A trans-synaptic IgLON adhesion molecular complex directly contacts and clusters a nicotinic receptor

Domain: neuroscience
Published: 2026-01-22
Editorial outcome: minor_revision

Abstract
The clustering of neurotransmitter receptors at appropriate postsynaptic sites is essential for controlling synaptic transmission. While most known mechanisms involve receptor binding with cytoplasmic scaffolds, recent evidence highlights the importance of extracellular interactions that directly target receptors. Using Caenorhabditis elegans, we identified a trans-synaptic complex that involves RIG-5 and ZIG-8, two adhesion molecules of the immunoglobulin (Ig) superfamily and orthologous to Drosophila DIPs and Dprs, and mammalian IgLONs. Our results show that RIG-5 and ZIG-8 are anchored in the pre- and postsynaptic membranes, respectively, and interact in vivo via their first Ig domains. Furthermore, ZIG-8 directly binds a α7-like acetylcholine receptor (AChR), known as ACR-16, via a cis-interaction between its Ig2 domain and the base of the extracellular AChR domain. This study provides direct evidence that trans-synaptic IgLON interactions can organize neurochemical synapses and suggests that the IgLONs may directly interact with ionotropic receptors in the mammalian nervous system.

Introduction
Chemical neurotransmission relies on the precise anchoring and the clustering of postsynaptic receptors directly opposite specific neurotransmitter release sites. This process is tightly controlled by cell surface and secreted molecules that interconnect the molecular components of the synapse and ensure accurate alignment between the presynaptic and postsynaptic domains1,2. However, the diversity of molecular players that govern receptor clustering is becoming increasingly complex, raising new questions about the mechanisms regulating synapse formation and homeostasis.

One prevailing mechanism of postsynaptic receptor clustering involves the recruitment of intracellular protein scaffolds, composed of multimodular proteins of the postsynaptic density that multimerize and anchor postsynaptic receptors through intracellular binding. Synaptic transmembrane proteins localize these scaffolds, resulting in the formation of large molecular assemblies that span the postsynaptic membranes. In the mammalian brain, prototypical examples of these cytoplasmic scaffolds include PSD95 (PostSynaptic Density protein 95), SAP97, Homer and Shank at excitatory synapses, and Gephyrin and Collybistin at inhibitory synapses3,4.

More recently, an increasing number of components of the synaptic extracellular matrix have been demonstrated to serve as scaffolding molecules and help recruit synaptic receptors through extracellular interactions (reviewed in ref. 5). For example, secreted neuronal pentraxins (Nptx), and the transmembrane-bound neuronal pentraxin receptor (Nptxr) associate with AMPARs via their pentraxin domains in vitro, and cluster AMPARs in vivo, likely through self-multimerization6,7. Similarly, Cerebellin 1 (Cbln1), Cerebellin 4 (Cbln4) and the C1ql2-3 complement-related molecules are secreted from presynaptic terminals and bind the atypical GluD1-2 glutamate receptor delta subunits and the GluK2-4 kainate receptor subunits, respectively8–10. Both Cerebellins and C1ql2-3 are part of large complexes that are stabilized through their own oligomerization and bridge pre- and postsynaptic components.

Interestingly, both mechanisms can be used at the same synapse. In the nematode C. elegans, the extracellular matrix MADD-4/Punctin is secreted by motoneurons and localizes two different ionotropic acetylcholine receptors (AChRs) at excitatory neuromuscular junctions (NMJs) via two distinct mechanisms. The levamisole-sensitive acetylcholine receptors (L-AChRs) are heteropentameric AChRs that are activated by the nematode-specific cholinergic agonist levamisole11. L-AChRs interact with a bona fide extracellular complex consisting of two secreted proteins, LEV-9 and OIG-4, and the ectodomain of a transmembrane protein, LEV-10; all expressed by muscle cells12–14. These proteins form L-AChR-containing microclusters that are recruited and stabilized at synapses, most likely through direct interaction with Punctin in the synaptic cleft15. The nicotine-sensitive AChRs (N-AChRs) are homopentameric receptors composed of the ACR-16 subunit, the ortholog of the mammalian α7 AChR subunit16. Their synaptic localization depends on their interaction with an intracellular scaffold. At the cholinergic NMJ, Punctin activates the netrin receptor UNC-40/DCC and localizes the transmembrane heparan sulfate proteoglycan receptor Syndecan17–19. This dual signaling triggers the subsynaptic recruitment of the scaffolding proteins LIN-2/CASK and FRM-3/FARP1-2, which physically bind the large intracellular loop of the ACR-16 receptor and localize it at the NMJ.

In C. elegans, acetylcholine is not only used at NMJs but is the major neurotransmitter throughout the nervous system. Neurons express a large diversity of AChRs. In particular, the gene acr-16 is readily expressed in several neurons but the mechanisms controlling its subcellular localization are unknown. To identify novel mechanisms of synapse organization, we performed an innovative genetic analysis of ACR-16-containing neuron-neuron synapses. Here we describe a critical role of two cell adhesion molecules, namely RIG-5 (neuRonal IG CAM-5) and ZIG-8 (Zwei IG domain protein-8), which are the sole IgLON orthologs in C. elegans. IgLONs are Ig-domain rich extracellular proteins that have often been suggested to act as synaptic adhesion molecules across phyla, but their actual synaptic functions have remained largely unexplored. We found that RIG-5 and ZIG-8 are specifically localized to pre- and postsynaptic membranes, respectively, and interact trans-synaptically through their N-terminal Ig1 domains. Most notably, we demonstrated that ZIG-8 recruits AChRs through a direct physical cis-interaction involving its Ig2 domain and the base of the AChR ectodomain. This is an unprecedented mechanism controlling the synaptic localization of an AChR through a direct extracellular interaction with synaptic adhesion molecules.

Results
The ACR-16 acetylcholine receptor forms neuronal clusters
To visualize ACR-16 in living animals, we used a knock-in allele expressing a Scarlet-tagged version of ACR-16 (Supplementary Fig. 1a)17. As previously described, ACR-16-Scarlet appeared at NMJs as characteristic spreading clusters along the ventral and dorsal sides of the animals. In addition, we also visualized large, round and sharply defined clusters in the ventral nerve cord (Supplementary Fig. 1b). These two types of clusters followed distinct tracks. Interestingly, transcriptomic data from single-neuron analyses indicate that acr-16 is expressed in several neurons within the ventral nerve cord (Supplementary Fig. 1c)20. This nerve structure is a major neuropil consisting of a bundle of ~40 neurites and hundreds of en passant synapses that extends from the head to the tail (Fig. 1a)21. To investigate whether the round ACR-16 clusters are formed in neurons, we performed tissue-specific degradation of ACR-16 by utilizing the Auxin-Inducible Degradation (AID) system22. We introduced the AID cassette in the acr-16::Scarlet locus using CRISPR/Cas9 and triggered proteasomal degradation of ACR-16-AID-Scarlet in body wall muscle cells and neurons (Supplementary Fig. 1a). Degrading ACR-16-AID-Scarlet in body wall muscle cells eliminated the spreading clusters, confirming their presence at NMJs (Fig. 1b). Conversely, degrading ACR-16-AID-Scarlet in neurons caused the disappearance of the round clusters, indicating their neuronal origin. Simultaneous degradation in both neurons and muscle cells completely removed ACR-16-AID-Scarlet, demonstrating highly efficient degradation. For this study, we focused primarily on the neuronal clusters of ACR-16-AID-Scarlet and, unless otherwise specified, we induced ACR-16-AID-Scarlet degradation specifically in body wall muscle cells.

ACR-16 forms postsynaptic clusters at AVE to AVA synapses
To identify the neurons of the ventral nerve cord in which ACR-16 clusters, we first examined single-neuron transcriptomic data and identified four classes of acr-16 expressing neurons that potentially synapse in the ventral nerve cord based on neuronal wiring data (Supplementary Fig. 1c)20,21. In parallel, we constructed a fluorescent transcriptional reporter driven by a 5.8kb upstream promoter sequence of acr-16 (Supplementary Fig. 1a). Its expression pattern was analyzed in the NeuroPAL strain, which facilitates neuron identification through unique color labels and positions23. We confirmed acr-16 expression in two classes of neurons that synapse in the ventral nerve cord: the AVA, a pair of interneurons that control locomotion, and the DB-type of cholinergic motoneurons (Supplementary Fig. 1c, d).

Using fluorescently-tagged ACR-16, we counted on average 116 neuronal clusters along the ventral cord (Fig. 1c, Supplementary Fig. 1e). To specifically evaluate the number of clusters formed in the AVA and DB neurons, we developed a versatile strategy based on split GFP reconstitution in the cytoplasm of specific neurons, which we named NeuroSIL (Neuron-type Specific Illumination, Fig. 1d)24. We inserted three copies of the spGFP11 sequence into the acr-16::aid::Scarlet locus via CRISPR/Cas9 (acr-16::aid::Scarlet::spgfp11) and expressed the complementary spGFP1-10 moiety in the AVA or in the DB neurons (Supplementary Fig. 1a)25,26. Using NeuroSIL, the red Scarlet fluorescence labels the full complement of ACR-16 clusters, while the GFP fluorescence is reconstituted only in the neurons expressing spGFP1-10. In the AVA neurons, NeuroSIL revealed on average 108 ACR-16-Scarlet and GFP positive clusters (Fig. 1c, and Supplementary Fig. 1f). Because some clusters showed only Scarlet fluorescence and thus were not formed in the AVAs, we also tested NeuroSIL in the DBs (Fig. 1c, e). This experiment revealed on average 7 GFP+ Scarlet clusters in the DB neurons. We concluded that the DBs and AVAs are the two neuron types in which ACR-16 form clusters, with the latter contributing to the majority of these clusters (Fig. 1f).

