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Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC/DCC controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABA_ARs) at inhibitory neuromuscular junctions. Here we show that activation of UNC assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK, that binds the synaptic adhesion molecule NLG-1/Neuroligin and physically connects GABA_ARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4, which controls the localization of GABA_ARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABA_ARs through the UNCdependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain.

The precise patterning of synapses arises from a series of spatially and temporally regulated developmental events that include axon navigation, contact recognition of pre- and postsynaptic partners and synaptic differentiation. Axonal navigation is guided by multiple extracellular guidance cues that activate signaling receptors on the surface of the growth cone. Similarly, synapse formation relies on tightly regulated structural and molecular changes that are instructed upon interaction of synaptic adhesion molecules. Intriguingly, numerous evidences also point to a role of axon guidance molecules in synaptic differentiation and plasticity^1,2,3, reviewed in Poon et al.⁴. Yet, it remains unclear for a given receptor what dictates the switch between a role in neurite guidance and a function in synaptogenesis or synaptic plasticity. Here, we identified the molecular mechanisms implementing the role of the netrin receptor DCC (Deleted in Colorectal Cancer) in the control of synaptic GABA_A receptor content in C. elegans.

Pioneer work in C. elegans initially identified three genes, unc-5, unc-6, and unc, that control the directionality of circumferential cell migrations and axon guidance⁵ (reviewed by Chisholm et al.⁶). Subsequently, netrins were identified as diffusible chemotropic factors of the laminin superfamily homologous to UNC-6^7,8. The vertebrate netrin receptors DCC and UNC5 were recognized according to their homology to C. elegans UNC and UNC-5, respectively^9,10. DCC is a type I transmembrane receptor that belongs to the immunoglobulin superfamily (IgSF). Netrin binding to the DCC ectodomain causes receptor dimerization, which brings in close proximity the cytosolic regions of the receptors and enables their dimerization. This provides a docking platform to recruit activators of multiple signaling pathways, usually leading to an attractive response towards high netrin concentrations (reviewed by Boyer and Gupton¹¹). In the presence of UNC5, netrin would trigger the formation of DCC/UNC5 heterodimers that mediate repulsive behaviors¹². This long-range chemotatic gradient model has recently been revisited after analysis of axonal growth in mice where netrin expression was inactivated in specific subregions of the developing nervous system^13,14,15. In these studies, the phenotypes were more consistent with a short-range haptotactic guidance model involving the interaction of growing axons with netrin present in the local environment. Detailed analysis of single axon outgrowth in C. elegans, together with in silico modeling, suggest that UNC-6/netrin biases the distribution of UNC/DCC at the membrane surface and stimulates an UNCdependent protrusive activity towards growth direction^16,17.

Scarce reports also demonstrated a role of netrin/DCC signaling at synapses in the mature brain. In cortical pyramidal neurons, DCC is thought to be involved in LTP (long-term potentiation) upon activation of the Src kinase and phosphorylation of the NMDA receptor¹. Consistently, deletion of DCC in the adult forebrain results in shorter dendritic spines, behavioral defects, and loss of long-term potentiation. Activity-dependent secretion of netrin was recently involved in DCC-dependent potentiation of excitatory glutamatergic transmission in the hippocampus via Ca2+-dependent recruitment of GluA1-containing AMPA receptors¹⁸.

The dual role of DCC in both development and synaptic homeostasis is evolutionarily conserved. In C. elegans, the UNC-6 netrin is secreted by ventral cells, and is thought to form a ventral-to-dorsal gradient^5,19. C. elegans cells and axons utilize the polarized distribution of UNC-6 to orient circumferential migrations using the UNC and UNC-5 receptors²⁰. A well-documented UNCdependent migration process is the outgrowth of body-wall muscle cell expansions towards motoneurons²¹. During post-embryonic development the muscle cells, which are located in four lateral quadrants along the animal’s body, extend projections called “muscle arms” to contact motoneurons at the medial ventral and dorsal cords and form en passant neuromuscular junctions (NMJs). UNC drives muscle arm extension in response to the Punctin/MADD-4 guidance cue that is secreted by developing motoneuron axons²². On the ventral side, MADD-4 functions redundantly with UNC UNC activation triggers the remodeling of the actin network and involves multiple actors including the Rho guanine-nucleotide exchange factor (GEF) Trio-homolog UNC, members of the WAVE actin-polymerization complex and UNC, a component of focal adhesion complex²³. The role of UNC/DCC was also carefully analyzed in the migration of several neurons (reviewed by Chisholm et al.⁶).

