.e.m., n=3 independent experiments of 3–5 independent lines, total number of animals tested: 187–300). (D) oig-4 is expressed predominantly in body-wall muscles (BWM). An artificial operon containing the gfp sequence under the control of an oig-4 genomic fragment drives GFP expression in BWM (arrows). Arrowheads indicate two processes emanating from a pair of head neurons. Non-specific fluorescence is due to the autofluorescence of the intestine (asterisk). Scale bar=10 μm.

Figure 3

OIG-4 is secreted and clustered at neuromuscular junctions (NMJs) while the mutated protein GFP-OIG-4(G84R) is retained intracellularly. (A) The GFP-OIG-4 translational fusion but not the mutated GFP-OIG-4(G84R) rescues the oig-4 mutant phenotypes. Graph represents the percentage of dead animals after overnight exposure to levamisole 0.6 mM (mean±s.e.m., n=3 independent experiments of 3–5 independent lines, total number of animals tested: 100–300). Ex: extrachromosomal transgene; Is: genome integrated transgene; expression was achieved using the oig-4 promoter (Poig-4) or the muscle-specific promoter Pmyo-3 (muscle). (B) GFP-OIG-4 is secreted. The GFP-OIG-4 translational fusion expressed by the muscle promoter Pmyo-3 in transgenic oig-4 mutants is detected in coelomocytes. Coelomocytes (dotted circle) are visualized by Nomarski (i, iii) and epifluorescence microscopy (ii, iv) in wild-type (i, ii) and transgenic animals (iii, iv). Asterisk indicates intestine autofluorescence; scale bar=10 μm. (C) GFP-OIG-4 forms clusters at NMJs GFP-OIG-4 were detected in the nerve ring (nr), and in both the dorsal and ventral nerve cords (dc, vc) using anti-GFP immunofluorescence staining (i). GFP-OIG-4 clusters are juxtaposed to the pre-synaptic cholinergic boutons visualized by immunostaining of the vesicular acetylcholine transporter (VAChT) UNC-17 (ii–iv) and colocalize with L-AChR clusters stained by antibodies against the UNC-38 subunit (v–vii). Scale bar=10 μm. (D) GFP-OIG-4(G84R) is retained in the muscle cell bodies (i, ii) and does not reach NMJs visualized by anti-UNC-17 immunostaining (iii). Arrows indicate muscle cells. Scale bar=10 μm.

Figure 4

L-AChRs are properly expressed but not detected at the NMJs of oig-4 mutants. (A) The UNC-38 L-AChR subunit cannot be detected by immunostaining at the dorsal cord of oig-4 mutants (ii) when compared with wild-type (WT) animals (i). The pre-synaptic varicosities in oig-4 mutants (iv) form properly as in wild type (iii). ACR-16 N-AChR and UNC-49 GABA receptor distribution in oig-4 mutants (vi, viii) is not affected compared with wild type (v, vii). Scale bar=10 μm. (B) Distribution of the knocked-in UNC-63-YFP L-AChR subunit scored in vivo in oig-4 mutant populations. In the reference knock-in strain (i) UNC-63-YFP is detected in ventral (VC) nerve cord and dorsal nerve cord (not shown). In the oig-4 mutant background UNC-63-YFP is either still detected as weak clusters (ii, group a) or not clustered in the cords (iii, group b) (AF: autofluorescence from intestine). Scale bar=10 μm. (C) Quantification of groups a and b (see B) in WT and oig-4 mutant background. The graph represents results of two independent experiments after blind scoring of 100 animals per experiment for each genotype. (D) Western blot analysis of UNC-29 L-AChR subunit expression. UNC-29 levels were normalized to tubulin A. (Percentage of wild-type levels in the oig-4(kr39) and oig-4(kr193) was 104±6% (n=4) and 134±40% (n=4), respectively, mean±s.d.) Bar indicates the 50 kDa marker.