The AVA and DB neurons receive numerous synaptic inputs from several cholinergic neurons in the ventral nerve cord, as evidenced by electron microscopy (Fig. 1g)21,27. Notably, the AVE interneurons establish synapses with the AVA neurons in the first quarter of the ventral nerve cord (Fig. 1f). To investigate the pattern of presynaptic sites in AVE neurons, we built an active zone reporter using CLA-1/Clarinet28. Our results revealed the juxtaposition of approximately 16 active zones and ACR-16-AID-Scarlet clusters in the first quarter of the ventral nerve cord, indicating that ACR-16 forms postsynaptic clusters at AVE to AVA synapses (Fig. 1c, g, h). Using the same methodology, we examined a presynaptic AVB reporter, as AVB neurons have been reported to make approximately 27 synapses on AVA by electron microscopy21,27. However, we did not detect strong juxtaposition of the pre- and postsynaptic signals in this context, suggesting that ACR-16 is not present at AVB to AVA synapses (Supplementary Fig. 1g). Owing to the lack of specific promoters for certain neuron types, such as AVD, which forms many synapses on AVA, we could not probe every potential presynaptic neuron of the AVAs and DBs21,27. Nevertheless, our data identify that ACR-16 form postsynaptic clusters at least at AVE to AVA synapses.

The RIG-5 and ZIG-8 IgLONs associate with ACR-16
To elucidate the molecular mechanisms controlling ACR-16 synaptic localization, we performed a genetic screen for mutants with altered patterns of ACR-16-AID-Scarlet clustering, and identified several mutations in the rig-5 and zig-8 genes (Supplementary Fig. 2a, b). These genes encode orthologs of mammalian IgLON cell adhesion molecules, also known as DIPs (Dpr Interacting Protein) and Dprs (Defective Proboscis extension Response) in Drosophila29,30. RIG-5 and ZIG-8 are composed of three and two extracellular Ig-like domains, respectively, such as DIPs and Dprs, and are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI) anchoring31,32. Using CRISPR/Cas9, we engineered BFP-RIG-5 and GFP-ZIG-8 translational reporters by tagging them with fluorescent proteins immediately after their signal peptides. Strikingly, both BFP-RIG-5 and GFP-ZIG-8 exhibited round puncta that overlapped extensively with ACR-16-AID-Scarlet clusters along the ventral nerve cord (Fig. 2a). Despite the abundance of cholinergic synapses in the ventral nerve cord, BFP-RIG-5 and GFP-ZIG-8 were only present along with ACR-16-AID-Scarlet, and not at other types of synapses (Fig. 2a, Supplementary Fig. 1h). This evidence strongly supports the conclusion that RIG-5 and ZIG-8 are cell adhesion molecules specifically associated with ACR-16 clusters.

RIG-5 and ZIG-8 control each other’s localization
A peculiar feature of IgLONs is their involvement in multiple homo- and heterophilic interactions between members of the family, in trans across different cells30,33. For several dimers, the structural basis of these interactions has been solved through crystallography, revealing a central hydrophobic patch of amino acids in the first N-terminal Ig1 domains as crucial for binding30,34,35. RIG-5 and ZIG-8 were demonstrated to form heterodimers by interacting in their Ig1 domains, as evidenced by crystallography and binding analysis, akin to mammalian and Drosophila IgLONs31. Given the in vitro interaction between RIG-5 and ZIG-8, we tested whether this interaction applies in vivo in the synaptic cleft. Firstly, we performed a GFP Reconstruction Across Synaptic Partners (GRASP) experiment by fusing RIG-5 and ZIG-8 to split GFP fragments. The resulting spgfp11::rig-5 and spgfp1-10::zig-8 double knock-in strain exhibited robust GFP fluorescence that precisely overlapped with ACR-16-AID-Scarlet neuronal puncta, thereby indicating the proximity of RIG-5 and ZIG-8 in the synaptic cleft (Fig. 2b).

Secondly, we assessed the impact of rig-5 deletion on GFP-ZIG-8 localization. To completely abolish the rig-5 gene activity, we engineered a full deletion mutant using CRISPR/Cas9 (Supplementary Fig. 2a). In this rig-5(0) mutant, GFP-ZIG-8 failed to concentrate at synapses in the ventral nerve cord, instead exhibiting a diffuse distribution along the neurites (Fig. 2c). Similarly, analyzing the distribution of a GFP-RIG-5 reporter in a zig-8(0) deletion allele revealed a dramatic loss of synaptic concentration (Supplementary Fig. 2b, Fig. 2d). This underscores the essential roles of both RIG-5 and ZIG-8 in maintaining their synaptic localization.

Thirdly, we introduced point mutations in residues within the Ig1 domains previously demonstrated to disrupt the interaction between RIG-5 and ZIG-831. We engineered a gfp::rig-5(F75D) allele and observed that both GFP-RIG-5(F75D) and Scarlet-ZIG-8 exhibited diffuse distributions along the neurites (Fig. 2e)31. This suggested that disrupting the interaction between RIG-5 and ZIG-8 affects their synaptic localization. In a reciprocal experiment, we engineered a Scarlet::zig-8(L77E) allele, which also resulted in a diffuse distribution of both Scarlet-ZIG-8(L77E) and GFP-RIG-5 along neurites (Fig. 2e). Likewise, GFP-RIG-5(F75D) and Scarlet-ZIG-8(L77E) failed to concentrate at synapses in a gfp::rig-5(F75D); Scarlet::zig-8(L77E) double mutant (Fig. 2e). Taken together, these experiments strongly support the notion that the interaction between RIG-5 and ZIG-8 in the synaptic cleft is crucial for their synaptic localization.

RIG-5 and ZIG-8 control ACR-16 clustering
Having identified RIG-5 and ZIG-8 in a genetic screen for mutants in ACR-16 localization, we aimed to precisely characterize their synaptic function (Supplementary Fig. 2a). In the rig-5(0) and zig-8(0) null mutants, as well as in the double rig-5(0); zig-8(0) mutant, ACR-16-AID-Scarlet failed to cluster at neuron-neuron synapses (Fig. 3a). In contrast, the clustering of an ACR-16-AID-Scarlet reporter occurred normally at NMJ in rig-5(0) and zig-8(0) mutants (Supplementary Fig. 3a). This result is consistent with the known clustering mechanism of ACR-16 in muscle cells, which involves an intracellular scaffold composed of FRM-3/FARP and LIN-2/CASK17. To verify that the neuronal phenotype was independent of the Scarlet tag, we analyzed the rig-5(0) and zig-8(0) mutations using an ACR-16-AID-GFP knock-in reporter, and found similar clustering defects at neuronal synapses (Supplementary Fig. 3b). Additionally, given that the RIG-5(F75D) and ZIG-8(L77E) variants lose the ability to interact in vivo and accumulate at synapses, we investigated the impact of these mutations on ACR-16-AID-Scarlet distribution. As expected, ACR-16-AID-Scarlet did not cluster at synapses in these missense mutants (Fig. 3b, c). We concluded that RIG-5 and ZIG-8 control the concentration of ACR-16 specifically at neuron-neuron synapses of the ventral nerve cord, but not at NMJs.

Conversely, we investigated whether RIG-5 and ZIG-8 could concentrate at synapses in the absence of acr-16. We found that GFP-RIG-5 and GFP-ZIG-8 formed dimmer puncta at synapses, but also displayed a diffuse pattern between synapses in acr-16 mutants (Fig. 3d, e). This suggests that ACR-16 is not critical for recruiting RIG-5 and ZIG-8 to synapses but may play a role in stabilizing their complex.

Synaptic adhesion molecules function during development and at mature synapses. To investigate whether RIG-5 and ZIG-8 have a developmental role, we analyzed larval stage 1 (L1) C. elegans. In newly born L1 larvae, BFP-RIG-5, GFP-ZIG-8 and ACR-16-AID-Scarlet highly colocalized in a punctate pattern, as in adult C. elegans (Fig. 2a, Supplementary Fig. 4a). Moreover, we observed that rig-5(0) and zig-8(0) mutations prevented ACR-16-AID-Scarlet clustering (Supplementary Fig. 4b). Additionally, GFP-RIG-5 exhibited a diffuse pattern in the zig-8(0) mutant (Supplementary Fig. 4c). Similarly, GFP-ZIG-8 showed a diffuse pattern in the rig-5(0) mutant (Supplementary Fig. 4d). The consistent findings observed during both larval and adult stages suggest that RIG-5 and ZIG-8 control ACR-16 clustering throughout synapse development and maintenance.