Besides its canonical functions in guidance, UNC plays direct roles in C. elegans synaptogenesis. Netrin signaling was shown to control synaptic connectivity between the two interneurons AIY and RIA²⁴. In this system, UNC guides the migration of postsynaptic neuron RIA and drives presynaptic differentiation in the AIY neuron in response to local secretion of UNC-6 by a glial cell. Downstream signaling modulates actin assembly and recruits presynaptic components²⁵. Recently, UNC-6 was involved in the male-specific maintenance of synapses between the sensory neuron PHB and the AVG interneuron²⁶.

We recently demonstrated that UNC plays a role in the postsynaptic organization of inhibitory NMJs of C. elegans²⁷. In C. elegans, each body-wall muscle cell receives excitatory and inhibitory innervation from cholinergic and GABAergic motoneurons. The evolutionarily conserved CePunctin/MADD-4 protein is identified as an anterograde synaptic organizer that specifies the cholinergic versus GABAergic identity of postsynaptic domains²⁸. CePunctin belongs to a family of poorly characterized extracellular matrix proteins, the ADAMTS-like proteins that contain multiple thrombospondin type-1 repeat (TSR) and immunoglobulin (Ig) domains as well as structurally unsolved domains in common with the ADAMTS family²⁹. The functions of its vertebrate orthologs Punctin1/ADAMTSL1 and Punctin2/ADAMTSL3 are ill-defined, but Punctin2 is expressed in the brain and was identified as a susceptibility gene for schizophrenia³⁰. Ce-punctin generates long (L) and short (S) isoforms by use of alternative promoters. MADD-4B/Punctin S and MADD-4L/Punctin L are differentially secreted by GABAergic and cholinergic neurons and trigger postsynaptic clustering of type A GABA receptors (GABA_ARs) and acetylcholine receptors (AChRs), respectively. At the inhibitory NMJ, MADD-4B-dependent clustering of GABA_ARs involves at least two molecular pathways (for review, see Zhou and Bessereau, )³¹. First, MADD-4B binds and clusters the synaptic adhesion molecule NLG-1/neuroligin in front of GABAergic boutons^27,32. Second, it binds, recruits, and likely activates the netrin receptor UNC/DCC, which in turn promotes the interaction of GABA_ARs with neuroligin through a non-characterized mechanism²⁷. Since UNC also controls the growth of muscle arms, the inhibitory NMJ represents an interesting paradigm to compare the molecular pathways required for the guidance and the synaptic functions of UNC

Using a genetic strategy, we identify here two proteins, FRM-3 and LIN-2, that implement the function of UNC for the recruitment of GABA_ARs at inhibitory NMJs. We show that UNC recruits FRM-3, a FERM (p, Ezrin, Radixin, Moesin) protein orthologous to FARP1/2, by a physical interaction between the intracellular P3 domain of UNC and the FERM-FA tandem of FRM In turn, FRM-3 recruits LIN-2, the ortholog of CASK (Calcium calmodulin dependent Serine/Threonine kinase), which might provide a hub to physically connect the GABA_ARs, FRM-3/FARP and NLG-1/neuroligin. All these processes are orchestrated by the main synaptic organizer MADD-4B/Punctin S, which both controls the synaptic localization of GABA_ARs through NLG-1/neuroligin and the synaptic content of GABA_ARs through an UNCdependent intracellular scaffold.

Mutations of frm-3/FARP and lin-2/CASK cause a strong decrease of synaptic GABA_ARs

To identify synaptic proteins that control GABA_ARs clustering, we mutagenized a C. elegans knock-in strain expressing fluorescently tagged GABA_ARs and performed a visual screen for abnormal fluorescence pattern. In this strain, the tagRFP-coding sequence was inserted in the unc locus, fusing tagRFP to the extracellular N-terminus shared by the three GABA_AR subunits UNCA, B and C. Among a wide range of phenotypes, we identified two strains with very strong decrease of synaptic fluorescence. Causative mutations were identified by whole genome sequencing, genetic mapping and rescue experiments.