Figure 5

L-AChRs are functional but diffusely distributed at the muscle membrane of oig-4 mutants. (A, B) L-AChRs are functional in oig-4 mutants. Response to pressure ejection of levamisole in voltage-clamped ventral muscle cells. Black arrows mark the 100 ms application onset for 5 × 10⁻⁴ M levamisole. The graph indicates the mean±s.e.m. of the levamisole-elicited current amplitude (366.7±36.6 for N2, n=7; 393.7±20.1 for oig-4(kr39), n=6, P=0.1605; 509.4±34.0 for oig-4(kr193), n=7, P=0.0175). (C–F) Evoked currents recorded from body-wall muscles after ventral nerve cord stimulation. (D) Evoked response amplitudes are 515.9±57.90 for oig-4(+), n=14 versus 445.0±72.76 for oig-4(kr39), n=7 and 659.2±104.5 for oig-4(kr193), n=11. The evoked response time-to-peak (E) and decay half-time (F) are increased in oig-4 mutants as compared to wild type (time-to-peak: 1.58±0.14 ms, n=13 for oig-4(+) versus 2.53±0.20 ms, P=0.0053, n=7 in oig-4(kr39) background, and 2.09±0.11 ms, P=0.0051, n=10 in oig-4(k193) background). Decay half-time: 3.45±0.22, n=15 for wild type and 6.49±0.55 ms, P=0.0007, n=8 for oig-4(kr39) and 6.07±0.35 ms, P=0.0002, n=11 for oig-4(kr193). Electrically evoked responses were obtained in an unc-49(e407);acr-16(ok789) background to eliminate currents due to GABAR and N-AChR activation. ^*P<0.05, ^**P<0.01, ^***P<0.001. Error bars are s.e.m.

Figure 6

LEV-10 and LEV-9 are not properly localized at the neuromuscular junctions of most oig-4 mutants. (A) Immunostaining of LEV-10 at the dorsal cord of WT and oig-4 mutants. (B) Detection of the knocked-in T7-LEV-9 protein using anti-T7 immunostaining. Scale bar=10 μm. (C) Western blot analysis of LEV-10 expression normalized to total protein content. (Percentage of wild-type levels in the oig-4(kr39) and oig-4(kr193) was 87±7% (n=4) and 114±24% (n=4) respectively, mean±s.d.) Bar indicates the 100 kDa marker. (D) The T7-LEV-9 protein immunoprecipitated from total worm extracts is weakly detected in oig-4 mutants. (E) Weak L-AChR clusters can be detected by immunostaining in rare oig-4(kr39) mutant animals (v) and colocalize with remaining LEV-9-T7 clusters (iv–vi). (F) The weak clusters of T7-LEV-9 infrequently detected in oig-4(kr39) mutants (v), colocalize with remaining staining of the LEV-10 protein (iv–vi). Scale bar=10 μm.

Figure 7

The synaptic localization of GFP-OIG-4 requires L-AChRs while LEV-10 and LEV-9 are partially dispensable. (A) GFP-OIG-4 is no longer clustered in the unc-29(x29) null mutant background (iv). However, small clusters of GFP-OIG-4 can be detected in lev-10(kr26) and lev-9(ox177) null mutants. Pre-synaptic VAChT is labelled using anti-UNC-17 antibodies. Scale bar=10 μm. (B) Western blot analysis of GFP-OIG-4 expression in the unc-29 mutant background (percentage of the wild-type GFP-OIG-4 levels in the unc-29 mutant background was 78.3±10.8% (mean±s.d., n=3)). (C) GFP-OIG-4 expressed in muscle cells under the control of the Pmyo-3 promoter is still secreted and accumulates in coelomocytes of WT and unc-29 null mutants. A wild type-like GFP-OIG-4 translational fusion expressed by the muscle promoter Pmyo-3 in transgenic oig-4;unc-29 mutants is detected in coelomocytes. Coelomocytes (dotted circle) are visualized by Nomarski (i, iii) and epifluorescence microscopy (ii, iv) in unc-29(+) (i, ii) and unc-29 null mutant background (iii, iv). Asterisk indicates autofluorescence of intestine. Scale bar=10 μm.

Figure 8

The OIG-4 interacts physically with L-AChRs and LEV-10 and stabilizes the L-AChR/LEV-10 interaction. (A) Detection of the UNC-29 L-AChR subunit after immunoprecipitation of GFP-OIG-4 using anti-GFP antibodies from WT and transgenic lines expressing GFP-OIG-4. UNC-29 no longer co-precipitates when GFP-OIG-4 is expressed in a lev-10(kr26) null mutant background (n=3 independent experiments). (B) LEV-10 co-immunoprecipitates with OIG-4-GFP as above. The interaction is lost in an unc-29(x29) null mutant background (n=3 independent experiments). (C) LEV-10 is co-immunoprecipitated with UNC-63-YFP using anti-GFP antibodies. Co-precipitation is lost in an oig-4(kr39) mutant background (n=3 independent experiments).