The development of neuronal networks involves multiple coordinated steps, including neuronal specification, migration, axon growth and guidance, synaptic partner recognition, and synapse formation and maintenance. To explore the potential involvement of RIG-5 and ZIG-8 in the initial phases of neuronal network establishment, we assessed rig-5(0) and zig-8(0) single mutants for defects in neuronal specification, guidance and maintenance of the AVE, AVA and DB neurons. Our results revealed that these mutants did not exhibit noticeable defects in these processes (Supplementary Fig. 5a–e). These results are consistent with previous studies showing that RIG-5 and ZIG-8 contribute to nervous system development and maintenance, but in strict redundancy with other cell surface receptors and extracellular matrix components36–38. Taken together, our data suggest that RIG-5 and ZIG-8 control the clustering of ACR-16 nicotinic receptors at specific cholinergic synapses.

RIG-5 is presynaptic, and ZIG-8 postsynaptic
Our analysis has identified ACR-16 clusters in the AVA and DB neurons, notably at AVE to AVA synapses. This allowed us to investigate in which neurons rig-5 and zig-8 function. Using a neuronal gene expression atlas, we found that either rig-5, zig-8, or both are expressed in AVAs, DBs and most of their known synaptic partners (Supplementary Fig. 1c)20. Specifically, rig-5 was expressed in the AVE, AVD, DVA and AVA neurons, while zig-8 was expressed in the AVE, AVA and DB neurons. The DB neurons exclusively express zig-8, suggesting a model in which RIG-5 might be presynaptic, and ZIG-8 might be postsynaptic at synapses onto AVAs and DBs.

To test this hypothesis, we performed a series of cell-specific rescue experiments, in single rig-5(0), zig-8(0) or double rig-5(0); zig-8(0) mutant backgrounds, in which ACR-16 does not cluster properly (Fig. 3a). In these rescue experiments, we observed the recovery of distinct classes of synapses depending on the genetic background. Firstly, we expressed BFP-RIG-5 in the AVE neurons of a rig-5(0) mutant and found that BFP-RIG-5 had a punctate distribution typical of AVE to AVA synapses, forming more than 20 puncta in the first quarter of the ventral nerve cord (Fig. 4a, d, Fig. 1c, f). These BFP-RIG-5 puncta robustly colocalized with ACR-16-AID-Scarlet, suggesting that presynaptic expression of RIG-5 could rescue postsynaptic ACR-16 localization. Secondly, we introduced BFP-ZIG-8 in the AVAs of a zig-8(0) mutant. We observed an average of 80 puncta containing both BFP-ZIG-8 and ACR-16-AID-Scarlet along the ventral nerve cord. They likely correspond to synapses where ACR-16 clusters in AVA neurons, from various presynaptic neurons, including but not limited to AVE neurons (Fig. 4b, d, Fig. 1c, f). This experiment suggested that expression of ZIG-8 in the postsynaptic AVA neuron could promote ACR-16-AID-Scarlet clustering. Thirdly, we co-expressed BFP-RIG-5 in the AVE neurons and GFP-ZIG-8 in the AVA neurons in the double rig-5(0); zig-8(0) mutant. We observed a sharp punctate distribution of BFP-RIG-5, GFP-ZIG-8, and ACR-16-AID-Scarlet, consistent with the synaptic pattern observed at AVE to AVA connections (Fig. 4c, d, Fig. 1c, f).

To confirm the synaptic location of these puncta, we expressed BFP-RIG-5 in the AVEs along with an AVE-specific presynaptic marker, and Scarlet-ZIG-8 in the AVA, in the double rig-5(0); zig-8(0) mutant. We observed that BFP-RIG-5 and Scarlet-ZIG-8 formed puncta juxtaposed to the AVE presynaptic marker, supporting the formation of functional synapses (Fig. 4e). To probe whether the orientation of the RIG-5—ZIG-8 trans-synaptic complex is important, we reversed their expression, introducing Scarlet-ZIG-8 in the presynaptic AVE neuron, and BFP-RIG-5 in the postsynaptic AVA neuron. Strikingly, we did not observe puncta of BFP-RIG-5 or Scarlet-ZIG-8 in this genetic background (Fig. 4f). This finding underscores that RIG-5 must be expressed in the presynaptic neuron and ZIG-8 in the postsynaptic neuron for correct localization to their respective pre- or postsynaptic membranes and efficient interaction at synapses. Overall, this set of cell-specific rescue experiments indicates that RIG-5 is presynaptic, and ZIG-8 postsynaptic, at least at AVE to AVA synapses.

Notably, the number of puncta observed in rescue experiments involving transgenic overexpression is comparable, though not identical, to those quantified using endogenous knock-in reporters. For instance, in the rig-5; zig-8 double mutant background, expression of BFP-RIG-5 in AVE and Scarlet-ZIG-8 in AVA neurons resulted in 29.9 ± 6.9 AVE-to-AVA puncta (Fig. 4c, d), whereas under physiological conditions, only 16 ± 3 AVE-to-AVA synapses were observed (Fig. 1c, h). Such increase is likely caused by transgenic overexpression, suggesting that the precise temporal and quantitative regulation of RIG-5 and ZIG-8 is essential for establishing the correct number of synapses.

The Ig2 domain of ZIG-8 recruits ACR-16
RIG-5 and ZIG-8 are adhesion proteins composed of 3 and 2 Ig-like domains, respectively. We analyzed the function of truncated RIG-5 and ZIG-8 proteins generated by CRISPR/Cas9 gene genome engineering, except for deletions of their Ig1 domains, which prevented their expression and were therefore not studied. Since ZIG-8 is required at the postsynaptic membrane, we investigated the role of its Ig2 domain in ACR-16-AID-Scarlet localization. Strikingly, in the absence of its Ig2 domain (gfp::zig-8(ΔIg2)), ZIG-8 ΔIg2 formed synaptic clusters that overlapped with BFP-RIG-5, yet ACR-16-AID-Scarlet remained diffuse along the neurites (Fig. 5a). To exclude that this could be caused by the shortening of the protein, we replaced the ZIG-8 Ig2 domain with the human CD4 Ig2 domain (gfp::zig-8(ΔIg2)::CD4(Ig2)) and observed that ACR-16-AID-Scarlet also failed to cluster properly (Fig. 5a). These results indicate that the Ig2 domain of ZIG-8 is critical for recruiting ACR-16. In contrast, fragments of RIG-5 showing deletions in the Ig2, Ig3, or both Ig2 and Ig3 domains, were detected at synapses and effectively promoted ACR-16-AID-Scarlet concentration in the presence of full-length ZIG-8 (Supplementary Fig. 2 and 6a–c).

Our results were consistent with a direct interaction between ZIG-8 and ACR-16. To test this hypothesis, we ran a structure prediction analysis using AlphaFold-Multimer. ACR-16 adopted a typical AChR structure, forming a pentamer composed of five subunits arranged around a central pore (Fig. 5b and Supplementary Fig. 7a)39. Interestingly, this analysis indicated potential interactions between ZIG-8 and each individual ACR-16 subunit at the base of the large N-terminal extracellular domain. The predicted interface involves several unconventional side-to-side, strand-to-strand hydrogen bond interactions, with the beta-strand G of ZIG-8 interacting with the beta-strand 9 of ACR-16 (Fig. 5c). This coupling is likely further stabilized by additional hydrogen bond and hydrophobic interactions involving atoms in the main and side chains of surrounding residues. For example, we observed that T201 and L199 side chains of ACR-16 sit in a mostly hydrophobic cavity formed between the A and G strands of ZIG-8’s Ig2 domain (Fig. 5d). Based on this, we predict that a bulkier side chain at these positions, such as an arginine at T201, would break this interaction.

To validate this predicted ZIG-8—ACR-16 interaction interface, we introduced a T201R point mutation into acr-16::aid::Scarlet (acr-16(T201R)::aid::Scarlet). To test whether ACR-16(T201R) was still functional, we used a loss-of-function mutation in unc-29, which encodes an essential subunit of the heteromeric L-AChRs present at the NMJ. When homomeric and heteromeric AChRs were non-functional in an unc-29; acr-16 double mutant, animals were paralyzed, whereas the unc-29 single mutant exhibited mild locomotion defects and the acr-16 single mutant displayed normal locomotion (Supplementary Fig. 8). Importantly, the acr-16(T201R) mutant showed normal locomotion and did not exacerbate locomotion defects when combined with the unc-29 mutation. These results indicated that the acr-16(T201R) mutation did not impair ACR-16 expression and function. To probe the interaction of the ACR-16(T201R)-AID-Scarlet variant with ZIG-8, we examined its clustering at neuron-neuron synapses in vivo. We found that the ACR-16(T201R)-AID-Scarlet variant failed to properly cluster at synapses labeled with BFP-RIG-5 and GFP-ZIG-8 (Fig. 5e). Our data strongly suggest that the T201R mutation in acr-16 disrupts its ability to interact with ZIG-8 in vivo at synapses.