One strain contained a mutation in the frm-3 gene, which codes for cytosolic proteins orthologous to the mammalian neuronal proteins FARP1 and FARP2 (Fig. 1a and Supplementary Fig. 1a). Two FRM-3 isoforms are generated by alternative splicing that share a common N-terminal part containing a FERM (p, Ezrin, Radixin, Moesin) and FA (FERM-Adjacent) domains. FRM-3A does not contain any recognizable domain in its C-terminal part. The frm-3B transcript was more recently annotated in WormBase and not characterized to our knowledge. FRM-3B contains a GEF domain and two PH domains as in mammalian FARPs. The kr mutation isolated in our screen introduces a G to A point mutation that inactivates a splicing donor site. For subsequent characterization of frm-3, we used the null allele frm-3(gk), further referred to as frm-3(0), which deletes most of the FERM coding region and introduces an early STOP (Fig. 1a). The second strain that was analyzed contained a mutation in the lin-2 gene, which encodes a membrane-associated guanylate kinase (MAGUK) orthologous to CASK. The lin-2 locus generates two isoforms by the use of two different promoters. LIN-2A contains a CaM-kinase domain, two LIN domains, a PDZ domain, a SH3 domain and a guanylate kinase domain. LIN-2B lacks the N-terminal CaM-kinase domain, and one of the LIN domain (Fig. 1b). The lin-2(kr) retrieved in our screen contained a deletion spanning the nt, which caused a frameshift from valine with subsequent introduction of an early stop codon. Interestingly, frm-3 and lin-2 were formerly shown to impact UNC content at synapses, but the phenotypes that we observed seemed much more dramatic than previously reported³³.

To confirm our observations, we analyzed GABA_ARs by immunostaining³⁴ and detected a strong loss of UNC staining at synapses in both frm-3(0) and lin-2(n) mutants as compared with wild-type (WT) animals (Fig. 1c). We then used the RFP::unc allele to perform a quantitative analysis of synaptic receptor content. In both mutants, fluorescence was decreased by about 80 % in synaptic regions as compared with WT. GABA_ARs were barely detectable, forming extremely small puncta at GABAergic boutons (Figs. 1d–e and Supplementary Fig. 1d). By contrast, the number and size of presynaptic GABA boutons were unaltered based on the quantitative analysis of the synaptic marker SNBGFP specifically expressed in GABAergic motoneurons (Fig. 1d and Supplementary Fig. 1b–c).

In C. elegans, muscle cells send projections to the motoneurons, named muscle arms, and establish en passant synapses at the dorsal and ventral nerve cords. To exclude that GABA_AR decrease might be explained by muscle arm development defects, we analyzed muscle morphology in the frm-3(0) mutant and found that the number and the morphology of muscle arms were normal (Supplementary Fig. 1e–f). The frm-3 mutation could hamper the biosynthesis or stability of GABA_ARs. We therefore measured the protein level of RFP-tagged UNC by western blot and we found that the amount of UNC was normal in frm-3(0) mutant (Supplementary Fig. 1g-h). These results are consistent with previously published electrophysiological recordings showing that bath application of GABA elicits similar currents in wild-type and frm-3(0) mutants³³. To evaluate the functional consequence of frm-3 and lin-2 inactivation on GABAergic transmission, we recorded GABA-evoked currents in muscle upon optogenetic stimulation of GABA motoneurons. As compared with the WT, we observed a 75% and 90% decrease of GABA-evoked currents in frm-3 and lin-2 null mutants, respectively (Fig. 1f–g). Altogether, these data indicate that FRM-3 and LIN-2 are essential for the localization of GABA_ARs at inhibitory NMJs.

To further characterize the contribution of lin-2 to GABA_AR clustering, we analyzed several lin-2 alleles. In lin-2(n), where amino acids of LIN-2A as well as part of LIN-2B N-terminus were deleted, the synaptic fluorescence level of RFP-UNC was reduced by 75%, similar to frm-3(0) mutants (Fig. 1h). Similarly, in lin-2(n), where the last 22 amino acids of the C-terminus common to LIN-2A and LIN-2B were deleted, GABA_ARs were also decreased by 70% at either 20 °C or 25 °C (Fig. 1h), despite the reported thermosensitive vulva-less phenotype of these mutants. Yet the lin-2(e) mutant, which has been used as a reference allele in previous studies³³, only caused a 40% reduction in RFP-UNC fluorescence. Partial information on the lin-2(e) allele suggests that LIN-2A coding sequence is altered but that LIN-2B might still be expressed, since LIN-2B coding sequence and part of the LIN-2B promoter region might be intact (Fig. 1b)^35,36. To further characterize the contribution of individual lin-2 isoforms to GABA_AR clustering, we expressed LIN-2A or LIN-2B in the muscle cells of lin-2(n) mutants and monitored RFP-UNC fluorescence levels. Expressing either LIN-2A or LIN-2B completely rescued the lin-2(n) mutant phenotype, indicating that the domains within the short LIN-2B isoform are sufficient for GABA_AR clustering (Fig. 1h). Altogether, these data indicate that LIN-2 acts cell autonomously to control the clustering of GABA_ARs.