To investigate whether ZIG-8 can directly cluster ACR-16 in vivo, we took advantage of the fact that ACR-16 is also expressed in muscle cells, but does not rely on RIG-5 and ZIG-8 for its synaptic localization. Rather, it involves an interaction of the large intracellular loop of ACR-16 with an intracellular scaffold comprising the FRM-3 multimodular protein. In frm-3 null mutants, ACR-16-Scarlet is still present at the plasma membrane of muscle cells but fail to concentrate at NMJs and is not detected by fluorescence microscopy (Fig. 6b)17. To test whether ZIG-8 is sufficient to cluster ACR-16, we engineered a mNeonGreen (mNG)-tagged chimeric protein between the ectodomain of the adhesion molecule NLG-1/neuroligin, which we previously demonstrated to localize to GABAergic NMJs of C. elegans, and the Ig2 domain of ZIG-8 with its GPI anchor signal (Fig. 6a, c)18. Strikingly, expression of the mNG-NLG-1-ZIG-8(Ig2) chimera in the muscle cells of frm-3 mutants caused the clustering of ACR-16-Scarlet at GABAergic NMJs. To confirm that the formation of these clusters did involve the ZIG-8—ACR-16 extracellular interface, we performed two control experiments. First, we introduced the T201R mutation in ACR-16-Scarlet and observed that mNG-NLG-1-ZIG-8(Ig2) no longer recruited ACR-16 at GABAergic NMJs (Fig. 6d, f–h). Second, we replaced the Ig2 domain of ZIG-8 by its Ig1 domain in the chimera and, although the mNG-NLG-1-ZIG-8(Ig1) chimera still localized to GABAergic NMJs, it failed to cluster ACR-16 (Fig. 6e, f–h).

Taken together, these results support a direct mechanism whereby the Ig2 domain of ZIG-8 binds the base of the ACR-16 extracellular domain in cis on the postsynaptic membrane to cluster ACR-16 at neuron-neuron synapses.

Methods
Strains
All experiments were performed at 20°C. Strains were grown on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP5083. The wild-type reference strain was C. elegans N2 Bristol. All strains and alleles used in this study are described in Supplementary Table S1 and S2, respectively.

Geneticscreen
Mutagenesis. Mutagenesis was performed using the EN7643 strain (krSi81[Pmyo-3::TIR1::bfp]; kr463[acr-16::aid::scarlet]). Animals were exposed to 47mM ethyl methanesulfonate (EMS; Sigma) for 4h at 20°C. Following treatment, populations were maintained at 15°C, 20°C, or 23°C to promote staggered hatching. A total of five independent mutagenesis experiments were conducted, and 12,912 C. elegans haploid genomes were screened.

Isolation of mutants. Clonal F2 progeny of mutagenized EN7643 animals, grown on auxin-supplemented NGM plates, were examined using a Nikon AZ100 Multizoom microscope. Animals exhibiting abnormalities in the ACR-16::AID::Scarlet expression pattern were isolated and categorized into four phenotypic classes: absence of signal, weak puncta, diffuse signal, and other distribution defects. Mutants exhibiting diffuse signal along neurites were prioritized, as this phenotype was consistent with impaired ACR-16 clustering. Animals with homozygous mutations were isolated in the F3 progeny.

Mapping, whole genome sequencing and validation of mutations.
Homozygous mutant strains were backcrossed twice to the parental EN7643 strain. After the second backcross, five independent F2 recombinant mutants were pooled for genomic DNA extraction and whole-genome sequencing (Eurofins and Novogene). A local bioinformatic pipeline derived from the CloudMap pipeline was used to identify candidate causative mutations and regions of genetic linkage, indicated by clusters of EMS-induced variants across large chromosomal segments84,85. Candidate mutations were validated by recreating the mutation in the parental EN7643 background and/or by reverting the mutation to the wild-type sequence in the mutant strain. Of the 53 mutant strains isolated, 12 exhibited a diffuse ACR-16::Scarlet distribution phenotype. These included five independent alleles of rig-5 and zig-8: rig-5(kr573[Q16*]), rig-5(kr849[P191S]), zig-8(kr850[Q92*]), zig-8(kr851[A68T]) and zig-8(kr852[I239F]) (Supplementary Fig. 2).

Allele generation by CRISPR/Cas9 genome engineering
All knock-in alleles were generated according to a standard protocol86. crRNA was designed using Benchling software and synthesized by Integrated DNA Technologies (IDT). To form a triplex, a mix of 2.8 μL crRNA (34 μM), 5 μL tracrRNA (18 μM) and 0.5 μL Cas9 nuclease (10 μL/μg) (IDT) was incubated for 15 min at 37°C. For deletions, two crRNA were used at both ends of the target sequence, with a triplex made of 1.4 μL of each crRNA. A mix containing 2.2 μL single-strand repair template (1 μg/μl) or 500ng double-strand repair template, with 800ng of pRF4 plasmid, and molecular biology grade water up to 20 μL was added to the triplex. This mix was injected into adult C. elegans. Worms with a roller phenotype in the next generation were isolated and tested by PCR. All gene edits were confirmed by sequencing. Finally, the edited strains were outcrossed once to remove nonspecific background mutations. A list of crRNA is provided in Supplementary Table S3.

Plasmids
The plasmids constructed for this study are described in Supplementary Table S4. All constructs were verified by Sanger sequencing from the GATC company. Plasmid sequences are available upon request.

Tissue-specific expression
For tissue-specific expression, the promoters used were as follows:
- Pmyo-3: body wall muscle cells
- Punc-17: cholinergic motoneurons
- Punc-129: D-type motoneurons (DA and DB type)
- Ppept-3: AVE neurons87
For specific expression in AVA and AVB neurons, the Cre-loxP recombination strategy was used, with promoter combination as follows: Pflp-18 and Pgpa-14 (AVA neurons), Ptkw-40 and Plgc-55 (AVB neurons)25,88,89. Promoter boundaries are described in Supplementary Table S5.
The protein sequences of rig-5 and zig-8 are provided in Supplementary Table S7. The cDNAs were cloned by RT-PCR from mixed-stage C. elegans RNAs.

Generation of single-copy insertion alleles
Single-copy insertion alleles were generated by the miniMos method90. Briefly, adult C. elegans were injected with a mix containing 15 ng/μL of the plasmid of interest (containing promoters and open reading frames fused to fluorescent proteins), 50 ng/μL pCFJ601 (Mos1 transposase), 10 ng/μL pMA122 (negative selection marker Phsp16.2::peel-1), and 2.5 ng/μL pCFJ90 (Pmyo-2::mCherry). Neomycin (G418) was added to plates 24h after injection at 1.5 μg/μL final concentration. Candidate plates were heat-shocked for 2h at 34°C. Worms with an insertion were isolated and homozygosed.

Multicopy and extrachromosomal array lines
Extrachromosomal array alleles were generated by injecting a mix of plasmids of interest as depicted in the Supplementary Table S2.
For acr-16 transcriptional reporter (krIs83[Pacr-16::aid::mNG], Supplementary Fig. 1b), a strain bearing a Pacr-16::aid::mNG extrachromosomal array was randomly integrated by X-ray irradiation (40 Gy).

Auxin-induced cell specific degradation
For all images except in Fig. 4e, f, Fig. 6, Supplementary Fig. 1b, Supplementary Fig. 3a, Supplementary Fig. 4 and Supplementary Fig. 5, worms were grown on auxin plates to degrade ACR-16-AID-Scarlet at NMJs. To induce protein degradation, adult C. elegans were transferred to auxin plates, and their progeny was analyzed. Auxin plates were prepared by adding auxin (indole-3-acetic acid, Sigma-Aldrich) from a 400 mM stock solution in ethanol to NGM, at the final concentration of 1 mM22.

Neuron-type Specific Illumination (NeuroSIL)
ACR-16 was tagged with AID and Scarlet sequences, along with three copies of spGFP11 (ACR-16-AID-Scarlet-spGFP11). The spGFP1-10 moiety was expressed in the cytoplasm of AVA or DB neurons using selected combinations of promoters, as described above. All ACR-16 clusters displayed Scarlet labelling but only ACR-16 clusters formed in the AVAs or DBs, exhibited GFP fluorescence upon reconstruction. Two to four transgenic lines were observed, and/or used for quantification: Fig. 1e (2 lines), Fig. 1h (4 lines) and Supplementary Fig. 1f (2 lines).

Microscopy imaging and quantification
For spinning disk imaging, young adult hermaphrodites were used, except in Supplementary Fig. 4, in which L1 larvae were imaged 1-2h after hatching. Live worms were mounted on 2% agarose dry pads with 2% (for young adults) or 5% (for L1 larvae) polystyrene beads (Polybeads, Polysciences) in M9 buffer. Worms were observed using an Andor spinning disk system (Oxford Instruments) installed on a Nikon-IX86 microscope (Olympus) equipped with a 60×/NA 1.42 oil-immersion objective and an Evolve electron-multiplying charge-coupled device camera. Each animal was imaged with IQ software (APIS Informationstechnologien) or Metamorph software (version 7.10.5.476) as a stack of optical sections (0.2 μm apart) across the whole thickness of the ventral or dorsal nerve cords. All images were processed using Fiji software (v2.0) and correspond to the sums of all slices with signal along the Z axis91,92.
Acquisition settings were the same across genotypes for quantitative analysis. Image quantification was performed using a macro in Fiji. For total fluorescence intensity measurements, a 50 μm (wide) × 3 μm (high) rectangle containing solely the nerve cord (first quarter) was cropped and projected as a sum in Z. The mean intensity was then projected in the x direction (orthogonal to the cord), and the intensity was measured as the area under the curve, after background exclusion93. Data are presented as a percentage of the average fluorescence relative to that of the wild type.
For puncta density and intensity quantification, a CORSEN analysis was used in Fig. 3d, e94. Due to the diffuse localization pattern observed in Fig. 2c–e, Fig. 3a–c, Fig. 5a, e, puncta density was quantified as follows: the cropped image (50 μm wide × 3 μm high) was converted to a one-dimensional axis along the y axis in Fiji (ImageJ). Plot profiles were generated for each image and saved as csv files. To determine puncta density, the csv files were computed by an R script to apply a threshold, define the background signal and count the number of peaks above this threshold95. The threshold was the same across genotypes for each quantitative analysis.
In Figs. 1c and 4a-d, the total number of puncta along the ventral nerve cord was quantified manually. For rescue experiments, one to three lines transgenic lines were observed, and/or used for quantification: Fig. 4a (1 line), Fig. 4b (1 line), Fig. 4c (2 lines), Fig. 4e (3 lines) and Fig. 4f (2 lines). Quantifications were performed on a single day to ensure consistency, whereas qualitative assessments of the rescues were repeated on three separated days to confirm reproducibility. The quantification of BFP-tagged proteins could not be assessed automatically at neuron-neuron synapses due to a low signal-to-noise ratio. For microscopy images show in Fig. 1b, h; 2a, b and Supplementary Fig. 1b, g-h; 3b; 4a-d, the observations were repeated on three different days.
For colocalization analysis, images were captured along 50 μm of the dorsal nerve cord anterior to the vulva. Fluorescence intensity along the cord was evaluated with the Plot Profile Fiji plugin. For each channel, values along the x axis were normalized to the maximal intensity value. Data are presented as minimum to maximum values for animals of each genotype. Colocalization between two channels was analyzed using Pearson’s correlation coefficient as previously described18. For quantification, data are presented as boxplots showing lower and upper quartiles (box), mean (square), median (centerline) and standard deviation (whiskers). Statistical tests were performed using RStudio version 2022.12.0 (R version 4.0.5).
Image quantification for each experiment was performed on the same day using standardized acquisition settings, thereby ensuring accurate and comparable measurements across genotypes. Consistency of phenotypes among genotypes was verified across at least three independent days of observation, confirming that the observed results were reproducible. All attempts at replication were successful. Blinding was not performed because data analysis was automated through macros applied to large datasets, minimizing the potential for user bias.
The NeuroPAL (Neuronal Polychromatic Atlas of Landmarks) strain was used to identify acr-16-expressing neurons based on a Pacr-16::aid::mNG transcriptional reporter in head, body and tail regions, by comparison to the reference manual23.

Structure prediction and analysis
Protein complex structure predictions were performed with AlphaFold-Multimer, using the ColabFold implementation version 1.5.2 running Alphafold release 2.3.196–98. For the prediction, we used the default settings, including the use of template information, relaxation of the predicted structures using amber force fields, and use of both paired and unpaired multiple sequence alignments. We ran alphafold predictions of the ACR-16 pentamer, and various ectodomain complexes of ACR-16 and ZIG-8, all returning models that were in close agreement. Alphafold reported high confidence values for the interfaces: the iPTM value for the ACR-16 pentamer, the 1:1 complex of ACR-16 and ZIG-8 and the ACR-16 pentamer with a ZIG-8 molecule were 0.849, 0.865 and 0.845. An example of the sequence coverage, pLDDT, and predicted alignment error (PAE) plots are shown in Supplementary Fig. 7b. Analysis of the structures and creation of composite models (including the ZIG-8-RIG-5 crystal structure) were performed using the molecular graphics program PyMOL (v. 3.0.4)31,99.

Locomotion test
Worm locomotion was assessed on plates by gently tapping the head and tail of each animal using a platinum wire mounted on a pick, as well as following mechanical tapping of the Petri dish. The assessments were conducted by three experimenters who were blinded to the genotype.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Source data are provided with this paper. The plasmids and C. elegans strains generated in this study are available from the corresponding authors upon request. Source data are provided with this paper.