FRM-3 and LIN-2 localize at synapses independently from neuroligin

To analyze the subcellular distribution of FRM-3 and LIN-2, we built a series of single-copy transgenes to express fluorescently tagged isoforms of FRM-3 and LIN-2 in muscle. FRM-3A and LIN-2A muscle-specific reporters formed puncta along the nerve cords (Fig. 2a–c). FRM-3A-GFP and RFP-LIN-2A highly colocalized (Fig. 2d). FRM-3 and LIN-2 puncta localized at GABAergic synapses (Fig. 2a), but were also found in between GABAergic synapses (Fig. 2a, d). Consistently, FRM-3B-GFP and GFP-LIN-2A were also present at cholinergic NMJs (Supplementary Fig. 2a–c).

We previously showed that NLG-1/neuroligin controls the synaptic targeting of GABA_AR²⁷. We confirmed this observation using a novel uncpHluorin knock-in strain, which visualizes only GABA_ARs at the plasma membrane. In nlg-1(0) null mutant animals, the overall fluorescence level of pHluorin-UNC was decreased by 50% and small GABA_AR clusters were fragmented and diffused away from GABA synapses (Fig. 2e–g). We then tested whether NLG-1 controls FRM-3 and LIN-2 positioning at GABAergic synapses. In nlg-1(0) mutants, FRM-3B-GFP, and GFP-LIN-2A were present at excitatory and inhibitory synapses. The fluorescence level of FRM-3B-GFP was unchanged in nlg-1(0) mutants while the RFP-LIN-2 fluorescence level was slightly reduced (Fig. 2h–k). Conversely, NLGGFP fluorescence level was nearly WT in lin-2(0) or frm-3(0) mutants (Fig. 2l). Altogether these data suggest that NLG-1 is not required for synaptic targeting of FRM-3 but might participate to the localization of LIN-2 at GABAergic synapses.

Postsynaptic UNC/DCC recruits FRM-3 independently from LIN-2

We previously showed that UNC/DCC signaling promotes the recruitment of GABA_ARs onto NLG-1 clusters²⁷. Using rfp- and pHluorin-unc knock-in stains, we confirmed that the amount of either total or surface GABA_ARs was reduced in multiple loss-of-function unc allele mutants, including the reference allele e further referred to as unc(0) (Fig. 3a–b and Supplementary Fig. 3a–c). We hypothesized that FRM-3 and LIN-2 might be involved in this mechanism. We first tested if UNC/DCC could regulate the synaptic content of FRM-3 and LIN In unc(0) null mutants, RFP-LIN-2A and FRM-3B-GFP were reduced by approximately 50% and 70%, respectively (Fig. 3a–d), whereas their localization was not changed (Fig. 3e–f). This decrease was not a consequence of GABA_AR decrease since FRM-3B-GFP and GFP-LIN-2 were properly expressed in unc(0) mutants (Supplementary Fig. 4a–e).

FRM-3 and LIN-2 might be both independently regulated by UNC, or one of these two components might be regulated by UNC, and in turn recruit the second one. To distinguish these two hypotheses, we investigated how LIN-2 and FRM-3 regulate each other. We found a strong loss of LIN-2 in frm-3(0) mutants (Fig. 3g–i). By contrast, the FRM-3B isoform, or the FERM-FA domains only, formed proper synaptic puncta in lin-2(n) mutant animals (Fig. 3h, j–k). These observations suggest that UNC controls the synaptic localization of FRM-3, which in turn recruits LIN

We then tested the hierarchy between UNC and FRM Both proteins colocalized at NMJs (Fig. 3l). However, although the removal of UNC reduced FRM-3, the loss of FRM-3 did not affect UNC localization or content (Fig. 3m–n). These data suggested that UNC primarily localizes at synapses and subsequently recruits FRM-3 at synapses. To strengthen this hypothesis, we overexpressed in muscle cells a myristoylated fusion of the intracellular domain of UNC, which acts as constitutively active receptor. Myr-UNC distributes at the entire surface of the cell and causes the budding of exuberant membrane processes^27,37. In unc(0) mutants, FRM-3B-GFP was weakly detected in non-synaptic regions. Co-expression of myr-UNC caused strong accumulation of FRM-3 at the membrane in the budding regions where myr-UNC accumulated (Fig. 3o, p). Altogether, these data suggest that activation of UNC is sufficient to recruit FRM