Code availability
The code used to perform analyses of synapse density is deposited on Zenodo, and is accessible via (https://doi.org/10.5281/zenodo.17571550)95.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Mialon et al. identify a novel mechanism for the clustering of acetyl choline receptors (AChRs) at neuronal synapses. Using a series of sophisticated manipulations in C. elegans, they convincingly show that extracellular interactions mediated by adhesion molecules of the IgLON family localize the ACR-16 AChR at the synapse. They find that RIG-5, a presynaptic IgLON, and ZIG-8, a postsynaptic IgLON, interact via their N-terminal Ig1 domains to form a trans-synaptic complex. In addition, ZIG-8 interacts in cis with ACR-16 via its Ig2 domain. Using AlphaFold multimer modelling, the authors identify a single amino acid substitution in ACR-16 that is sufficient to perturb its interaction with the ZIG-8 Ig2 domain, resulting in a loss of ACR-16 clustering at synapses. Finally, the authors show that the interaction with the ZIG-8 Ig2 domain is sufficient to mislocalize ACR-16 to GABAergic postsynaptic compartments. Taken together, this study identifies a novel mechanism for localizing AChRs at synapses, which may have implications for mammalian nervous systems. The rationale for the experiments is clearly explained, results are clearly described, elegant assays such as extra- and intracellular split-GFP complementation are used, and the experiments are performed to a high standard. I have only one major comment and several minor ones for improvement of the manuscript.\nMajor comment\n1. The authors test the interaction between ACR-16 or ACR-16(T201R) and ZIG-8 in a co-immunoprecipitation assay in HEK293T cells (Fig. 5B). The blot shown is not entirely convincing and appears to have some smudges on the left side of the RFP blot. The IP of ACR-16 in the condition where it is coexpressed with ZIG-8 (right lane) is weak, resulting in very low levels of coimmunoprecipitated ZIG-8. This blot should be repeated to convincingly show the interaction between ACR-16 and ZIG-8, and absence of the interaction between ACR-16(T201R) and ZIG-8. In addition to showing the pulldown lanes, the authors should also include the input lanes, to show presence of ACR-16 and ZIG-8 across the various conditions.\nMinor comments\n1. Mutations in rig-5 and zig-8 were identified in a genetic screen. From supplementary table S2 it was not clear to me whether these were the only candidates, or top candidates, or two of multiple candidates, to affect ACR-16 localization. A bit more background on this screen and the identification of rig-5 and zig-8 would be helpful.\n2. Fig. 4D (cell type-specific rescues) shows that expression of ZIG-8 in the AVA neurons leads to a strong increase in apparent synaptic puncta density. The reason for this is not entirely clear. From the image in Fig. 4B, there actually appears to be a decrease in ZIG-8 puncta density and an increase in diffuse ZIG-8 signal. Is the increase in puncta density due to the more diffuse distribution of ZIG-8? Are these additional clusters synaptic? Or could this indicate that ZIG-8 can induce additional ACR-16 clusters, partly independent of presynaptic RIG-5? Or could the apparent increase in ACR-16 clusters be due to the presence of RIG-5 in other presynaptic neurons besides the AVE neurons? Please discuss.\n3. Related to the comment above: the number of ACR-16 puncta in AVE-to-AVA synapses is ±16 in Fig. 1C and ±29 in Fig. 4D (rescue with presynaptic RIG-5 in AVE and postsynaptic ZIG-8 in AVA neurons). Please comment on these differences.\n4. There are many AChRs in C. elegans, but only 2 IgLON orthologs. Do the authors think the interaction with IgLONs might also occur with other AChRs or is this specific for ACR-16? Is the interacting region in ACR-16 (T201) conserved in other AChRs?\n5. Any insight on whether RIG-5 and ZIG-8 affect AChR function would further strengthen the manuscript. I understand electrophysiology is not trivial, but possibly other approaches exist? E.g. does AlphaFold multimer modelling show changes in AChR structure in the presence of ZIG-8?", "reviewer_2": "Precise subcellular localization and clustering of NTRs at post-synaptic sites is crucial for proper neuronal function, but how this is achieved, and specifically the roles that extracellular proteins play in this process, is poorly understood. In C.elegans, Ach is the most widely used NT. ACR-16 (the α7-like AChR subunit), was previously shown to cluster in the NMJ by intracellular protein scaffolds, but how it clusters in neuron-neuron synapses remains unknown.\nIn this study, Mialon and colleagues focus on specific neurons in the worm nerve cord, and find that the IgLON homologs RIG-5 and ZIG-8 localize to pre- and postsynapes, respectively, and trans-neuronally interact (via Ig1) to control the localization of one another. Moreover, ZIG-8 directly binds and recruits ACR-16 via its Ig2 domain. Thus, they uncover a novel mechanism controlling the synaptic localization of an AChR via extracellular interactions with IgSF synaptic molecules.\nThis is a very elegant study, well-constructed and addressing the majority of the critical aspects required for the authors to establish their hypothesis. It is carefully designed and highly convincing, and its potential implications on other neuronal systems and higher organisms – given the conserved nature of all molecules involved – is of a broad interest to the molecular neuroscience community. I actually think that if the authors would have written the paper in a bit more broader manner, and perhaps focusing on synaptic targeting rather than NTR localization, this study could be published in the top journals. That said - in its current state and with minor textual changes and improvements of figure presentation - this is a top paper for Nature Comm.\n1) Annotation of the figures in many places is lacking and they are sometimes confusing or difficult to interpret (in some cases even after reading the entire legend). The system is not straightforward and involves many types of neurons that is difficult for the non-worm expert to follow. There are some schemes throughout which try to clarify, but they are not clear enough (e.g., 1D doesn't show AVE, 1F is tiny and hard to understand - zoom in would help). Better annotation of the images themselves would also be helpful (e.g. S Fig. 5, and other places - two types of arrows indicate two different neurons - but the legend doesn't say which type points to what).\n2) The intro is, in my opinion, a bit tedious. Too many facts and gene names. It's a stylish preference - so completely up to the authors - but one could make the paper lighter and more interesting to the general reader.\n3) Tissue specific manipulation - again worm specific, but I could not find specifics on how the authors control the tissue-specificity of the AID experiments (Fig. 1B) – how is the auxin administered/introduced to the animal?\n4) The screen - no hard data or information is provided about the genetic screen in which zig8 and rig5 were identified.\n5) \"dimmer\" puncta in 3E - not evident in the fluorescent images.\n6) \"Taken together, our data strongly suggest that the main function of RIG-5 and ZIG-8 is to cluster the nicotinic receptor ACR-16 at specific cholinergic synapses.\" - I find this a bit too strong of a statement.\n7) In figure 4 - what does the change in the number of puncta mean? this is not really evident in the fluorescent images BTW. Also - how does the number of puncta relate to the number of synapses (in figure 1) - this is unclear. ALSO - if Zig8 and Rig5 interact independently of Acr16 then why does it matter which of the proteins is in the pre- or post- synapse? This relates to 4E/F\n8) coIP does not equal with physical interaction but rather presence in the same complex.\n9) are these worms \"normal\"?\nI want to reiterate - this is an excellent and very elegant paper. One of its main discoveries is the way by which igSF proteins 'match' pre and post synaptic partners - which is by clustering neurotransmitter receptors. Super cool, super relevant - definitely accept."}, "author_response": "We agree with our reviewer that the technical quality of the co-IP is not optimal. This is largely due to the intrinsic difficulty of expressing AChRs in heterologous systems, and in particular ACR-16, which requires the co-expression of the RIC-3 chaperone. Despite numerous attempts to optimize transfection conditions, expression times, and plasmid combinations, we were unable to obtain convincing co-immunoprecipitation results due to persistent expression issues. We therefore turned to an alternative strategy and invested considerable effort in developing a flow cytometry–based assay. We attempted to produce ACR-16 in insect cells and generated recombinant ZIG-8 fused to streptavidin, which was further detected by fluorescent biotin. Unfortunately, once again, ACR-16 expression was too low to yield a signal above background.\nSince we believe that the ZIG-8—ACR-16 interaction is strongly supported by in silico evidence (AlphaFold-Multimer iPTM score >0.8), point mutagenesis (disruption of ACR-16 (T201R) clustering at neuronal synapses (Fig. 5e) but not at NMJs (Sup Fig. 8)), and the sufficiency of the ZIG-8 Ig2 domain for ACR-16 clustering when ectopically expressed in muscle (Figure 6c-e), we have decided to remove the co-IP data from the revised manuscript. We have therefore removed panel B from Figure 5 and renamed the remaining panels. We have removed the following text from the main text:\n- 1 -\n“Our results were compatible with a direct interaction between ZIG-8 and ACR-16. To test this hypothesis, we performed a co-immunoprecipitation experiment in HEK cells expressing both proteins. To prevent ZIG-8 from being released into the culture medium via cleavage of the GPI anchor, we replaced the C-terminal sequence of ZIG-8 containing the GPI anchor site with the transmembrane domain of human CD4 (GFP-ZIG-8-CD4(TM)). In addition, we coexpressed the RIC-3 chaperone to ensure proper intracellular trafficking of ACR-16-Scarlet38. The pull down of ACR-16-Scarlet efficiently co-immunoprecipitated GFP-ZIG-8-CD4(TM), hence supporting the existence of a physical interaction between ZIG-8 and ACR-16 (Figure 5B).”\n“Consistently, the ACR-16(T201R)-AID-Scarlet receptor failed to co-immunoprecipitate GFP-ZIG-8-CD4(TM) in the HEK cell assay (Figure 5B).”\nMinor comments\n1. Mutations in rig-5 and zig-8 were identified in a genetic screen. From supplementary table S2 it was not clear to me whether these were the only candidates, or top candidates, or two of multiple candidates, to affect ACR-16 localization. A bit more background on this screen and the identification of rig-5 and zig-8 would be helpful.\nWe thank the reviewer for the opportunity to elaborate on our genetic screen. In response, we have added a dedicated paragraph in the Methods section (line 816). Major changes are highlighted in yellow in the article file.\n“Genetic screen\nMutagenesis. Mutagenesis was performed using the EN7643 strain (krSi81[Pmyo-3::TIR1::bfp]; kr463[acr-16::aid::scarlet]). Animals were exposed to 47 mM ethyl methanesulfonate (EMS; Sigma) for 4 hours at 20 °C. Following treatment, populations were maintained at 15 °C, 20 °C, or 23 °C to promote staggered hatching. A total of five independent mutagenesis experiments were conducted, and 12,912 C. elegans haploid genomes were screened.\nIsolation of mutants. Clonal F2 progeny of mutagenized EN7643 animals, grown on auxin-supplemented NGM plates, were examined using a Nikon AZ100 Multizoom microscope. Animals exhibiting abnormalities in the ACR-16::AID::Scarlet expression pattern were isolated and categorized into four phenotypic classes: absence of signal, weak puncta, diffuse signal, and other distribution defects. Mutants exhibiting diffuse signal along neurites were prioritized, as this phenotype was consistent with impaired ACR-16 clustering. Animals with homozygous mutations were isolated in the F3 progeny.\nMapping, whole genome sequencing and validation of mutations. Homozygous mutant strains were backcrossed twice to the parental EN7643 strain. After the second backcross, five independent F2 recombinant mutants were pooled for genomic DNA extraction and whole-genome sequencing (Eurofins and Novogene). A local bioinformatics pipeline derived from the CloudMap pipeline was used to identify candidate causative mutations and regions of genetic linkage, indicated by clusters of EMS-induced variants across large chromosomal segments83-84. Candidate mutations were validated by recreating the mutation in the parental EN7643 background and/or by reverting the mutation to the wild-type sequence in the mutant strain. Of the 53 mutant strains isolated, 12 exhibited a diffuse ACR-16::Scarlet distribution phenotype. These included five independent alleles of rig-5 and zig-8: rig-5(kr573[Q16*]), rig-5(kr849[P191S]), zig-8(kr850[Q92*]), zig-8(kr851[A68T]) and zig-8(kr852[I239F]) (Supplementary Figure 2).”\n2. Fig. 4D (cell type-specific rescues) shows that expression of ZIG-8 in the AVA neurons leads to a strong increase in apparent synaptic puncta density. The reason for this is not entirely clear. From the image in Fig. 4B, there actually appears to be a decrease in ZIG-8 puncta density and an increase in diffuse ZIG-8 signal. Is the increase in puncta density due to the more diffuse distribution of ZIG-8? Are these additional clusters synaptic? Or could this indicate that ZIG-8 can induce additional ACR-16 clusters, partly independent of presynaptic RIG-5? Or could the apparent increase in ACR-16 clusters be due to the presence of RIG-5 in other presynaptic neurons besides the AVE neurons? Please discuss.\nWe thank the reviewer for this insightful comment. In these rescue experiments, we likely observe distinct classes of synapses depending on the genetic background: AVE > AVA connections in Figure 4a, c and e; and synapses onto AVA from various presynaptic neurons—including but not limited to AVE—as shown in Figure 4b. Although the number of puncta observed in the rescue conditions (Figure 4) is broadly similar to those quantified under physiological conditions, they are not identical (Figure 1c). For instance, in Figure 4b, where BFP-ZIG-8 is expressed in AVA neurons of a zig-8 mutant, we counted 80.7 ± 11.7 puncta, whereas one might expect approximately 108 ± 10 synapses based on the endogenous context.\nWe believe this discrepancy primarily arises from differences in the mode of expression of adhesion molecules. In contrast to the fluorescent knock-in reporters used throughout the study, the rescue constructs rely on transgenic overexpression driven by cell-specific promoters (Pept-3 for AVE neurons or a combination of Pgpa-14 and Pflp-18 for AVA neurons). Furthermore, in Figure 4b, ZIG-8 is tagged with a blue fluorescent protein (tagBFP), which is inherently dimmer and more difficult to detect than commonly used green or red fluorescent proteins. This may contribute to the more diffuse appearance of the BFP-ZIG-8 signal.\nIn principle, a more physiological approach to dissect cell-specific requirements could involve auxin-mediated degradation of endogenous proteins. However, this strategy depends on cytosolic degron tagging, which is not feasible for GPI-anchored proteins such as ZIG-8 and RIG-5. Consequently, cell-specific rescue using transgenic expression was the most viable option for these experiments.\nWe have improved Figure 4’s display and added two sentences to clarify the experiment (lines 283 and 291):\n“In these rescue experiments, we observed distinct classes of synapses depending on the genetic background.”\n“We observed an average of 80 puncta containing both BFP-ZIG-8 and ACR-16-AID-Scarlet along the ventral nerve cord. They likely correspond to synapses where ACR-16 clusters in AVA neurons, from various presynaptic neurons, including but not limited to AVE neurons (Figure 4b, d, Figure 1c, f).”\nMoreover, we have added a discussion of this point in the revised manuscript (line 311) :\n“Notably, the number of puncta observed in rescue experiments involving transgenic overexpression is comparable, though not identical, to those quantified using endogenous knock-in reporters. For instance, in the rig-5; zig-8 double mutant background, expression of BFP-RIG-5 in AVE and Scarlet-ZIG-8 in AVA neurons resulted in 29.9 ± 6.9 AVE-to-AVA puncta (Figure 4c, d), whereas under physiological conditions, only 16 ± 3 AVE-to-AVA synapses were observed (Figure 1c, h). Such increase is likely caused by transgenic overexpression, suggesting that the precise temporal and quantitative regulation of RIG-5 and ZIG-8 is essential for establishing the correct number of synapses.”\n3. Related to the comment above: the number of ACR-16 puncta in AVE-to-AVA synapses is ±16 in Fig. 1C and ±29 in Fig. 4D (rescue with presynaptic RIG-5 in AVE and postsynaptic ZIG-8 in AVA neurons). Please comment on these differences.\nPlease see our previous answer.\n4. There are many AChRs in C. elegans, but only 2 IgLON orthologs. Do the authors think the interaction with IgLONs might also occur with other AChRs or is this specific for ACR-16? Is the interacting region in ACR-16 (T201) conserved in other AChRs?\n- 3 -\nThe conservation of a ZIG-8-AChR interaction is a very interesting question that we have sought to address. To this end, we performed a Clustal aligment of a ACR-16 region spanning from the β-sheet 7 to loop C with its closest paralogs (ACR-15, -7 and -10)PMID: 34388373. Residues at the predicted interface with ZIG-8 are highlighted in yellow, and the T201 residue is marked in red. This alignment shows poor conservation of the interacting residues, suggesting that the predicted interaction between ZIG-8 and ACR-16 is specific.\nACR-16 DEQKCFFKFGSWTYDGYKLDLQPA------TGGFDISEYISNGEWALPLTTVERNEKFYDCCPEPY 60\nACR-15 DEQVCYFKFGSWTYTRDKIQLE--------KGDFDFSEFIPNGEWIIIDYRTNITVKQYECCPEQY 58\nACR-7 DEQICFMKFGSWTYHGFALDLRLDVV-KGQEPSADLSTYITNGEWHLLSAPARREEKFYKCCKEPY 65\nACR-10 DDQVCYLKFGSWTYHGLALDLSIIAEEDDSELSIDLSTYTPSGEWHLTKAPAVKDVKYNSCCKEPY 66\n*:* *::******* ::* . *:* : .*** : . * .** * * \nThe interaction between ACR-16 and ZIG-8 was robustly predicted using AlphaFold-Multimer. In principle, a similar strategy could be used to assess potential interactions between ZIG-8 and other AChRs. However, such analyses are complicated by the existence of approximately 30 AChR subunits, which can assemble into heteromeric complexes of up to five distinct subunits, with interaction interfaces possibly spanning multiple subunits.\nEven if ZIG-8 does not bind other C. elegans AChR through the same interface, this raises the broader issue of whether interactions involving IgLONs and ionotropic receptors may be relevant across species. Interestingly, during the course of our revision, a preprint from the Huganir lab reported binding of two rodent IgLONs to AMPA receptors and suggested a role in modulating receptor mobility. However, the study did not identify the binding interface. We have added a brief discussion on this report (line 503).\n“Interestingly, a recent preprint reports the direct binding of two rodent IgLONs (OPCML and NTM) to AMPA receptors. This interaction may occur at synapses and regulate AMPAR mobility. However, the precise binding interface has not yet been identified79.”\n5. Any insight on whether RIG-5 and ZIG-8 affect AChR function would further strengthen the manuscript. I understand electrophysiology is not trivial, but possibly other approaches exist? E.g. does AlphaFold multimer modelling show changes in AChR structure in the presence of ZIG-8?\nWhether ZIG-8 modulates the function of ACR-16 is an intriguing question. We agree with the reviewer that electrophysiological analysis is technically challenging. Moreover, whereas AlphaFold-Multimer can reveal plausible binding modes and suggest how such interactions might alter receptor conformation, it does not directly predict functional outcomes, such as changes in ion conductance, ligand affinity, gating, or desensitization kinetics.\nTo explore this further, we used AlphaFold-Multimer v3 to model both an ACR-16 homopentamer (magenta) and a complex between the ACR-16 homopentamer and the Ig2 domain of ZIG-8 (shades of green). The two predicted structures were aligned using ChimeraX. The resulting comparison suggests that ZIG-8 binding does not induce substantial conformational changes in the extracellular domain of ACR-16 and the ion pore, nor does it obstruct the agonist-binding pocket located at the top of the receptor.\n- 4 -\nSideview Topview\nACR-16homopentamer\nACR-16homopentamerincomplexwithZIG-8Ig2domain\nGiven the potential functional relevance of this interaction, we will maintain this brief discussion in our manuscript (line 432):\n“Due to their distinct binding specificity to AChRs and unique expression patterns in the mammalian brain, immune system, and other tissues, Lynx prototoxins have been proposed to modulate the function of specific AChR subtypes in a regionally specific manner. However, they have not been shown to control AChR localization. In C. elegans, it might be interesting in the future to test whether ZIG-8 binding modulates the function of ACR-16 in a similar manner, although ZIG-8 does not interfere with the ACR-16 agonist-binding site based on in silico prediction.”\nReviewer #2 (Remarks to the Author):\n1) Annotation of the figures in many places is lacking and they are sometimes confusing or difficult to interpret (in some cases even after reading the entire legend). The system is not straightforward and involves many types of neurons that is difficult for the non-worm expert to follow. There are some schemes throughout which try to clarify, but they are not clear enough (e.g., 1D doesn't show AVE, 1F is tiny and hard to understand - zoom in would help). Better annotation of the images themselves would also be helpful (e.g. S Fig. 5, and other places - two types of arrows indicate two different neurons - but the legend doesn't say which type points to what).\nWe thank the reviewer for this advice. We have modified the figures and legends accordingly. Here are the main revisions:\n- We have used a consistent code for showing neuromuscular junctions (arrows) and neuron-neuron synapses (arrowheads) throughout the figures, including Supplementary Figure 5.\n- In Figure 1d, we have added a schematic illustrating NeuroSIL expression in DB neurons. We did not label the AVA presynaptic neuron “AVE” because AVA neurons receive inputs from various presynaptic neurons, including but not limited to AVE neurons. Instead, we have modified Figure 1g color display to highlight that AVE is presynaptic to AVA, along with other neurons.\n- In Figure 1f, we have enlarged schematics of C. elegans for improved clarity.\n- In Figure 4, we have revised the presentation of genetic backgrounds and corresponding synaptic patterns.