FERM-FA domain of FRM-3 is required in the muscle cell to control GABA_AR clustering

We investigated the function of the different FRM-3 domains for GABA_AR clustering. Muscle-specific expression of either FRM-3A or FRM-3B in frm-3(0) mutants rescued the synaptic content of GABA_ARs (Fig. 4a). Both isoforms encompass the same N-terminal moiety that contains the predicted FERM and FA domains. Expression of these domains was sufficient to rescue the loss of GABA_ARs in frm-3(0) mutants, while the FERM domain only or the C-terminus of FRM-3B containing the RhoGEF and the two PH domains failed to rescue (Fig. 4a). Consistently, FRM-3A, FRM-3B, and FERM-FA domains were properly targeted to synapses while the FERM domain alone and the C-terminus of FRM-3B were diffusively localized in the muscle near the nerve cord (Fig. 4b). The C-terminus of FRM-3A was present in the nucleus of muscle cells, probably through unmasking of a cryptic nuclear localization signal (Supplementary Fig. 5a–b). Altogether, these data indicate that the FERM-FA domains of FRM-3 are necessary and sufficient to promote GABA_AR clustering.

A recent study reported that the FERM-FA domains of the Drosophila protein Yurt form oligomers, and that multimerization of Yurt supports its function in cell polarity³⁸. Oligomerization relies on several hydrophobic residues within the F3 lobe of the FERM domain, which are conserved in FRM-3 (Supplementary Fig. 1a). We investigated whether the FERM-FA domains of FRM-3 might oligomerize using GST-pull down assays. GST-tagged FERM-FA domains efficiently pulled down HA-tagged FERM-FA (Fig. 4c). As a positive control, we verified that GST-tagged LIN-2 pulled down the HA-tagged FERM-FA domains (Fig. 4c), consistently with a previously reported yeasthybrid interaction between LIN-2 and FRM-3³³. Therefore, the FERM-FA domains of FRM-3 can oligomerize, which might in turn assemble a submembrane lattice promoting GABA_AR clustering.

UNC physically interacts with FRM-3 through its P3 domain

Our data show that activation of UNC can recruit FRM-3 to the plasma membrane. We tested if these two proteins form a complex, first by co-immunoprecipitation experiments using strains expressing UNC and different FRM-3 versions. UNC co-immunoprecipitated with FRM-3B full length or the FERM-FA domains (Fig. 4d–e). Second, we used in vitro GST pulldown assay and detected the direct binding of FERM-FA to the UNC intracellular domain (Fig. 4g). Interestingly, the FERM domain of myosin-X binds the mammalian DCC through its C-terminal P3 domain^39,40. This domain folds into an α-helix which contains hydrophobic residues critical for binding to the FERM domain of myosin X^39,40. Despite poor conservation of the primary sequence, this α-helix can be readily predicted in UNC with a remarkable conservation of hydrophobic residues between UNC and DCC (Fig. 4f)³⁷. To test if P3 is involved in the UNC/FRM-3 interaction, we repeated the in vitro GST pulldown after deleting P3 from the UNC intracellular domain and could no longer detect an interaction with the FERM-FA domain of FRM-3 (Fig. 4g).

The P3 domain of DCC is necessary for intracellular multimerization in vitro and for axonal attraction in Xenopus spinal neurons⁴¹. To test a putative role of P3 at GABA synapses, we replaced the P3 domain of UNC by an HA-tag using CRISPR/Cas9 (Fig. 5a). As compared with unc(e) null mutants that are small and have severe locomotion and egg-laying defects, unc∆P3 mutants had normal body length and exhibited much less defects in locomotion and egg-laying (Fig. 5b). To quantitively measure the remaining activity of UNC∆P3, we scored post-embryonic muscle arm growth and ventral guidance of the mechanosensory AVM axon, which were shown to rely mostly on UNC^20,23. The unc∆P3 mutants had on average 2 muscle arms per muscle cells, as compared with only 1 in unc null mutants and 4 in the wild type (Fig. 5c, d). AVM defects were detected in 15 % of the ∆P3 animals, as compared with 36 % in null mutants (Fig. 5e, f). Hence, deletion of the P3 domain only partially impairs the function of UNC for attraction guidance.