\n- We have added details on the rig-5 and zig-8 isoforms used and the deletions made in the main text (line 333), in the Methods (line 873), and in the legend of Supplementary Figure 2. In addition, we have provided the protein sequences of rig-5 and zig-8 in a new Supplementary Table S7.\nMain text: “In contrast, fragments of RIG-5 showing deletions in the Ig2, Ig3, or both Ig2 and Ig3 domains, were detected at synapses and effectively promoted ACR-16-AID-Scarlet concentration in the presence of full-length ZIG-8 (Supplementary Figure 2 and 6a-c).\nMethods: “The protein sequences of rig-5 and zig-8 are provided in Supplementary Table S7. The cDNAs were cloned by RT-PCR from mixed-stage C. elegans RNAs.”\n“Supplementary Figure 2 (related to Figure 2). The rig-5 and zig-8 loci. (A) Genomic organization of rig-5 and the positions of different tags and mutants used in this study. We used the longest isoform (C36F7.4e.1), considering the second methionine in the transcript as a translation start site, as it generates a strongly predicted signal peptide (see Supplementary Table S7 for sequence numbering and reference30). (B) zig-8 locus, tags and mutants, including an insertion used in this study. Genome annotation of C. elegans reports a single transcript for zig-8 (Y39E4B.8.1), which encodes a protein with UniProt accession number G5ED00 (Supplementary Table S7). Boxes represent exons with sequences of the Ig domains colored in orange. Ig domains were identified using the SMART web-based tool (Simple Modular Architecture Research Tool, hcp://SMART.embl-heidelberg.de). Complete coding sequence were deleted for the rig-5 Ig1 and Ig3 domains (rig-5(bab526) and rig-5(bab520) alleles). In contrast, only partial deletions were created for the rig-5 Ig2 and zig-8 Ig1 domains due to the presence of large introns (rig-5(bab518) and zig-8(bab528) alleles). Additionally, a small portion of the coding sequence in the zig-8 Ig2 domain was retained because of CRISPR design constraints (zig-8(kr727) allele). Arrowheads: point mutations; brackets: deletions.\nSupplementary Table S2 contains a brief description of rig-5 and zig-8 alleles, including deletion boundaries.”\nFinally, we adjusted the abstract length, subheading, and figure display to align with the journal’s style guidelines.\n2) The intro is, in my opinion, a bit tedious. Too many facts and gene names. It's a stylish preference - so completely up to the authors - but one could make the paper lighter and more interesting to the general reader.\nThe most innovative finding of our study is the unexpected binding of a postsynaptic receptor via its extracellular domain. In this context, we found it particularly interesting to precisely compile known instances of such extracellular interactions - a rare mechanism involving only a small number of synaptic adhesion molecules. While we acknowledge that this introduction may seem tedious, we believe our work stands out meaningfully within this body of literature.\n- 6 -\n3) Tissue specific manipulation - again worm specific, but I could not find specifics on how the authors control the tissue-specificity of the AID experiments (Fig. 1B) – how is the auxin administered/introduced to the animal?\nIn response to this comment, we have revised the relevant sentence in the Methods section (line 893). The updated wording is highlighted in brown below:\n“Auxin-induced cell specific degradation\nFor all images except in Fig. 4e, f, Fig. 6, Supp. Fig.1b, Supp. Fig. 3a, Supp. Fig. 4 and Supp. Fig. 5, worms were grown on auxin plates to degrade ACR-16-AID-Scarlet at NMJs. To induce protein degradation, adult C. elegans were transferred to auxin plates, and their progeny was analyzed. Auxin plates were prepared by adding auxin (indole-3-acetic acid, Sigma-Aldrich) from a 400 mM stock solution in ethanol to NGM, at the final concentration of 1 mM22.”\n4) The screen - no hard data or information is provided about the genetic screen in which zig8 and rig5 were identified.\nAs noted in our response to Reviewer 1 point 1 (minor comments), details on the genetic screen have now been included in the Methods section.\n5) \"dimmer\" puncta in 3E - not evident in the fluorescent images.\nThe dimmer appearance of GFP-RIG-5 puncta in the acr-16(ok789) mutant is supported by a reduced fluorescence intensity per puncta, quantified from a set of 15–16 images and shown in the right panel of Figure 3e. This difference is also visually apparent: when using the ‘fire’ lookup table, the center of GFP-RIG-5 puncta appears white in the wild type, but shifts to yellow in the acr-16(ok789) mutant, indicating lower intensity. To enhance visual clarity for the reader, we have now included the ‘fire’ lookup table directly in Figure 3 and other Figures.\n6) \"Taken together, our data strongly suggest that the main function of RIG-5 and ZIG-8 is to cluster the nicotinic receptor ACR-16 at specific cholinergic synapses.\" - I find this a bit too strong of a statement.\nWe have toned down this statement (line 268):\n\" Taken together, our data suggest that RIG-5 and ZIG-8 control the clustering of ACR-16 nicotinic receptors at specific cholinergic synapses.\"\n7) In figure 4 - what does the change in the number of puncta mean? this is not really evident in the fluorescent images BTW.\nWe thank the reviewer for this insightful comment. The ambiguity surrounding the rescue experiments was also noted by Reviewer #1 (Point 2). Please refer to our detailed response and the corresponding revisions in the main text, and Figure 4, we have made.\nAlso - how does the number of puncta relate to the number of synapses (in figure 1) - this is unclear.\nWe thank the reviewer for the opportunity to clarify this point. First, in the rescue experiments, the number and distribution of puncta closely resembled the pattern typically observed at synapses between AVEs and AVAs, or at synapses formed onto AVAs (Figure 4a-c). We have now added this clarification to the manuscript (line 283):\n“In these rescue experiments, we observed the recovery of distinct classes of synapses depending on the genetic background.”\n- 7 -\nSecond, we directly demonstrate recovery of AVE>AVA synapses using an AVE-specific presynaptic marker (Figure 4e), as noted on line 299. We have modified a sentence to clarify the conclusion of this experiment (in brown):\n“To confirm the synaptic location of these puncta, we expressed BFP-RIG-5 in the AVEs along with an AVE-specific presynaptic marker, and Scarlet-ZIG-8 in the AVA, in the double rig-5(0); zig-8(0) mutant. We observed that BFP-RIG-5 and Scarlet-ZIG-8 formed puncta juxtaposed to the AVE presynaptic marker, supporting the formation of functional synapses (Figure 4e).”\nALSO - if Zig8 and Rig5 interact independently of Acr16 then why does it matter which of the proteins is in the pre- or post- synapse? This relates to 4E/F\nWhen RIG-5 is expressed in the presynaptic neuron and ZIG-8 in the postsynaptic neuron, their interaction is detected. In contrast, reversing the expression pattern does not yield a stable interaction. One possibility is that the RIG-5—ZIG-8 interaction can be stabilized at synapses upon interaction with other synaptic proteins. Interestingly, RIG-5 is a likely candidate for engaging presynaptic components. However, this remains speculative, and we prefer not to extend the interpretation further at this stage.\n8) coIP does not equal with physical interaction but rather presence in the same complex.\nWe agree. Due to technical limitations, we have decided to remove this specific experiment from the revised manuscript. Please refer to our response to the reviewer #1’s first point.\n9) are these worms \"normal\"?\nThe behavioral consequences of the acr-16, rig-5, and zig-8 mutations remain an open question. We have not observed any obvious defects in forward or backward movement on agar plates. Noteworthy, complete loss of ACR-16 does not cause obvious locomotory defects (PMID: 15917232). Because rig-5 and zig-8 mutations impact the connectivity of the locomotory circuit, we cannot rule out that subtle modifications of locomotion might be detectable by detailed quantitative analysis and calcium imaging of neuronal activity. While this would be interesting, we believe it is beyond the scope of our current work.\nI want to reiterate - this is an excellent and very elegant paper. One of its main discoveries is the way by which igSF proteins 'match' pre and post synaptic partners - which is by clustering neurotransmitter receptors. Super cool, super relevant - definitely accept."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the extensive effort the authors have put in to further demonstrate the interaction between ZIG-8 and ACR16 and agree with their decision to remove the western blot data from the manuscript - the interaction is convincingly supported by other lines of evidence. The added clarifications in response to the reviewers' comments have further improved an already excellent and exciting study - this manuscript is ready for publication.", "reviewer_2": "In this revised version of the manuscript the authors addressed most of my comments of what I already thought was a very good manuscript. I do have only one suggestion - the title of the last section of the results \"ZIG-8 Ig2 domain binds the ACR-16 extracellular domain\" is not, in my opinion, totally supported by the data. Is it the most plausible? likely yes. But I don't think - especially now without the coIP (and even with it) that the authors can truly make this claim. If you want to maintain this claim then perform SPR experiments on isolated proteins. This is not necessary in my opinion but then tweak down this statement."}, "author_response": "RESPONSE TO REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nI appreciate the extensive effort the authors have put in to further demonstrate the interaction between\nZIG-8 and ACR16 and agree with their decision to remove the western blot data from the manuscript -\nthe interaction is convincingly supported by other lines of evidence. The added clarifications in response\nto the reviewers' comments have further improved an already excellent and exciting study - this\nmanuscript is ready for publication.\nResponse:\nWe thank the reviewer for taking the time to review our manuscript, and for her/his insightful comments.\nReviewer #2 (Remarks to the Author):\nIn this revised version of the manuscript the authors addressed most of my comments of what I already\nthought was a very good manuscript. I do have only one suggestion - the title of the last section of the\nresults \"ZIG-8 Ig2 domain binds the ACR-16 extracellular domain\" is not, in my opinion, totally supported\nby the data. Is it the most plausible? likely yes. But I don't think - especially now without the coIP (and\neven with it) that the authors can truly make this claim. If you want to maintain this claim then perform\nSPR experiments on isolated proteins. This is not necessary in my opinion but then tweak down this\nstatement.\nResponse:\nWe agree that a biophysical assay using purified proteins would provide definitive proof of a direct\ninteraction between ACR-16 and ZIG-8. However, we appreciate that the reviewer concurs that a direct\ninteraction remains the most plausible explanation for our findings. We have modified the title of the last\nsection of the results as suggested by the reviewer.\n« The Ig2 domain of ZIG-8 recruits ACR-16 »"}]

Decision letter